You are on page 1of 694

SRIE LIVROS 28

PORIFERA RESEARCH BIODIVERSITY, INNOVATION AND SUSTAINABILITY

Editors Mrcio Reis Custdio Gisele Lbo-Hajdu Eduardo Hajdu Guilherme Muricy

RIO DE JANEIRO MUSEU NACIONAL 2007

Universidade Federal do Rio de Janeiro Reitor Alosio Teixeira Museu Nacional Diretor Srgio Alex K. Azevedo Comisso de Publicaes do Museu Nacional Editores Miguel Angel Monn Barrios, Ulisses Caramaschi

ISBN 978-85-7427-023-4

Editores de rea Adriano Brilhante Kury, Alexander Wilhelm Armin Kellner, Andrea Ferreira da Costa, Ctia Antunes de Mello Patiu, Ciro Alexandre vila, Dbora de Oliveira Pires, Guilherme Ramos da Silva Muricy, Izabel Cristina Alves Dias, Joo Alves de Oliveira, Joo Wagner de Alencar Castro, Marcela Laura Monn Freire, Marcelo de Arajo Carvalho, Marcos Raposo, Maria Dulce Barcellos Gaspar de Oliveira, Marlia Lopes da Costa Fac Soares, Rita Scheel Ybert, Vnia Gonalves Loureno Esteves Normalizao Vera de Figueiredo Barbosa MUSEU NACIONAL Universidade Federal do Rio de Janeiro Quinta da Boa Vista, So Cristvo, 20940-040 Rio de Janeiro, RJ, Brasil Reviso Fernando Moraes, Raphael Augusto Sims Belleza, Gisele Lbo-Hajdu, Guilherme Muricy Diagramao e arte-final Mrcio Reis Custdio e Beatriz Waller Capa Beatriz Waller Comisso Editorial do Volume Mrcio Reis Custdio Gisele Lbo-Hajdu Eduardo Hajdu Guilherme Muricy Patrocinadores FAPERJ, CNPq, CAPES Apoio Universidade Federal do Rio de Janeiro, Universidade do Estado do Rio de Janeiro, Universidade de So Paulo, PETROBRAS Impresso: IMOS Grfica e Editora Impresso no Brasil / Printed in Brazil

Ficha catalogrfica P836 Porifera research : biodiversity, innovation and sustainability / editors Mrcio Reis Custdio ... [et al.]. Rio de Janeiro : Museu Nacional, 2007 694 p. ; il. ; 28 cm. (Srie Livros ; 28) Inclui bibliografia ISBN 978-85-7427-023-4 1. Esponjas. I. Custdio, Mrcio Reis. II. Museu Nacional (Brasil). III. Srie. CDD 593.4

Preface
This book began to be assembled in the frame of the 7th International Sponge Symposium, held in Armao dos Bzios (Rio de Janeiro, Brazil) in May 2006. Under different names, and with a history almost four decades long now, this series of meetings started in London (1968), followed by Paris (1978), Woods Hole (1985), Amsterdam (1993), Brisbane (1998), Rapallo (2002) and Armao dos Bzios. These are the worlds main international scientific events centered on Porifera. The 7th ISS was the first of this series held in Latin America, and the Museu Nacional of the Universidade Federal do Rio de Janeiro (MN-UFRJ) and the Sociedade dos Amigos do Museu Nacional (SAMN) were honored to organize it. In what seems to be a welcome trend in these meetings, this seventh edition was also the largest so far. Almost 260 participants from 35 countries contributed with 308 presentations, distributed in 14 sessions, and covering all aspects of sponge basic and applied research. But we sincerely hope that these numbers will be superseded in 2010, when the sponge research community is expected to meet again in Spain. Following the lead of our fellow Italians, Maurizio Pansini, Roberto Pronzato, Giorgio Bavestrello and Renata Manconi, editors of the previous volume Sponge Science in the New Millenium (2002), we decided not to divide the book by subject matters. This organization reflects the current tendency of multidisciplinary work in biological sciences, in which ever more studies use different approaches, drawn from several disciplines, to address their hypotheses. Like in all previous volumes, the data presented here will be a valuable, updated reference of the present knowledge for those working with this still largely unknown and fascinating group of animals. Titles bridging a variety of disciplines tend to be unattractive to those conducting sharply focused research. Nevertheless, the focus of ISS meetings has to adjust to a time where borders between disciplines become more and more blurred, not to speak of borders between Phyla! Nonetheless, Porifera constituted the magnet for all the contributions presented in the 7th ISS, and so it is with those published in the following pages of this book. Porifera Research alone would not convey the excitement of organizing the meeting and editing the book, neither would it be fair to all of you who packed your back-packs and suitcases on every continent on Earth to travel to the far meeting ground at Bzios. To reflect this, and in respect to Brazils nearly synonymous significance, first subtitle emerged Biodiversity. But, natures treasures alone are no guarantee of wealth to any nation, and among the many biodiverse countries represented by those who participated in the 7th ISS, most are developing and struggle to generate wealth to their peoples. Accordingly, an unavoidable target became second subtitle - Innovation. Hoping we can all adjust to an era of growing environmental concern, partly as a consequence of fear of the global consequences of increased warming of the planet, our third subtitle wishes to convey the ideals of the editors, as far as exploration of natural resources are concerned Sustainability. In this way, we reached our motto Biodiversity, innovation and sustainability. The book Porifera research: biodiversity, innovation and sustainability begins with a series of twelve invited contributions, not meant to match the books motto, and spanning most fields of research on Porifera, from the paleontology of old Pre-Cambrian rocks to DNA barcoding of recent sponges and its potential effects on the classification of the Phylum. Following, 61 articles are listed in alphabetical order of first authors family name, again spanning a vast spectrum of disciplines. Differently from the other books in the series, this is not strictly a Proceedings volume. We decided to open the possibility for those who were unable to attend the 7th ISS, as well as those who participated, to publish results other than those presented in the meeting. In this way, we expect to present a broad perspective of the contemporary knowledge and future research trends in the group. The 73 manuscripts published in this book contain the work of over 230 coauthors, and were evaluated by more than 100 anonymous peer reviewers, in a process that took over a year. During these procedures, we attempted to assure that contrasting ideas and opinions could be published. To provide a wide distribution of all articles, the whole book is available in PDF format for download without restrictions from the Porifera Brasil website: www.poriferabrasil.mn.ufrj.br. It would have been impossible to organize this book without the help of many persons and sponsors. Our deepest acknowledgements go to the authors of the articles published along these pages, for their scientific contributions, which are the heart of the book. We are also grateful to all reviewers, who spent their time and experience correcting or making clearer the rationales (and often the language) of submitted manuscripts. Their work greatly improved the quality of the book, and we thank you all for your cooperation and patience, and hope that you enjoy the final product as we did. The financial support by some sponsors was essential for the realization of the 7th ISS and publication of this book. Special thanks go to FAPERJ, CAPES, CNPq and CENPES/ PETROBRAS. The logistic support of some institutions was also pivotal. Thanks here go to Museu Nacional (Universidade Federal do Rio de Janeiro), Universidade do Estado do Rio de Janeiro (UERJ), Universidade de So Paulo (USP), Sociedade dos Amigos do Museu Nacional, Hotel Prola Bzios and ABVTUR. We also warmly thank all members of the steering committee: Antnio Sol-Cava, Beatriz Mothes, Carla Silva, Carla Zilberberg, Ceclia Volkmer-Ribeiro, Cla Lerner, Cristiano Coutinho, Michelle Klautau, Radovan

Borojevic, Roberto Berlinck and Solange Peixinho; as well as Andrezj Pisera, Marinella Laport and Sally Leys for additional support. Finally, we could not possibly forget the several volunteers who helped us in preparing and running the conference: Andr Rossi, Barbara Andrea, Carla Zilberberg, Daniela Batista, Daniela Lopes, Emiliano Calderon, Emlio Lanna, Fernanda Azevedo, Fernanda Cavalcanti, Fernando de Moraes, Guilherme Maia, Gustavo da Silva, Karina Hajdu, Leandro Monteiro, Mara de Oliveira, Mariana Carvalho, Maurcio de Campos, Suzi Ribeiro and Viviane Santos. Looking back in time, two moments were crucial for the making of the 7th ISS, and consequently for the publication of this book. First of these, the gathering in Rio de Janeiro, back in 1987, of Professors Nicole Boury-Esnault, Solange

Peixinho, Radovan Borojevic and Antnio Sol-Cava, to teach a course on sponge biology to a group of young undergraduates of Universidade Federal do Rio de Janeiro. Among these, three editors of this book. Remembering these first steps is a much deserved honor we are obliged to render. Secondly, the many demands for a meeting in Rio de Janeiro, starting in Amsterdam, at the occasion of the 4th ISS in 1993, a time when the four editors of this volume were still half way in their PhDs. We hope that this book will not only provide an update of achievements in most fields of inquiry regarding sponges, but also be a fertile ground for the birth of new questions, debates and ideas for future endeavours on Porifera Research spread worldwide.

The Editors
Mrcio Reis Custdio Gisele Lbo-Hajdu Eduardo Hajdu Guilherme Muricy

Table of contents

Invited Articles
Pedro M. Alcolado Reading the code of coral reef sponge community composition and structure for environmental bio-monitoring: some experiences from Cuba ....................................................... 3-10 Matilde Sylvia Beresi Fossil sponges of Argentina: a review ...........................................................................................11-21 Nicole Boury-Esnault, Chantal Bzac Morphological and cytological descriptions of a new Polymastia species (Hadromerida, Demospongiae from the North-West Mediterranean Sea ............................................................. 23-30 Maria Cristina Diaz, Robert W. Thacker, Klaus Rtzler, Carla Piantoni Two new haplosclerid sponges from Caribbean Panama with symbiotic filamentous cyanobacteria, and an overview of sponge-cyanobacteria associations ....................................... 31-39 Alexander V. Ereskovsky Sponge embryology: the past, the present and the future ............................................................. 41-52 Sally P. Leys Sponge coordination, tissues, and the evolution of gastrulation .................................................. 53-59 Renata Manconi, Roberto Pronzato Gemmules as a key structure for the adaptive radiation of freshwater sponges: a morphofunctional and biogeographical study ........................................................................................... 61-77 Gradimir N. Misevic, Camille Ripoll, Jonathan Norris, Vic Norris, Yann Guerardel, Emmanuel Maes, Gerard Strecker, Pascal Ballet, Yannis Karamanos, Lazar T. Sumanovski, Octavian Popescu, Nikola Misevic Evolution of multicellularity in Porifera via self-assembly of glyconectin carbohydrates .......... 79-88 Werner E.G. Mller, Isabel M. Mller Porifera: an enigmatic taxon disclosed by molecular biology/cell biology ............................... 89-106 Jean Vacelet Diversity and evolution of deep-sea carnivorous sponges ........................................................107-115 Ceclia Volkmer-Ribeiro South American continental sponges: state of the art of the research .......................................117-121 Gert Wrheide, Dirk Erpenbeck, Christian Menke The Sponge Barcoding Project: aiding in the identification and description of poriferan taxa ........................................................................................................................................... 123-128

ii

Research Articles
Brbara R. Andra, Daniela Batista, Cludio L.S. Sampaio, Guilherme Muricy Spongivory by juvenile angelfish (Pomacanthidae) in Salvador, Bahia State, Brazil .............. 131-137 William C. Austin, Kim W. Conway, J. Vaughn Barrie, Manfred Krautter Growth and morphology of a reef-forming glass sponge, Aphrocallistes vastus (Hexactinellida), and implications for recovery from widespread trawl damage .................... 139-145 Enrique vila, Jos Lus Carballo, Jos Antonio Cruz-Barraza Symbiotic relationships between sponges and other organisms from the Sea of Cortes (Mexican Pacific coast): same problems, same solutions ........................................................ 147-156 Francesca Azzini, Barbara Calcinai, Carlo Cerrano, Giorgio Bavestrello, Maurizio Pansini Sponges of the marine karst lakes and of the coast of the islands of Ha Long Bay (North Vietnam)........................................................................................................................ 157-164 Kristina Bayer, Susanne Schmitt, Ute Hentschel Microbial nitrification in Mediterranean sponges: possible involvement of ammoniaoxidizing Betaproteobacteria .................................................................................................... 165-171 Leontine E. Becking, Yoichi Nakao, Nicole J. de Voogd, Rob W.M. van Soest, Nobuhiro Fusetani, Shigeki Matsunaga Perplexing distribution of 3-alkylpyridines in haplosclerid sponges ....................................... 173-178 Sergey I. Belikov, Oksana V. Kaluzhnaya, Heinz C. Schrder, Isabel M. Mller, Werner E.G. Mller Lake Baikal endemic sponge Lubomirskia baikalensis: structure and organization of the gene family of silicatein and its role in morphogenesis ........................................................... 179-188 Marco Bertolino, Laura Schejter, Barbara Calcinai, Carlo Cerrano, Claudia Bremec Sponges from a submarine canyon of the Argentine Sea ......................................................... 189-201 Barbara Calcinai, Francesca Azzini, Giorgio Bavestrello, Laura Gaggero, Carlo Cerrano Excavating rates and boring pattern of Cliona albimarginata (Porifera: Clionaidae) in different substrata ..................................................................................................................... 203-210 Emiliano Nicolas Calderon, Carla Zilberberg, Paulo Csar de Paiva The possible role of Echinometra lucunter (Echinodermata: Echinoidea) in the local distribution of Darwinella sp. (Porifera: Dendroceratida) in Arraial do Cabo, Rio de Janeiro State, Brazil ...................................................................................................................211-217 Maurcio Campos, Beatriz Mothes, Cla Lerner, Joo Lus Carraro, Inga Ludmila Veitenheimer-Mendes Sponges (Porifera, Demospongiae) from Bransfield strait, off Joinville Island, collected by Brazilian Antarctic Program - PROANTAR ....................................................................... 219-232 Victor Ribeiro Cedro, Eduardo Hajdu, Hilda Helena Sovierzosky, Monica Dorigo Correia Demospongiae (Porifera) of the shallow coral reefs of Macei, Alagoas State, Brazil ........... 233-237

iii

Carlo Cerrano, Barbara Calcinai, Cristina Gioia Di Camillo, Laura Valisano, Giorgio Bavestrello How and why do sponges incorporate foreign material? Strategies in Porifera ...................... 239-246 Andia Chaves-Fonnegra, Sven Zea Observations on reef coral undermining by the Caribbean excavating sponge Cliona delitrix (Demospongiae, Hadromerida) .................................................................................... 247-254 Mark Chiappone, Leanne M. Rutten, Steven L. Miller, Dione W. Swanson Large-scale distributional patterns of the encrusting and excavating sponge Cliona delitrix Pang on Florida Keys coral substrates ......................................................................... 255-263 Steve de C. Cook Clarification of dictyoceratid taxonomic characters, and the determination of genera ............ 265-274 Bruno Cosme, Solange Peixinho A new species of Stelletta (Astrophorida: Demospongiae) with a redescription and distribution range expansion for Stelletta kallitetilla in the Southwestern Atlantic Region .... 275-280 Cristiano C. Coutinho, Guilherme de Azevedo Maia Mesenchymal cells in ancestral spongiomorph urmetazoa could be the mesodermal precursor before gastrulation origin ......................................................................................... 281-295 Alan R. Duckworth, Carsten Wolff, Elizabeth Evans-Illidge Developing methods for commercially farming bath sponges in tropical Australia ................ 297-302 Hermann Ehrlich, Hartmut Worch Sponges as natural composites: from biomimetic potential to development of new biomaterials .............................................................................................................................. 303-312 Elthon G. Ferreira, Diego V. Wilke, Paula C. Jimenez, Tiago A. Portela, Edilberto R. Silveira, Eduardo Hajdu, Cludia Pessoa, Manoel O. de Moraes, Letcia V. Costa-Lotufo Cytotoxic activity of hydroethanolic extracts of sponges (Porifera) collected at Pedra da Risca do Meio Marine State Park, Cear State, Brazil ............................................................. 313-318 Christopher J. Freeman, Daniel F. Gleason, Rob Ruzicka, Rob W.M. van Soest, Alan W. Harvey, Greg McFall A biogeographic comparison of sponge fauna from Grays Reef National Marine Sanctuary and other hard-bottom reefs of coastal Georgia, U.S.A. ......................................... 319-325 Elena I. Gerasimova, Alexander V. Ereskovsky Reproduction of two species of Halichondria (Demospongiae: Halichondriidae) in the White Sea.................................................................................................................................. 327-333 Deborah J. Gochfeld, Carmen Schlder, Robert W. Thacker Sponge community structure and disease prevalence on coral reefs in Bocas del Toro, Panama ..................................................................................................................................... 335-343 Elisaveta L. Gonobobleva Basal apparatus formation in external flagellated cells of Halisarca dujardini larvae (Demospongiae: Halisarcida) in the course of embryonic development.................................. 345-351

iv

Eduardo Hajdu, Daniela A. Lopes Checklist of Brazilian deep-sea sponges .................................................................................. 353-359 Isabel Heim, Michael Nickel, Franz Brmmer Molecular markers for species discrimination in poriferans: a case study on species of the genus Aplysina .......................................................................................................................... 361-371 Isabel Heim, Jrg U. Hammel, Michael Nickel, Franz Brmmer Salting sponges: a reliable non-toxic and cost-effective method to preserve poriferans in the field for subsequent DNA-work ......................................................................................... 373-377 Friederike Hoffmann, Eberhard Sauter, Oliver Sachs, Hans Ry, Michael Klages Oxygen distribution in Tentorium semisuberites and in its habitat in the Arctic deep sea ....... 379-382 Valeria Itskovich, Sergey Belikov, Sofia Efremova, Yoshiki Masuda, Thierry Perez, Eliane Alivon, Carole Borchiellini, Nicole Boury-Esnault Phylogenetic relationships between freshwater and marine Haplosclerida (Porifera, Demospongiae) based on the full length 18S rRNA and partial COXI gene sequences .......... 383-391 Michelle Kelly, Michael Ellwood, Lincoln Tubbs, John Buckeridge The Lithistid Demospongiae in New Zealand waters: species composition and distribution................................................................................................................................ 393-404 Anne Kuusksalu, Madis Metsis, Tnu Reintamm, Merike Kelve Construction and characterization of a cDNA library from the marine sponge Chondrosia reniformis .............................................................................................................. 405-412 Emilio Lanna, Leandro C. Monteiro, Michelle Klautau Life cycle of Paraleucilla magna Klautau, Monteiro and Borojevic, 2004 (Porifera, Calcarea) ................................................................................................................................... 413-418 Nathan Lemoine, Nicole Buell, April Hill, Malcolm Hill Assessing the utility of sponge microbial symbiont communities as models to study global climate change: a case study with Halichondria bowerbanki ....................................... 419-425 Daniela Marques, Marise Almeida, Joana Xavier, Madalena Humanes Biomarkers in marine sponges: acetylcholinesterase in the sponge Cliona celata .................. 427-432 Diana M. Mrquez, Sara M. Robledo, Alejandro Martnez Antileishmanial epidioxysterols from extracted sterols of the Colombian marine sponge Ircinia campana ........................................................................................................................ 433-437 Mirna Mazzoli-Dias, Suzi M. Ribeiro, Patricia Oliveira-Silva Foraminifera associated to the sponge Mycale microsigmatosa in Rio de Janeiro State, southeastern Brazil - an initial approach .................................................................................. 439-442 Elizabeth L. McLean, Paul M. Yoshioka Associations and interactions between gorgonians and sponges.............................................. 443-448 Larisa L. Menshenina, Konstantin R. Tabachnick, Daniela A. Lopes, Eduardo Hajdu Revision of Calycosoma Schulze, 1899 and finding of Lophocalyx Schulze, 1887 (six new species) in the Atlantic Ocean (Hexactinellida, Rossellidae) ........................................... 449-465

Fernando Moraes, Guilherme Muricy A new species of Erylus (Geodiidae, Demospongiae) from Brazilian oceanic islands............ 467-475 Beatriz Mothes, Manuel Maldonado, Rafael Eckert, Cla Lerner, Maurcio Campos, Joo Lus Carraro A new species of Characella (Demospongiae, Astrophorida, Pachastrellidae) from the south Brazilian continental shelf .............................................................................................. 477-482 Yulia I. Mukhina, Vadim V. Kumeiko, Olga I. Podgornaya, Sofia M. Efremova The events of metamorphosis in the demosponge Halisarca dujardini Johnston, 1842, studied with immunocytochemical method .............................................................................. 483-490 Marisa H. Nicolai, Ana Esteves, Marise Almeida, Madalena Humanes Haloperoxidase from the marine sponge Erylus discophorus (Schmidt, 1862) ....................... 491-496 Ronald Osinga, Michiel Kotterman Ferric iron promotes the formation of oscules: observations on sponges in aquaria ............... 497-502 Lorenzo Parma, Dario Fassini, Giorgio Bavestrello, Iain C. Wilkie, Francesco Bonasoro, Daniela Candia Carnevali Ecology and physiology of mesohyl creep in Chondrosia reniformis ..................................... 503-508 Solange Peixinho, Jlio Fernandez, Mara V. Oliveira, Simone Cares, Eduardo Hajdu Description of two new species of Acanthotetilla Burton, 1959 from NE Brazil, Southwestern Atlantic (Tetillidae, Spirophorida, Demospongiae) ........................................... 509-515 Henry M. Reiswig, Welton L. Lee A new species of Cladorhiza (Porifera: Cladorhizidae) from S. California (USA)................... 517-523 Pilar Ros, Javier Cristobo Sponges of genus Myxilla Schmidt, 1862, collected in Antarctic waters by Spanish Antarctic expeditions ................................................................................................................ 525-546 Pablo R.D. Rodriguez, Guilherme Muricy A new species of Cinachyra (Demospongiae: Tetillidae) collected by Project REVIZEE off Esprito Santo State, SE Brazil ........................................................................................... 547-553 Adriana Salgado, Thomz Vieiralves, Flvia R.M. Lamaro, Leonardo L.M. Assumpo, Dbora Gomes, Lia Jascone, Ana Luiza Valado, Rodolpho M. Albano, Gisele Lbo-Hajdu Field preservation and optimization of a DNA extraction method for Porifera ....................... 555-560 Susanne Schmitt, Markus Wehrl, Niels Lindquist, Jeremy B. Weisz, Ute Hentschel Morphological and molecular analyses of microorganisms in Caribbean reef adult sponges and in corresponding reproductive material ............................................................................. 561-568 Christine H.L. Schnberg, Ryota Suwa Why bioeroding sponges may be better hosts for symbiotic dinoflagellates than many corals......................................................................................................................................... 569-580

vi

Heinz C. Schrder, Anatoli Krasko, David Brandt, Matthias Wiens, Muhammad Nawaz Tahir, Wolfgang Tremel, Werner E.G. Mller Silicateins, silicase and spicule-associated proteins: synthesis of demosponge silica skeleton and nanobiotechnological applications ...................................................................... 581-592 Carla M.M. da Silva, Meiryelen V. da Silva, Bruno Cosme Redescription of the Brazilian endemic sponge Geodia glariosa (Demospongiae: Geodiidae), with new records on its geographic and bathymetric distribution ........................ 593-602 Antonio M. Sol-Cava, Gert Wrheide The perils and merits (or The Good, the Bad and the Ugly) of DNA barcoding of sponges a controversial discussion ........................................................................................ 603-612 Frank Spetland, Hans Tore Rapp, Friederike Hoffmann, Ole Secher Tendal Sexual reproduction of Geodia barretti Bowerbank, 1858 (Porifera, Astrophorida) in two Scandinavian fjords ....................................................................................................... 613-620 Robert W. Thacker, Maria Cristina Diaz, Klaus Rtzler, Patrick M. Erwin, Steven J.A. Kimble, Melissa J. Pierce, Sandra L. Dillard Phylogenetic relationships among the filamentous cyanobacterial symbionts of Caribbean sponges and a comparison of photosynthetic production between sponges hosting filamentous and unicellular cyanobacteria ............................................................................... 621-626 Carsten Thoms, Peter J. Schupp Chemical defense strategies in sponges: a review .................................................................... 627-637 Laura Valisano, Attilio Arillo, Giorgio Bavestrello, Marco Giovine, Carlo Cerrano Influence of temperature on primmorph production in Petrosia ficiformis (Porifera: Demospongiae) ........................................................................................................ 639-643 Rob W.M. van Soest, Fleur C. van Duyl, Connie Maier, Marc S.S. Lavaleye, Elly J. Beglinger, Konstantin R. Tabachnick Mass occurrence of Rossella nodastrella Topsent on bathyal coral reefs of Rockall Bank, W of Ireland (Lyssacinosida, Hexactinellida) ................................................................ 645-652 Eduardo Vilanova, Carla Zilberberg, Michele Kochem, Mrcio R. Custdio, Paulo A.S. Mouro A novel biochemical method to distinguish cryptic species of genus Chondrilla (Chondrosida, Demospongiae) based on its sulfated polysaccharides ..................................... 653-659 Author index.................................................................................................................................. 661-663 Subject index ................................................................................................................................. 665-679 Participants list ............................................................................................................................. 680-684

INVITED ARTICLES

Porifera research: Biodiversity, innovation and sustainaBility - 2007

Reading the code of coral reef sponge community composition and structure for environmental biomonitoring: some experiences from Cuba
Pedro M. Alcolado
Instituto de Oceanologa, Ave. 1ra, No. 18406, Playa, La Habana, Cuba. alcolado@ama.cu Abstract: The structure of exposed (non-cryptic) coral reef sponge communities could be considered as a potentially readable coded message reflecting their physical environment. The present paper describes explorations in Cuba of the potential use of sponge communities as bio-indicators. Clathria venosa is the sponge that most consistently has proved to be a bioindicator of urban based pollution in Cuban coral reefs due to its stenotopic character with regard to this stress source. Iotrochota birotulata forma musciformis was abundant close to the polluted Havana Bay, but not in other polluted sites, making it inconsistent as indicator. It has been quite rare in non-polluted waters. Cliona delitrix was represented in an area with great sewage influence. However it did not appear in some polluted sites probably because corals were extremely scarce and small. Scopalina ruetzleri was well represented close to bays with different degrees of urban based pollution. Cliona varians was well represented only in one polluted place. Multivariate analyses (cluster analysis, non-metric multidimensional scaled analysis) have proved to be very useful tools to clearly segregate sites with regard to level of pollution, and to identify factors and interactions determining community structure and composition. Abundance or dominance of Tectitethya crypta and Cliona vesparia (alpha stage) were typical of heavy sedimentation conditions; while Aplysina cauliformis tended to dominate in sites affected by both hurricanes and sedimentation (abundance increased by fragmentation). Meta-analysis of Shannons heterogeneity index H and Pielous equitability index J is proposed as a useful tool to classify and compare sites with regard to the way that sponges interpret their environment (degree of severity and predictability). Meta-analysis by means of a scatter graph with ranges of H at different depths provides a spatial framework for comparing and classifying sponge communities with regard to environment severity. Keywords: sponges, bio-indicators, coral reefs, Cuba

Introduction
Many papers have dealt with the factors and interactions that determine sponge distribution and community characteristics (partly reviewed by Sar and Vacelet 1973, Bergquist 1978, Wulff 2006), but few have been explicitly devoted to exploring the potential usefulness of sponge communities as bio-indicators for environmental bio-monitoring purposes. In the last few decades, the search for bio-indicators has become an urgent need in a world environment that is changing at an unprecedented rate. According to Alcolado (1984; with some added arguments), sessile taxa are suitable as potential environmental bio-indicators because: - They must be adapted to the environment due to their immobility. Thus, their abundance or their presence (or even absence) must reflect the average ecological conditions, or very recent strong stressful events. - Their composition and community structure are not affected by migrations or local displacements. - The exposed (non-cryptic) sponge communities, having passed the fish predation filter thanks to deterrence (Wulff 1997), are influenced more by the physical environment

than by ecological interactions within themselves (sensu Bradbury 1977). Cooperation rather than competition seems to be the rule among sponge populations (Sar 1970) and, according to Rtzler (1970), sponges are able to solve competition by entering into complex epizoic relationships, without detriment to their pumping and filtering activities. Reiswig (1973) adds that small sponge individuals (during the first year after settlement) are subject to severe mortality by competition with other sessile organisms, but when sponges reach greater volume competitors have little further effect. On the other hand, sponges overgrow corals much more frequently than the reverse, although when the reverse occurs, the sponge tissue shows no adverse effect (Jackson and Buss 1975). - The absence of food partitioning mechanisms influencing community structure. Such features favor sponges over many other zoological groups as potential indicators. That does not mean that there could not be some degree of influence of biological interactions, but apparently to a much lower extent than the physical environment (light, waves, sediments, pollution) in building up the community structure and composition. This

also makes community structure and composition easier to analyze and to understand in a bio-monitoring context. For these reasons, the structure of exposed (non-cryptic) coral reef sponge communities could be considered as a potentially readable coded message reflecting how sponges interpret their physical environment. Indeed, sponges have been suggested as potential environmental bio-indicators by Alcolado (1984, 1985, 1990, 1992, 1994, 1999), Alcolado and Herrera (1987), Muricy (1989, 1991), Zea (1994), Alcolado et al. (1994), Carballo et al. (1994, 1996), Carballo and Naranjo (2001), and Vilanova et al. (2004). Some attempts and successes in Cuba and other countries exploring the potential use of sponge communities as simpler, faster and lower cost bio-indicators (from a sponge life perspective) are discussed below. The results compiled in this review come from a great number of coral reef sites sampled around Cuba since 1976.

Discussion Indicator species


A few sponge species have been found to be associated with polluted or relatively unpolluted conditions in coral reefs (Table 1). Particularly, Clathria venosa (Alcolado, 1984) and Iotrochota birotulata forma musciformis (Duchassaing and Michelotti, 1864) have only been observed dominating in fore-reefs (10-20 m deep) affected by organic pollution (Alcolado and Herrera 1987) (Table 1; Fig. 1). The first species appeared to be markedly stenotopic of enriched inshore and coral reef waters and its occurrence has been very consistent in all polluted reefs evaluated or visited in northwestern Cuba (close to Havana Bay, Almendares and Quib rivers, and the town of Santa F), and according to Zea (1994), also in Santa Marta, Colombia. This species was previously reported by Hechtel (1965) on shells and piling in the enriched waters of Port Royal (southern shore of Kingston Harbor), Jamaica (as Microciona microchela n. sp.); and by van Soest (1984) in the fouling community

on dead corals and gorgonians at the bay and Hilton Hotel Landing of Curaao (as Rhaphidophlus raraechelae n. sp.). It was also found in the fouling communities of the concrete dock of Marina Barlovento (organically enriched site) and the seawall of a small organically polluted cove (Rada del Instituto de Oceanologa), both in the western Havana City, Cuba. However, I. birotulata forma musciformis was not consistently dominant or abundant in the visited Cuban polluted sites. Mycale microsigmatosa Arndt, 1927, which has been found dominating under domestic sewage stress in Brazil (Muricy 1989), was also found in a very polluted coastal lagoon at Jaimanitas Town, west of Havana city (muddy/ algal bottom) together with well developed Suberites aurantiaca (Duchassaing and Michelotti, 1964), Chondrilla aff. nucula Schmidt, 1862 and Halichondria melanadocia de Laubenfels, 1936. Both S. aurantiaca and C. aff. nucula are bacteriosponges (Rtzler 2002), which could explain their abundance in this lagoon. Holmes (1997, 2000), Holmes et al. (2000) and Rtzler (2002), comment on the increased abundance and activity of boring sponges in areas affected by urban based pollution. Indeed, Cliona delitrix Pang, 1973, a species reported as abundant in areas submitted to sewage pollution (Rose and Risk 1985, Chvez-Fonnegra and Zea 2006), was observed during four years by Marcos and Alcolado (unpublished observations) with significant relative abundance (%) at a fore-reef site close to both the polluted Quib River and a nearby sewage outfall (western Havana City). However, it was not found by Alcolado and Herrera (1987) at stations near Havana Bay, maybe due to the scarcity and small size of corals (dominated by Siderastrea radians Pallas, 1766). Another boring sponge, Cliona varians (Duchassaing and Michelotti, 1864), was well represented only in a polluted fore-reef close to both the Quib River and a nearby sewage outfall at western Havana City (Marcos and Alcolado unpublished observations). However, it was also common

Table 1: Potential indicator species and their respective inferred condition according to authors. D and M = Duchassaing and Michelotti. Dominant or abundant species Clathria venosa (Alcolado, 1984) Iotrochota birotulata f. musciformis (D. and M., 1864) Scopalina ruetzleri (Wiedenmayer, 1977) Cliona delitrix Pang, 1973 Mycale microsigmatosa Arndt, 1927 Amphimedon viridis D. and M., 1864 Aplysina fistularis (Pallas, 1766) Cliona caribbaea D. and M., 1864 Cliona vesparia (Lamarck, 1815) (alpha stage) Tectitethya crypta (de Laubenfels, 1949) Aplysina fulva (Pallas, 1766) Aplysina cauliformis (Carter, 1882) Indicated condition Organic pollution Organic pollution Moderate organic pollution Sewage pollution Sewage (bacterial) pollution Sewage pollution Sewage pollution Comparatively non-polluted Comparatively non-polluted Sedimentation plus wave stress Sedimentation stress Strong waves Eventual strong waves and sedimentation Author Alcolado and Herrera (1987) Alcolado and Herrera (1987) Alcolado and Herrera (1987); Muricy (1989); Zea (1994) Muricy (1989) Rose and Risk (1985) Muricy (1989) Muricy (1989) Alcolado (present paper, Fig. 1) Lpez-Victoria and Zea (2004) Alcolado (present paper) Alcolado and Gotera (1985) Wulff (1995) Alcolado (present paper)

Fig. 1: Relative abundances of potential pollution bioindicators species, presented as percentages of total sponge abundance (number of individuals), at stations located at different distances from two main pollution sources in the north-western Cuba (Havana Bay and Almendares River). Mariel Bay is not significantly polluted.

in non-polluted reef areas, which makes it inconsistent as a potential bio-indicator. Lpez-Victoria and Zea (2004) showed that the abundance of Cliona caribbaea is not related to pollution in San Andrs Archipelago, Colombia. Indeed, this species did not occur at sites close to the organically polluted Quib River and the nearby sewage outfall, but only in more distant sites (Marcos and Alcolado, unpublished observations). Other sponge species have been associated with factors other than pollution, namely sedimentation and wave stress (Table 1). Particularly, Aplysina cauliformis is apparently tolerant to strong waves, as can be deduced from its dominance in coral reef sites exposed to more frequent tropical storms (keys Juan Garca and Cantiles, southwestern Cuba). This can be due to its branching morphology, flexibility and elasticity, similar to what was suggested by Wulff (1995) for Aplysina fulva, also branching and with rather similar consistency. The suggested usefulness of the presence or abundance of some sponges as environmental indicators has been based much on expert observation and on inferences related to distance from known pollution sources, wave and wind exposure, visual evidence of varying intensity of sedimentation, etc. For that reason, to validate these results and make further progress, more evidence is necessary, obtained both from well designed experiments and from multivariate analysis in which factors are directly measured on appropriate temporal and spatial scales. Additionally, more sites in the Wider Caribbean, suffering various degrees of pollution, tropical storm frequency, exposure to waves and dominant winds, etc., are worth being researched to test the generality of the mentioned findings. It would be of particular

interest to determine if Clathria venosa feeds on bacteria with emphasis on enteric taxa, as does Clathria prolifera (Ellis and Solander, 1786) according to Claus et al. (1967).

Community indices
In agreement with other authors (Muricy 1989, Carballo et al. 1996, among others), Alcolado and Herrera (1987) found that species richness and Shannons heterogeneity index H were lower at more polluted sites (Fig. 2). Pielous equitability index J was also lower in the more polluted sites close to the mouth of Almendares River (Fig. 2). Given that a condition of significant stress can be inferred only when the dominance of some of the mentioned indicator species (Table 1) is coupled with low values of species richness or species heterogeneity (Alcolado et al. 1994), these univariate indices have to be taken into account as an important complement for environmental monitoring. The summing up of the numerical percentages of individuals belonging to species that are tolerant to the same kind of stressor (e.g., pollution, sedimentation, turbulence, etc.) could be useful as another potential community index for monitoring purposes, as done by Alcolado (1981) with gorgonians to infer relative turbulence intensity, and by Herrera-Moreno (1991), also with gorgonians, to infer relative organic pollution level. The usefulness and conceptual validity of diversity indices has been controversial (Hurlbert 1971, Peet 1974). However (without disregarding potential pitfalls), the herein explored diversity indices can be used and tested pragmatically and heuristically for bio-monitoring purposes in the context of environmental management, not specifically for advancing

Fig. 2: H and J in stations located at different distances from two main pollution sources in the north-western Cuba (Havana Bay and Almendares River). Mariel Bay is not significantly polluted.

science (but being increasingly supported by scientific research). The same validation and progress efforts can be applied to community indices.

Environmental severity and predictability inference graph


Preston and Preston (1975) deserve the credit for integrating and applying theoretical criteria from classic community ecology in a simple and practical scheme to infer the environmental severity and predictability (constancy) for comparative purposes. According to Margalef (1963, 1968) and Odum (1969), high species diversity and high equitability are generally associated with mature, late successional stages. On the other hand, Sanders (1969) and Slobodkin and Sanders (1969) hypothesize that severe environments generally permit less diversity to develop than favorable ones do. As suggested by Slobodkin and Sanders (1969) and supported by Preston and Preston (1975), the degree of stress is determined primarily by the degree of temporal predictability of environmental conditions and the degree of physiological stress imposed by the physical environment. After Preston and Preston (1975), by means of the values of H and J three different ecological situations can be inferred: high values of both indices suggest a favorable and predictable environment; a low H coupled to a high J indicates a constantly severe environment; and low values of both indices reflect an unpredictably severe environment. Due to the fact that this scheme excessively reduces the real variety of situations and leads to misinterpretation of

the specific case of extremely low values of both indices, it was modified by Alcolado (1992) for sponges (Fig. 3). This modification consisted of an inference diagram obtained from a scatter graph of pairs of H and J values from 112 sites. The resulting scatter area was divided into 11 environmental inference zones or classes reflecting corresponding ecological situations instead of Preston and Prestons (1975) original three zones or classes (constantly favorable, constant or temporally predictable stress and unpredictable stress). Except for the class 1 of the scale, which is a qualitative addition to the original scheme, the remaining classes resulted from subdividing the original overly inclusive classes. This resulted in a finer grain of environmental situations to infer, matching more closely the relatively wide range of withinclass environmental variability perceived in the field by the author within the original three classes (e.g., sponge size, coral size and cover, wind and wave exposure, etc.). The number of subdivisions preferred among different persons would certainly vary, as happens with the different temperature measurement scales (Celsius, Fahrenheit and Kelvin). The new scale comprises the following environmental severitypredictability classes: 1 = environment extremely stressed by both, a constant basal level of disturbance and intermittent unpredictable strong events (H = 0-1.3 natural bells; J = 00.5); 2 = very severe and unpredictable environment (H = 0-1.3 natural bells; J = 0.5-0.69); 3 = severe and unpredictable environment (H = 1.3-2.0 natural bells; J = 0.5-0.69); 4 = almost constantly severe environment (H = 1.3-2.0 natural bells; J = 0.7-0.8); 5 = constantly severe environment

(e.g., Cliona aprica, among sponges, Gorgonia flabellum Linnaeus, 1758, among gorgonians, and Acropora palmata Lamarck, 1816, among scleractinians). For that reason, both J an H show extremely low values. This combination, within Preston and Prestons (1976) original scheme, would suggest an unpredictable environment (with its constant component omitted). Alcolados (1992) inference diagram also differentiates the very favorable and constant environments of the deep reefs (e.g., at 20-30 m) within the rank 11, from those that are simply favorable and quasi-constant (rank 10). Nevertheless, it is advisable to be aware of specific situations of very longterm environmental stability where some species can escape from demographic control and become excessively dominant, and consequently diminishing H and J. This situation is common at reef sites deeper than 25 m. This phenomenon of community senescence is not contemplated in either of the two mentioned inference methods and has to be taken into account in supposedly extremely constant environments (e.g., deep reef zones, and reefs where hurricanes are very rare, as those of Bonaire and Tobago). The authors scale is proposed as an alternative reference (among other possible ones) and could be tested and improved with further research. More sites across the Wider Caribbean should be studied and included in the scatter graph to refine its spatial contour.

Scatter graphs for comparing community indices at different depths


Fig. 3: Inference diagram reflecting eleven ways in which sponges interpret their physical environment, derived from a meta-analysis with 112 coral reef stations. 1 = extremely severe with mixture of constant and unpredictable environment; 2 = very severe and unpredictable; 3 = severe and unpredictable; 4 = quasiconstantly severe; 5 = constantly severe; 6 = moderately severe and unpredictable; 7 = moderately severe and quasi-constant; 8 = moderately severe and constant; 9 = favorable and quasi-constant; 10 = constantly favorable; and 11 = very favorable and constant.

(H = 1.3-2.0 natural bells; J = 0.8-1); 6 = moderately and unpredictably severe environment (H = 2.0-2.5 natural bells; J = 0.5-0.69); 7 = moderately and almost constantly severe environment (H = 2.0-2.5 natural bells; J = 0.7-0.8); 8 = moderately and constantly severe environment (H = 2.0-2.5 natural bells; J = 0.8-1); 9 = favorable and almost constant environment (H = 2.5-2.9 natural bells; J = 0.7-0.8); 10 = favorable and constant environment (H = 2.5-2.9 natural bells; J = 0.8-1); and 11 = very favorable and constant environment (H >2.9 natural bells; J = 0.8-1). Rank 1 shows a (qualitative) situation that is not considered by Preston and Preston (1975). This is the case of the surf zones of some Cuban reefs, which have constant average (basal or chronic) conditions of fairly strong wave action, but which are also unpredictably affected by severe impacts of tropical storms. Under these circumstances, there is only one predominant species within each sessile taxon

Scatter graphs of variability of sponge diversity indices, population density and cover with regard to depth were obtained for many Cuban reef sites (Alcolado 1994, 1999). These graphs, which display the area (range) of variation of those indices with regard to depth, can be used as a reference pattern to infer in a comparative way the community condition within stress gradients, taking into account site depth, given that such indices do not necessarily behave in the same way along depth gradients. What is normally a moderate value of H for a given depth could be considered a high value for a lower depth. The upper border of the variation area (an ascending convex line with a slight diminution at depths greater than 25 m) reflects the best conditions registered at different depths for sponge species richness and species heterogeneity (Fig. 4), while the lower border (an asymptotically ascending curve) shows the worst environmental conditions at different depths (Alcolado 1994). Care must be taken at deep reef stations (about 30 m depth or more), as lower diversities can be caused by extremely constant and favorable conditions that lead to the dominance of competitively stronger species, and not by any stressor. The same recommendations given for the environmental severity and predictability inference graph are applicable here.

Classification and ordination


Classification (Fig. 5) and ordination (Fig. 6) analyses have proved to be useful when using sponge communities to separate sites with regard to degree of pollution and

Fig. 4: Example of meta-analysis as a scatter graph of H values at different depths, with classification bands of inferred environmental conditions (from 112 coral reef sites of Cuba). Care must be taken at stations about 30 m depth or more, as lower diversities can be determined by extremely constant and favorable conditions leading to dominance of very competitive species, and not by severe environmental conditions. Arrows indicate more polluted stations (10 m depth) and stations affected by sediments (deeper stations).

Fig. 6: MDS analysis segregating stations located at different distances from two main pollution sources (Havana Bay and Almendares River) with regard to degree of pollution (from left to right: very polluted, polluted, and little polluted). This analysis was done with quadratic transformation of sponge densities and Bray Curtis similarity Index.

Fig. 5: Cluster analysis segregating stations located at different distances from two main pollution sources (Havana Bay and Almendares River) with regard to degree of pollution (going downwards: very polluted, polluted, and little polluted). This analysis was done with quadratic transformation of sponge densities, Bray Curtis similarity Index, and un-weighted paired average clustering.

serve also to strengthen the validation and to reduce the pitfalls of potential indicator species and ecological indices that have been proposed, to a great extent based on observational and inference approaches. In the context of the application of the suggested biomonitoring methods, an aspect that deserves future effort is to assess the convenience of using sponge cover instead of sponge density, both from practical and scientific points of view. Finally, another matter of concern could be the need of sponge taxonomy skills for the implementation of the proposed bio-monitoring methods. In this sense, the potential indicator species are easy to identify in situ, and sampling for calculation of community indices would only require differentiation of species, and not necessarily identification to species level. With some practice, the identification of most common species can be learned and the sampling work can become even easier.

Acknowledgements
I would like to thank the National Museum of Rio de Janeiro, PETROBRAS, the UNDP/GEF Project SabanaCamagey and Dr. Robert N. Ginsburg (Ocean Research and Education Foundation) for making possible my participation in the 7th International Sponge Symposium. I am grateful to Dr. Janie Wulff, Dr. Georgina Bustamante and Marta Rivero for their valuable comments to the manuscript.

to explore the factors impinging on their structure and composition (Alcolado and Herrera 1987, Muricy 1989, 1991, Carballo et al. 1994, 1996, Carballo and Naranjo 2001, Bell and Barnes 2003, Vilanova et al. 2004, Marcos and Alcolado unpublished observations). Particularly, the Multidimensional Scaled analysis (MDS) has provided clear results (PRIMER version 5). Multivariate techniques are useful tools for identifying factors and interactions, and as such have to be applied complementarily with simpler, faster and lower cost univariate inference approaches in environmental monitoring. Multivariate analyses have to

References
Alcolado PM (1981) Zonacin de los gorgonceos someros de Cuba y su posible uso como indicadores comparativos de tensin

hidrodinmica sobre los organismos del bentos. Inf Cient-Tc Inst Oceanol Acad Cien Cuba 187: 1-43. Alcolado PM (1984) Utilidad de algunos ndices ecolgicos estructurales en el estudio de comunidades marinas de Cuba. Cien Biol 11: 61-77 Alcolado PM (1985) Estructura ecolgica de las comunidades de esponjas de Punta del Este, Cuba. Rep Invest Inst Oceanol Acad Cien Cuba 38: 1-65 Alcolado PM (1990) General features of Cuban sponge communities. In: Rtzler K (ed). New perspectives in sponge biology. Smithsonian Institution Press, Washington DC. pp. 351-357 Alcolado PM (1992) Sobre la interpretacin del medio marino mediante el empleo de los ndices de diversidad y equitatividad. Cien Biol 24: 124-127 Alcolado PM (1994) General trends in coral reef sponge communities of Cuba. In: van Soest RWM, van Kempen TMG, Braekman JC (eds). Sponges in time and space: biology, chemistry, paleontology. Balkema, Rotterdam. pp. 251-255 Alcolado PM (1999) Comunidades de esponjas de los arrecifes del Archipilago Sabana-Camagey, Cuba. Bol Invest Mar Cost 28: 95-124 Alcolado PM, Gotera GG (1985) Estructura de las comunidades de esponjas en arrecifes cubanos. Contrib Simp Cien Mar, VII Jornada Cient Inst Oceanol, XX Aniv 1: 11-15 Alcolado PM, Herrera A (1987) Efectos de la contaminacin sobre las comunidades de esponjas en el Litoral de La Habana, Cuba. Rep Invest Inst Oceanol Acad Cien Cuba 68: 1-17 Alcolado PM, Herrera-Moreno A, Martnez-Estalella N (1994) Sessile communities as environmental bio-monitors in Cuban coral reefs. In: Ginsburg RN (ed). Proceeding of the Colloquium on Global aspects of coral reefs: health hazards and history, 1993 RSMAS, University of Miami. pp. 27-33 Bell JJ, Barnes DK (2003) Effect of disturbance on assemblages: an example using Porifera. Biol Bull 3(1): 144-159 Bergquist PR (1978) Sponges. Hutchinson & Co., London Bradbury RH (1977) Independent lies and holistic truths: towards a theory of coral reefs communities as complex systems. Proc 3rd Int Coral Reef Symp, Miami 1: 2-17 Carballo JL, Snchez-Moyano JE, Garca-Gmez JC (1994) Taxonomic and ecological remarks on boring sponges (Clionidae) from the Strait of Gibraltar (southern Spain): tentative bioindicators? Zool J Linnean Soc 112: 407-424 Carballo JL, Naranjo SA, Garca-Gmez JC (1996) Use of marine sponges as stress indicators in marine ecosystems at Algeciras Bay (southern Iberian Peninsula). Mar Ecol Progr Ser 135: 109-122 Carballo JL, Naranjo S (2001) Environmental assessment of a large industrial marine complex based on a community of benthic filter feeders. Mar Poll Bull 44: 605-610 Claus G, Madri P, Kunen S (1967) Removal of microbial pollutants from waste effluents by the redbeard sponge. Nature 216: 712714 Chvez-Fonnegra A, Zea S (2006) Observation of reef coral undermining by the Caribbean excavating sponge Cliona delitrix (Demospongiae, Hadromerida). In: Custdio MR, Lbo-Hajdu G, Hajdu E, Muricy G (eds). 7th International Sponge Symposium Book of Abstracts (Armao dos Bzios, Brazil). Museu Nacional, Srie Livros, vol. 16. p. 324 Hechtel G (1965) A systematic study of the Demospongiae of Port Royal, Jamaica. Bull Peabody Mus nat Hist 20: 1-94

Herrera-Moreno A (1991) Efectos de la contaminacin sobre la estructura ecolgica de los arrecifes coralinos en el litoral Habanero. PhD Thesis. Instituto de Oceanologa, La Habana Holmes KE (1997) Eutrophication and its effect on bioeroding sponge communities. Proc 8th Int Coral Reef Symp, Balboa 2: 1411-1416 Holmes KE (2000) Effects of eutrophication on bioeroding sponge communities, with description of new West Indian sponges Cliona spp. (Porifera, Hadromerida: Clionaidae). Invert Biol 119: 125138 Holmes KE, Edinger EN, Hariyadi, Limmon GV, Risk MJ (2000) Bioerosion of Live Massive Corals and Branching Coral Rubble on Indonesian Coral Reefs. Mar Poll Bull 40(7): 606-617 Hurlbert SH (1971) The nonconcept of species diversity: A critique and alternative parameters. Ecology 52: 577-586 Jackson JBC, Buss L (1975) Allelopathy and spatial competition among coral reef invertebrates. Proc Natl Acad Sci USA 72: 51605163 Lpez-Victoria M, Zea S (2004) Current trends of space occupation by encrusting excavating sponges on Colombian coral reefs. Mar Ecol 26: 33-41 Margalef R (1963) On certain unifying principles in ecology. Am Nat 97: 357-374 Margalef R (1968) Perspectives in ecological theory. University of Chicago Press, Chicago Muricy G (1989) Sponges as pollution bio-monitors at Arraial do Cabo, Southeastern Brazil. Rev Bras Biol 49(2): 347-354 Muricy G (1991) Structure des peuplements de spongiaires autour de lgout de Cortiou (Marseille, France). Vie et Milieu 41(4): 205221 Odum EP (1969) The strategy ecosystem development. Sciences 164: 262-270 Peet RK (1974) The measurement of species diversity. Ann Rev Ecol Syst 5: 285-307 Preston EM, Preston JL (1975) Ecological structure in a West Indian gorgonian fauna. Bull Mar Sci 252: 248-258 Reiswig HM (1973) Population dynamics of three Jamaican Demospongiae. Bull Mar Sci 23: 191-226 Rose CS, Risk MG (1985) Increase in Cliona delitrix infestation of Montastraea cavernosa heads on an organically polluted portion of the Grand Cayman fringing reef. Mar Ecol 6(4): 345-363 Rtzler K (1970) Spatial competition among Porifera: solution by epizoism. Oecologia 5: 85-95 Rtzler K (2002) Impact of crustose clionid sponges on Caribbean reef corals. Acta Geo Hisp 37(1): 61-72 Sanders HL (1969) Benthic marine diversity and the stability-time hypothesis. Brookhaven Symp Biol 22: 71-81 Sar M (1970) Competition and cooperation in sponge populations. Symp Zool Soc Lond 25: 273-284 Sar M, Vacelet J (1973) cologie des Demosponges. In: Grass PP (ed). Trait de Zoologie, Vol. 3, Masson et Cie, Paris. pp. 462-576 Slobodkin LB, Sanders HL (1969) On the distribution of environmental predictability to species diversity. Brookhaven Symp Biol 22: 82-93 van Soest RWM (1984) Marine sponges from Curacao and other Caribbean localities. Part. III. Poecilosclerida. Stud Fauna Curacao Caribb Isl 66(199): 1-167 Vilanova E, Mayer-Pinto M, Curbelo-Fernandez MP, Gonalves da Silva SH (2004) The impact of a nuclear power plant discharge on the sponge community of a tropical bay (SE Brazil). In: Pansini M,

10

Pronzato R, Bavestrello G, Manconi R (eds). Sponge science in the new millennium. Boll Mus Ist Biol Univ Genova 68: 647-644 Wulff JL (1995) Hurricane effects on survival and orientation of large erect coral reef sponges. Coral Reefs 14: 55-61 Wulff JL (1997) Parrotfish predation on cryptic sponges of Caribbean coral reefs. Mar Biol 129(1): 41-52

Wulff JL (2006) Ecological interactions of marine sponges. Can J Zool 84(2): 146-166 Zea S (1994) Patterns of coral and sponge abundance in degraded versus still healthy coral reefs at Santa Marta Colombian Caribbean. In: van Soest RWM, van Kempen TMG, Braekman JC (eds). Sponges in time and space: biology, chemistry, paleontology. Balkema, Rotterdam. pp. 257-264

Porifera research: Biodiversity, innovation and sustainaBility - 2007

11

Fossil sponges of Argentina: a review


Matilde Sylvia Beresi
CONICET-CRICYT: Ianigla, Dto. de Geologa y Paleontologa, Avda Ruiz Leal s/n, 5500 Mendoza, Argentina. mberesi@lab.cricyt.edu.ar Abstract: This is a review on fossil sponges and sponge spicules reported from several regions in Argentina and in strata ranging in age from Early Cambrian to Tertiary. Sponges have been collected from marine sediments of the Puna, Cordillera Oriental and Sierras Subandinas basins, northern Argentina; Famatina Range; Precordillera terrane, San Rafael block, Neuqun basin and from lacustrine deposits of the North Patagonian Massif. Knowledge of the sponge fossil record is based on whole relatively rigid skeletons, fragments of skeletal nets and spicules seen in thin sections or recovered from acetic acid residues. Early to Middle Cambrian Porifera and Chancelloriids are known from the carbonate platform and slope facies of the Precordillera terrane. Specimens with body preservation of Protospongia, Diagoniella, Kiwetinokia, fragments of hexactinellid, and anthaspidellid sponges and sclerites of Chancelloria had been reported from Cambrian of the Precordillera. Remains of hexactinellid sponges, Pelicaspongiidae and Protospongiidae, have been found in Ordovician rocks of the Puna and of the Famatina System, western margin of Gondwana. Protospongia sp. and hexactinellid mesh were reported from Upper Cambrian-Lower Ordovician siliciclastic sediments in the Cordillera Oriental and Sierras Subandinas. The most significant fossil record of Lower-Middle Ordovician sponge faunas is from the carbonate platform of the San Juan Precordillera. Sponge faunas are dominated by orchoclad lithistid demosponge genera, although hexactinellids are known from loose spicules and root tufts, and calcareous heteractinid sponges are known from isolated octactine spicules and only one genus. Hexactinellid, calcarean and demosponge spicules were reported from diverse localities of the Precordillera. A Late Jurassic (Oxfordian) carbonate complex of the Neuqun Basin, west-central Argentina, contains siliceous sponges dominated by hexactinellids (Hexactinosa and Lyssakinosa). Palaeospongilla chubutensis, a fresh water sponge, was described from lacustrine Cretaceous deposits of the Chubut River valley. Oxeas and strongyles, belonging to the Family Spongillidae, have been mentioned from Tertiary sediments of the Paran basin, northeastern Argentina. Keywords: biostratigraphy, fossil sponges, Argentina.

Introduction
The actual knowledge of the record fossil in Argentina is based on relatively rigid whole skeletons, fragments of skeletal nets and spicules seen in thin sections or recovered from acid residues for obtaining conodonts. The fossil record of sponges comes from several geological provinces of Argentina with different lithologic, paleontologic and environmental characteristics. Sponge faunas were collected from marine sediments of the Puna, Cordillera Oriental, Sierras Subandinas basins of northern Argentina; Famatina Range, Precordillera Terrane, San Rafael Block, Neuqun Basin (western Argentina), from lacustrine deposits of the North Patagonian Massif, and from the Chaco-Paranaense Basin. Occurrences of sponge faunas have been reported from the Lower Paleozoic (upper-Lower Cambrian) to the Cenozoic (Tertiary). Only a few publications have dealt with sponge and sponge spicules in Argentina. Most of them concern the fauna from the Cambrian and Ordovician rocks of the Precordillera terrane. Associated with protospongiid spicules, well-preserved chancelloriid sclerites occur in the Cambrian carbonate platform of the Precordillera. Chancelloriids are Cambrian

enigmatic organisms constituting the monophyletic taxon Coeloscleritophora (Bengtson and Missarzhevsky 1981). Sclerites of chancelloriids (Family Chancelloridae) were first described from the Burgess Shale by Walcott (1920), who interpreted them as heteractinid sponges. This traditional view of the fossil group as sponges was accepted for more than 50 years. In the Precordillera, chancelloriid sclerites associated with spicules are a common and distinguishing features of the Lower to Middle Cambrian fossil fauna. Protospongia and Chancelloria crucensis (Rusconi, 1955) were the first Cambrian species of the Mendoza Precordillera. Cambrian Protospongia Salter, 1864 and hexactinellid spicules were mentioned by Pernas (1964), Devizia (1973), Bordonaro and Martos (1985), Heredia et al. (1987) and Beresi and Heredia (1995) from the San Isidro area in the Precordillera of Mendoza. Afterwards, assemblages tentatively identified as Kiwetinokia utahensis Walcott, 1920, protospongiid skeletal nets and Chancelloria eros Walcott, 1920 were reported from early-Middle Cambrian carbonates blocks of San Juan and Mendoza, Precordillera, by Beresi and Rigby (1994). Two small associated specimens assigned to Protospongia sp. occur in the western Precordillera of San

12

Juan (Beresi and Banchig 1997). A synthesis of the Cambrian sponge occurrence in the Argentine Precordillera was given by Beresi (2003a). Silicified sponge spicules from residues of conodonts from diverse Cambrian and Ordovician sections in the Precordillera, were described by Mehl and Lehnert (1997). Anthaspidellid skeletal fragments from the Middle Cambrian rocks of the Precordillera, in the province of San Juan, document the only known occurrence from South America (Beresi and Rigby 1994). Protachileum kayseri Zittel, 1877 from the San Juan province, was the first report of a Precordilleran sponge. Taxonomic studies have been concentrated in the rich sponge fauna of the warm carbonate platform (San Juan Formation) from the Lower-Middle Ordovician of the Precordillera basin (Beresi and Rigby 1993, Carrera 1996a, 1996b). The purpose of this paper is to review the occurrence of fossil sponges and their biostratigraphic distribution in the diverse geological provinces of Argentina.

Cambrian and Ordovician fauna from the Northwest Argentinian region


Siliciclastic sediments with minor carbonates dominate the Cambrian and Ordovician of Northern Argentina and the Central Andean Basin of South America. Cambrian and Ordovician sponges and spicules from Northwest Argentina provide additional paleontological data from the siliciclastic platforms of western Gondwana.

Puna
In the Puna region (Fig. 1A-B), a single specimen (Fig. 2M-N) of a complete Ordovician hexactinellid sponge was discovered. It has been collected from volcaniclastic rocks of the Las Vicuas Formation (Tremadocian) in Lari Creek, southwest of the Salar del Rincon area, Salta province, Argentina. The material was assigned to the new genus and species Larispongia magdalenae (Carrera, 1998), that belong to the family Pelicaspongiidae, and it is the first record of the family in western Gondwana.

Subandean Ranges
The first report on the occurrence of one completely preserved hexactinellid sponge is a part and counterpart of a round small sponge described as Diagoniella sp. (Beresi et al. 2006). This sponge (Fig. 2H) was reported for the first time from the Upper Cambrian-Lower Ordovician siliciclastic rocks of the Orcomato Formation, in the Candelaria Range, Sub-Andean Ranges, in Salta Province (Fig. 1B). The fossiliferous record of the unit suggests a CambrianOrdovician age. The sponge is preserved in greenishyellowish shales, as dark stained flattened rounded bodies (somewhat deformed and fragmentary).

Fig. 1: A. South America showing the position of Argentina. B. Location of the Basins of Argentina with sponge fauna: (Pu) Puna; (CO) Cordillera Oriental, (SS) Sierras Subandinas, (F) Famatina, (Pa) Paran basin, (P) Precordillera, (SR) San Rafael Block, (N) Neuqun basin, (MP) North Patagonian Massif. C. Map of Argentine showing the San Juan and Mendoza provinces. D. Sponge localities within the Precordillera Precordillera of San Juan and northern Mendoza provinces.

Eastern Cordillera
Hexactinellid meshes of Protospongia (Beresi et al. 2006) were recently reported from Lower Tremadocian siliciclastic sediments in the Eastern Cordillera, Salta Province (Fig. 1). The hexactinellid spicules were recovered from the lower

13

Fig. 2: A. Cambrian anthaspidellid sponge fragment, genus and species indeterminant from San Isidro, Mendoza. B-G. Coeloscleritophora: Chancelloria eros Walcott, 1920, sclerites from the upper LowerMiddle Cambrian of Zonda Range and San Isidro area. H. Diagoniella sp. from Salta province, SubAndean ranges. I. Cambrian fragment of bioclastic limestone showing sclerites of Chancelloria Walcott, 1920, San Isidro. J. Root tuft, large monaxons or monactine-like spicules, in bundle with small stauracts or hexacts (Tontal Range). K. Protospongia sp. showing stauractine-based skeleton and long rayed hexactines forming marginal spines along the left margin (Tontal Range). L. Protospongia sp. Esteban and Rigby, 1998, specimen PIL 14.192 from Pea Negra section, Famatina region. M-N. Larispongia magdalenae Carrera, 1998, holotype CEGH-UNC 17365 from Lari Creek, Puna region. N. Detail of the same specimen showing dermal hexactines surrounding major gaps.

levels of the Saladillo Formation, at the Angosto de la Quesera section, Eastern Cordillera. Spicules of Protospongia (Fig. 2K) are preserved on upper surfaces of yellow-brownish shales and sandstones belonging to the lower section of the unit, sharing the stratigraphic position with Tremadocian graptolites. The sedimentation of the greenish and yellowish shales and sandstones of the Saladillo Formation indicates a transition to an upper offshore-lower offshore environmental setting.

which have been described by Aceolaza and Toselli (1977) for the Chaschuil region. The material appears dispersed in carbonate concretions of the Suri Formation (Arenig).

La Rioja Province
The sponge material came from black siliceous graptolitic shales of the upper part of the Volcancito Formation of Lower Tremadocian age, outcropping in the Pea Negra location in the Famatina range (Fig. 1B). Fragments of a reticulated skeletal net of Protospongia species (A and B) were described by Esteban and Rigby (1998), in the siliclastic Famatina basin, western margin of Gondwana (Fig. 2L). The sponges are associated with planktonic graptolites and this level can be assigned to the Lower Tremadoc (Esteban and Gutierrez-Marco 1997).

Famatina system Catamarca Province


The only discovery of Ordovician sponges in this province corresponds to isolated hexactinellid spicules,

14

Precordillera (Cuyania) Terrane, Western Argentina


The Argentine Precordillera is situated along the forefront of the high Andes at approximately 28 to 37 S, and it is a major geologic province in northwestern Argentina. It contains a complete thick sequence of Early Paleozoic rocks. The Precordillera, as part of the Cuyania Terrane, was formed during the Andean (Tertiary) crustal shortening. This distinctive terrane can be recognized mainly on the basis of its key stratigraphic composition, involving biostratigraphic, sedimentary and magmatic events; its boundaries with adjacent geologic regions are abrupt (Ramos et al. 1986). In accordance with the Terrane concept, the present Precordillera, plus the San Rafael Block and San Jorge Limestones, integrate a unique geologic entity, the so-called Precordillera Terrane or Cuyania Terrane. Two hypotheses exist regarding the origin of the Precordillera: 1) the Precordillera represents a terrane of Laurentian origin that became attached to Gondwana (western Argentina) already during Ordovician times (Thomas and Astini 2003). It includes either the classical Precordillera as well as the San Rafael Block, to the south in the province of Mendoza, and the San Jorge Limestones cropping out in the Province of La Pampa, within the Sierras Pampeanas structural setting as an allochthonous terrane Cuyania, accreted to Gondwana during the lower Paleozoic. 2) the Precordillera is considered as an autochthonous Gondwanan fragment (Baldis et al. 1989, Aceolaza et al. 1999, 2002) displaced by simple transcurrence mechanics, from a hypothetical intermediate sector between South America, Africa, and Antarctica. Recently, U-Pb geochronology of detrital zircons indicated a Gondwanan provenance for Lower Cambrian and Upper Ordovician sandstones of the Precordillera of western Argentina, supporting the autochthonous Gondwanan nature of the Precordillera Terrane (Finney et al. 2003a, 2003b).

the Precordillera of San Juan and Mendoza. Well preserved sclerites are associated with protospongiid spicules (Fig. 2BG).

Cambrian assemblages
Two spicule assemblages occur in the Cambrian facies (Beresi and Rigby 1994, Beresi 2003a). The autochthonous assemblage corresponding to material collected from the upper Lower to Middle Cambrian platform sequence of the eastern Precordillera of San Juan. This assemblage consists of a variety of stauractines and sclerites of Chancelloria eros (Walcott, 1920). The Protospongiidae are represented by triradiate prodianes, pentactines and hexactines, all belonging to Kiwetinokia utahensis Walcott, 1920, Protospongia and anthaspidellid fragments. This fossil fauna represents the oldest assemblages known of Argentina. The allochthonous assemblage proceeds from the diverse Cambrian carbonate olistoliths of slope sequences of the western San Juan Precordillera and from the classical area of San Isidro, Mendoza Province. The assemblages consist of the first precordilleran Protospongia with body preservation, Diagoniella Rauff, 1894, Kiwetinokia Walcott, 1920 and Chancelloria and skeletal net with hexactines and monaxons (Beresi and Banchig 1997). Demosponges have a limited record in the Cambrian of the Precordillera. Typically anthaspidellid fragments with dendroclones (Fig. 2A) have been reported from the carbonate platform and slope sequences of the San Juan and Mendoza Precordillera (Beresi and Rigby 1994).

Ordovician Porifera
Deposits of Ordovician carbonate basins occur in the central and eastern Precordillera. The Lower-Middle Ordovician sediments of the Precordillera represent a drowning carbonate platform with a diverse and relatively complete fossil record. Well-preserved and diverse faunas of sponges have been collected from limestones of the San Juan Formation (Upper Tremadoc-Early Llanvirn) in the Precordillera basin of San Juan province (Fig. 1C-D). This fauna represents the most significant Ordovician sponge fauna known from South America and provides the first extensive record of sponges derived from a stable carbonate platform, constituting one of the most important Early Ordovician sponge associations of the world. Precordilleran sponge faunas are dominated by orchoclad lithistid demosponge genera, although hexactinellids are known from loose spicules and root tufts, and calcareous heteractinid sponges are known from isolated octactine spicules and only one genus. Spicules assemblages were reported from diverse localities of the Precordillera (Beresi and Esteban 2003, Carrera 2003). The San Juan Formation was deposited on an open carbonate shelf, bounded to the west by continental slope and oceanic basin deposits. The diverse marine fauna and the lack of specific structures indicative for shallow water

Cambrian Porifera
The Cambrian system of the Argentinian Precordillera is represented by a carbonate platform, in the east, and of a continental slope, in the west. Cambrian platform and slope facies, containing spicules and chancelloriids sclerites are located in the Precordillera of San Juan and northern Mendoza provinces, western Argentina (Fig. 1C-D). Cambrian sponges are known mainly from fragments of skeletal nets and dissociated spicules from the shallow carbonate platform sequences of the upper-Lower to Middle Cambrian in the eastern and central belts, and from the slope olistostromic sequences with allochthonous blocks in the western part of the Precordillera of San Juan Province. The spicules from the Upper Cambrian were collected in Tontal Range, San Juan Precordillera (Beresi and Banchig 1997) and in the La Cruz Olistolith, San Isidro area (Beresi and Heredia 1995), Precordillera of Mendoza Province. Sclerites of Chancelloria (Coeloscleritophora) occur in shallow carbonate platforms and allochthonous blocks in

15

conditions point to low energy, subtidal conditions within an open-platform environment during the entire sedimentation interval. The age of the San Juan Formation is well constrained by conodonts (Sarmiento 1985, Albanesi and Ortega 2002) spanning from the late Tremadocian (Paltodus deltifer Zone) to the early Darriwilian (Lenodus variabilis Eoplacognathus suecicus zones). The first report of a Precordilleran sponge was Protachileum kayseri Zittel, 1877 from the Talacasto Gulch, Precordillera of San Juan Province (Fig. 3J). Anthaspidellid genera first appear in the basal beds of the San Juan Formation (Upper Tremadoc) at the Niquivil Range, Eastern Precordillera of San Juan, associated with

reef-mound (Caas and Carrera 1993). The cosmopolitan Archaeoscyphia Hinde, 1889 and Rhopalocoelia Raymond and Okulitch, 1940 are the predominant genera in this sponge-algal association (Fig. 3H, 3E). Diverse and well-preserved sponge faunas are from the middle and upper part of the San Juan Formation (ArenigLower Llanvirn). In this carbonate platform the fauna is dominated by orchocladine lithistid demosponges. Their first taxonomic study was made by Beresi and Rigby (1993) and afterwards by Carrera (1996a, 1996b, 1998). Apart from orchocladine demosponges there are hexactine spicules, hexactinellid root tufts, and isolated octactine spicules that document the presence of the Heteractinida.

Fig. 3: Early and Middle Ordovician orchoclad lithistid sponges from the Precordillera of San Juan. (A-B-C-D-G-K; Beresi and Rigby 1993). A. Talacastonia chela Ianigla PI T-2. B. Tangential thin section. C. Anthaspidella annulata Ianigla PI T-49. D. Calycocoelia perforata Ianigla PI VI-2. E. Rhopalocoelia clarkii Raymond and Okulitch, 1940, Ianigla PI T-22. F. Hudsonospongia cyclostoma Raymond and Okulitch, 1940, Ianigla PI VI-2. G. Hudsonospongia talacastensis. Ianigla PI T-32. H. Archaeoscyphia minganensis Billings, 1859 Ianigla PI T-47. I. Incrassospongia ramis Carrera, 1996b, CEGH-UNC 9308. J. Protachileum kayseri Zittel, 1877, Ianigla PI H-43. K. Aulocopium sanjuanensis Ianigla PI VI-13.

16

The greatest generic and specific diversity of lithistid sponges occurs during the Lower Llanvirnian (Darriwilian) in the upper most part of the San Juan Formation. A total of 15 demospongiid genera and 20 species are described. This fauna shows a variety of external morphologies and body plan. Many new Ordovician species were described for the San Juan Formation by Beresi and Rigby (1993): Anthaspidella inornata, A. annulata (Fig. 3C), and A. alveola, Archaeoscyphia nana, Aulocopium sanjuanensis (Fig. 3K), Calycocoelia perforata (Fig. 3D), Hudsonospongia talacastensis (Fig. 3G), H. cyclostoma (Fig. 3F), Patellispongia robusta, Psarodictyum magna, Rhopalocoelia rama, among other species. The new species Talacastonia chela (Fig. 3A-B) was described from the classical Ordovician Talacasto section, Central Precordillera (Fig. 1B). Root tufts of hexactinellids also occur (see Table 1). New megamorinid genera as Rugospongia viejoensis (family Saccospongidae) (Carrera, 1996a) and the tricranocladine sponge Eoscheiella concave (Family Hindiidae Rauff, 1893) have been recovered from the top of the San Juan Formation in the Cerro Viejo, Huaco (Carrera 2007). Nexospongia sillaensis (family Nexospongiidae Carrera, 1996a) was described from the Cerro La Silla, Niquivil Range, Eastern Precordillera. In the upper levels of the San Juan Formation from the Early Llanvirnian, at the Cerro La Chilca section, the calcareous heteractinid Chilcaia bimuralis (Carrera, 1994) and a lithistid species Incrassospongia ramis (Carrera, 1996a) were described (Fig. 3I). Endemic genera such as Protachileum and Talacastonia Beresi and Rigby, 1993, from the Talacasto Gulch and Rugospongia Carrera, 1996 and Chilcaia from different localities of the San Juan Formation occur in the Precordillera. Ordovician sponges from the Precordillera show changes from algal-sponge (reef ecosystems) in the Early Arenig to stromatoporid associations in the Middle Arenig to anthaspidellid demosponge dominated associations in the Upper Arenig to Lower Llanvirn. From the Llanvirn up to the Upper Ordovician, the effects of diverse abiotic factors such as volcanic activity, sea level fluctuations and finally the global climatic cooling, could have contributed to the decrease of the sponges diversity. The diversification of the orchoclad demosponges in the Lower Ordovician carbonate platform of the Precordillera was similar to worldwide radiation pattern (Carrera and Rigby 1999).

assemblages were documented by Gnoli and Serpagli (1980) in the Pachaco section, western Precordillera. Calcarean and demosponge spicules assemblages recovered from residues of conodont samples of several Lower to Middle Ordovician sections of the San Juan Precordillera were described by Mehl and Lehnert (1997). The well preserved silicified spicules include: Polyactinellidae, Heteractinellidae (Calcarea) and hexactinellid and demosponge spicules. The species Dodecaactinella oncera Mehl and Lehnert, 1997, Sardospongia cynodonta Mehl and Lehnert, 1997, Praephobetractinia sp. and Eiffelia sp. were reported from Lower Ordovician (Arenig) strata of the San Juan Formation (Fig. 4A-E). These spicule assemblages were collected from reef-mound horizons and biostromes with sponges, stromatoporoids and receptaculitids of the San Juan Formation (Lower Arenig-Lower Llanvirn) and from the Gualcamayo and Las Aguaditas formations (Lower Llanvirn to Caradocian).

San Rafael Block


Sponge spicules are derived from residues of conodont samples from Middle Ordovician strata, in the geological province of the San Rafael Block, southern Mendoza Province, Argentina (Fig. 1C-D). Spicules (Fig. 4F-Q) have been recovered from the Ponn Trehu Formation, a clasticcarbonate sequence. Poriferan taxa (Beresi and Heredia 2000) include two spicule assemblages: 1) associations of exclusively heteractinellid spicules (sexiradiates) restricted to Arenigian allochthonous blocks of the Oepikodus evae Zone (Heredia 2001) from the shallow platform of the San Juan Formation; and 2) associations of hexactinelliid spicules, calcarean triaene and monaxons, from Upper Llanvirnian autochthonous limestones and carbonate sandstones of the Pigodus serra Zone and the P. anserinus Zone (Heredia 2001) from the outer platform and slope. The spicule associations of the Ponn Trehu Formation represent the most austral Ordovician assemblage described in the context of the Precordillera (Cuyania) Terrane.

Jurassic sponges from the Neuqun Basin


A late Jurasic (Oxfordian) carbonate complex was developed on the foreland side of the Neuqun Basin (Fig. 1B), west- central Argentina and form part of the Cordillera Principal. Shelf carbonates facies are exposed throughout Mendoza and Neuqun provinces. One of these facies consists of small siliceous sponge buildups of the La Manga Formation (Plicatilis Zone), well developed at the Ro Poti Malal section, in southern Mendoza Province. The siliceous sponges with moderate diversity are fossilized in their original shape and exhibit calcareous preservation. Sponge fauna is dominated by hexactinellids (Hexactinosida and Lyssacinosida, 95%) and lithistid demosponges (5%). Up to now, approximately 20% of the material has been preliminary determinated (Beresi 2003b).

Ordovician sponge spicules Precordillera of San Juan Province


The oldest spicule assemblage comes from the Lower Ordovician limestones (Oepikodus intermedius Zone) of the San Juan Formation. The Arenigian silicified spicule

17

Table 1: Biogeographic distribution of sponge taxa from the Cambrian and Ordovician Argentine Basins. Precordillera San Juan Mendoza Paleoenvironment Carbonate platform X X X X X X X X X X X X X X X X X X X X X X X X X X Carbonate platform Carbonate platform Carbonate platform Marine volcaniclastic X X Silicoclastic platform Silicoclastic platform Carbonate platform Carbonate platform Carbonate platform Carbonate platform Carbonate platform X X X X X Famatina System Northern Region X X X

Sponge taxa

DEMOSPONGIAE Family Anthaspidellidae Allosacus sp. Carrera, 1994 Anthaspidella alveola Beresi and Rigby, 1993 Anthaspidella annulata Beresi and Rigby, 1993 Anthaspidella inornata Beresi and Rigby, 1993 Archaeoscyphia minganensis Beresi and Rigby, 1993 Archaeoscyphia nan Beresi and Rigby, 1993 Archaeoscyphia pulchra Bassler, 1941 Aulocopium sanjuanensis Beresi and Rigby, 1993 Calycocoelia perforata Beresi and Rigby, 1993 Hudsonospongia cyclostoma Raymond and Okulitch, 1940 Hudsonospongia talacastensis Beresi and Rigby, 1993 Incrassospongia ramis Carrera, 1996 Patellispongia argentina Carrera, 1994 Patellispongia occulata Bassler, 1941 Patellispongia robusta Beresi and Rigby, 1993 Patellispongia magna sp. Protachilleum kayseri Zittel, 1877 Psarodictium magna Beresi and Rigby, 1993 Rhopalocoelia clarikii Raymond and Okulitch, 1940 Rhopalocoelia rama Beresi and Rigby, 1993 Rhopalocoelia regularis Raymond and Okulitch, 1940 Rhopalocoelia tenuis Carrera, 1994 Talacastonia chela Beresi and Rigby, 1993 Family Hindiidae Eoscheiella concave Carrera, 2007 Family Nexospongiidae Nexospongia sillaensis Carrera, 1996 Family Saccospongiidae Rugospongia viejoensis Carrera, 1996 HEXACTINELLIDA Family Pelicaspongiidae Larispongia magdalenae Carrera, 1998 Family Protospongiidae Protospongia sp. A Protospongia sp. B Diagoniella sp. Protospongia sp. Kiwetinokia utahensis Walcott, 1920 Family uncertain Root tuft HETERACTINIDA Chilcaia bimuralis Carrera, 1994 Dodecaactinella oncera Mehl and Lehnert, 1997 Sardospongia cynodonta Mehl and Lehnert, 1997 Praephobetractinia sp. Eiffelia sp. Octactine spicules Triactine spicules

X X X X

X X X

X X X

18

Fig. 4: Ordovician sponge spicules. A-E. Calcarean and demospongid spicules from the Lower-Middle Ordovician of the San Juan Precordillera (Mehl and Lehnert 1997). CEGHUNC 15951. A. Sardospongia cynodonta. B. Calcarean triactine. C. Demospongid oxea. D-E. Dodecaactinella oncera. F-Q. Spicules from the Ponn Trehu Formation, San Rafael Block (Beresi and Heredia 2000). F. Octactine spicule shows the distal and two of the tangential rays broken. G. Octactine spicule. H. The proximal-distal vertical ray shows a prominent node. I. Monaxon spicule appears to have been sheared diagonally by diagenetic processes. J. Calcarean triactine. K-Q. Scanning electron microscope (SEM) photomicrographs. K, M, N, P. Octactine spicules. L. monaxon spicule shows the central circular canal. O, Q. Hexactine spicules.

The following species have been identified: Laocoetis sp., Laocoetis procumbens and Laocoetis parallela (Hexactinosida Schrammen, 1903, Family Craticulariidae Rauff, 1893) (Fig. 5C-J); Cribrospongia sp., Cribrospongia clathrata and Cribrospongia cucullata (family Cribrospongiidae Roemer, 1864) (Fig. 5A-B) and Polygonatium sp. (Lyssacinosida Zittel, 1877). Sponges belonging to the Family Cribrospongiidae are cup-shaped (Cribrospongia reticulata), tubular and conical. Only a few specimens are triangular in shape and compressed (Cribrospongia cucculata). Fragments of cylindrical to

tubular sponges belong to the genus Laocoetis (=Craticularia Zittel, 1877; emend. Schrammen, 1937).

Cretaceous freshwater sponge from the North Patagonian Massif


The only freshwater sponge was described from Lower Cretaceous lacustrine sediments of the Chubut River Valley, in the Chubut Province, North Patagonian Massif (Fig. 1B). The sponge was determined as Palaeospongilla chubutensis by Ott and Volkheimer (1972). The encrusting sponge belongs to the monogeneric family Palaeospongillidae Volkmer-

19

Fig. 5: Oxfordian hexactinellid sponges at the Ro Potimalal section, Neuqun Basin (Beresi 1997) A. Cribrospongia cuccullata Quenstedt, 1878, lateral view. B. Dermal surface with craticulariid diplorhysis. C. Laocoetis procumbens Goldfuss, 1826, lateral view. D. Upper view of the osculum sponge showing in H. E. Upper view of the narrow osculum and folded wall in a cribrospongiid sponge. F. Lateral view of a cylindrical sponge. G. Laocoetis clathrata Goldfuss, 1833, lateral view. H. Longitudinal section of a tubular cribrospongiid sponge. I. Longitudinal section of a tubular cribrospongiid sponge. J. Upper view of the same sponge, showed in I. K-M. Palaeospongilla chubutensis Ott and Volkheimer, 1972. K. Megasclere with central canal. L. Gemmules. M. Gemmule and spicular texture of the sponge skelton. N-O. Tertiary megascleres, Paran basin.

Ribeiro and Reitner, 1991. It is characterized by acanthoxeas to acanthostrongyles gemmoscleres (Fig. 5K-O).

Remarks
Fossil sponges are known from several geologic basins with different lithologic, sedimentologic and environmental characteristics (Table 2). There are sponges and loose spicules representative of the Classes: Hexactinellida, Demospongida and Calcarea. The oldest sponge fauna known in Argentina is from the upper Lower Cambrian of the Precordillera, the youngest one occurs in the Middle Tertiary of the Paran Basin. Protospongiids characterized the western old Gondwana continent and are known from the Cambrian and Lower

Tertiary spicules of the Paran basin


Isolated oxeas and strongyles, possibly belonging to the species Trochospongilla repens (family Spongillidae) were collected from Tertiary (Miocene) pelitic sediments of the Paran basin, northeast of Argentina (Fig. 1). Borings of Cliona entrerriana and C. ameghinoi on calcareous shells of Ostrea patagonica have also been found in Tertiary sediments.

20

Table 2: Sponge taxa from the Mesozoic and Tertiary Argentine Basins. Taxon sponges and spicules types DEMOSPONGIAE Family Spongilliidae ? Trochospongilla repens Bonetto and Ezcurra de Drago, 1969 Family Palaeospongilliidae Paleoespongilla chubutensis Ott and Volkheimer, 1972 HEXACTINELLIDA Order Hexactinosida Family Craticulariidae Laocoetis paradoxa Goldfuss, 1833 Laocoetis procumbens Goldfuss, 1826 Laocoetis parallela Goldfuss, 1826 Laocoetis clathrata Goldfuss, 1826 Family Cribrospongiidae Cribrospongia sp. Cribrospongia cucullata Quenstedt,1878 Order Lyssacinosida Polygonatium sp.; Schrammen, 1937 Paran Agrio Chaco-Paranense North Patagonian Massif Middle Tertiary Cretaceous: Hauterivian Fluviomarine Lacustrine Formation Basin Age Environment

La Manga

Neuqun

Upper Jurassic Oxfordian

Marine, carbonate bioherms

Ordovician of the Puna, Eastern Cordillera and Sub-Andean Ranges of northern Argentina and the Famatina System, occurring also in Cambrian rocks of the Precordillera. Middle Ordovician sponge faunas are mostly dominated by demosponges, whereas calcareans and hexactinellids do not occur frequently in the warm carbonate platform of the Precordillera. Within the range of the Oxfordian Plicatilis Zone Hexactinellid sponges are common in the carbonate facies of the Neuqun basin, similarly to the Oxfordian European sponge facies. There is still a long way to go concerning the fossil sponges of Argentina, especially in the Cambrian of the Precordillera and northern regions, the Jurassic of Neuqun Basin and freshwater sponges from Mesozoic and Cenozoic lacustrine sediments.

Acknowledgements
The author thanks the Scientific Committee of the 7th International Sponge Symposium for the invitation to participate as Invited Lecturer in the session Palaeontology, and Dr. W. Volkheimer (Conicet-Ianigla, Mendoza) for critic reading of the manuscript. This paper is a contribution to the CONICET-Project: PIP 5222, Argentina.

References
Aceolaza FG, Toselli AJ (1977) Observaciones geolgicas y paleontolgicas sobre el Ordovcico de la zona de Chaschuil, Provincia de Catamarca. Acta Geol Lilloana 14: 55-81

Aceolaza FG, Toselli AJ (1999) Argentine Precordillera: allochthonous or autochthonous Gondwanic? Zentralbl Geol Palontol Teil I 7/8: 743-756 Aceolaza FG, Miller H, Toselli AJ (2002) Proterozoic-Early Paleozoic evolution in western South America - a discussion. Tectonophysics 354: 121-137 Albanesi GL, Ortega G (2002) Advances on conodont-graptolite biostratigraphy of the Ordovician system of Argentina. In: Aceolaza FG (ed). Aspects of the Ordovician System in Argentina, Serie Correlacin Geolgica 16. INSUGEO, Tucumn. pp. 143165 Baldis BA, Bordonaro OL, Armella C, Beresi MS, Cabaleri N, Peralta SH, Bastias H (1989) La cuenca Paleozoica inferior de la Precordillera Argentina. Correl Geol 6: 101-121 Bassler RS (1941) The Nevada early Ordovician (Pogonip) sponge fauna. US Natl Mus Proc 91(3126): 91-102 Bengston S, Missarzhevski VV (1981) Coeloscleritophora - a major group of enigmatic Cambrian metazoans. US Geological Survey, Open-file report 81(743): 19-21 Beresi MS (2003a) Cambrian sponge spicules and chancelloriids of the Argentine precordillera: a review. Geol Acta 1(1): 73-84 Beresi MS (2003b) Oxfordian sponge association from the Neuqun basin, Mendoza, west-central Argentina. J S Am Earth Sci 16:105112 Beresi MS, Aceolaza G, Nievas S (2006) Cambro-Ordovician sponges and spicule assemblages from Northwest Argentina: additional data from the siliciclastic platforms of western Gondwana. Neues Jahrbuch Geol Palontol Monatsh 7: 403-420 Beresi MS, Banchig AL (1997) First hexactinnellid spicules from the Late Cambrian olistolith of the Los Sombreros Formation, Tontal Range, San Juan Precordillera, Argentina. Rev Esp Paleontol 12(2): 141-150

21

Beresi MS, Esteban SB (2003) Sponge fauna and spicules assemblages from the Ordovician of Argentina: a review. In: Aceolaza FG (ed). Aspects of the Ordovician System in Argentina, Serie Correlacin Geolgica 16. INSUGEO, Tucumn. pp. 71-85 Beresi MS, Heredia SE (1995) Asociacin de espculas de porferos cmbricos en la Precordillera de Mendoza. Ameghiniana 32: 401405 Beresi MS, Heredia SE (2000) Sponge spicule assemblages from the Middle Ordovician of Ponn Trehu, southern Mendoza, Argentina. Rev Esp Paleontol 15(1): 37-48 Beresi MS, Rigby JK (1993) The Lower Ordovician sponges of San Juan, Argentina. Geol Stud 39: 1-64 Beresi MS, Rigby JK (1994) Sponges and Chancelloriids from the Cambrian of western Argentina. J Paleontol 68(2): 208-217 Beresi MS, Banchig AL (1997) First hexactinellid spicules of the Late Cambrian Olistolith, of the Los Sombreros Formation, Tontal Range, San Juan Precordillera, Argentina. Rev Esp Paleontol 12(2): 141-150 Bonetto AA, Ezcurra de Drago LD (1969) Systematics notes about the genus Uruguaya Carter (Porifera, Spongillidae). Physis 28(77): 351-358 Bordonaro OL, Martos L (1985) El gnero Chancelloria en el Cmbrico de San Juan. Reunin de Comunicaciones Paleontolgicas, Asociacin Paleontolgica Argentina, San Juan: 6-7 Caas F, Carrera MG (1993) Early Ordovician microbial-spongereceptaculitid bioherms of the Precordillera basin, Western Argentina. Facies 29: 169-178 Carrera MG (1994) An Ordovician sponge fauna from San Juan Formation, Precordillera basin, Western Argentina. Neues Jahrb Geol Palontol Abhand 191(2): 201-220 Carrera MG (1996a) Ordovician Megamorinid sponges from San Juan Formation, Precordillera, Western Argentina. Geobios 29(6): 643-650 Carrera MG (1996b) Nuevos porferos de la Formacin San Juan (Ordovcico), Precordillera Argentina. Ameghiniana 33(3): 335342 Carrera MG (1998) First ordovician sponge from the Puna region, northwestern Argentina. Ameghiniana 35(2): 205-210 Carrera MG (2007) The oldest hindiid demosponge from the Darriwilian (Middle Ordovician) of the Argentine Precordillera: evolutionary implications for the Tricranocladines. J Paleontol 81(4): 754-759 Carrera MG, Rigby JK (1999) Biogeography of the Ordovician sponges. J Paleontol 73: 26-37 Devizia C (1973) Estudio geolgico del sector de San Isidro, Departamento Las Heras Mendoza. PhD Thesis, Museo de La Plata. La Plata Esteban SB, Gutirrez-Marco JC (1997) Graptolitos del Tremadoc del Sistema de Famatina (Argentina). V Reunin Internacional Proyecto 351 PICG Paleozoico Inferior del Noroeste de Gondwana, Libro de Resmenes. pp. 59-63 Esteban SB, Rigby JK (1998) Hexactinellid sponges from the Lower Tremadocian Volcancito Formation, Famatina Range, Northwestern Argentina. Geol Stud 43: 1-7 Finney SC, Gleason J, Gehrels G, Peralta S, Aceolaza G (2003) Early Gondwanan connection for three Argentine Precordillera terrane. Earth Planet Sci Lett 205: 349-359 Finney SC, Gleason J, Gehrels GE, Peralta SH, Vervoort JD (2003b) U/Pb geochronology of detrital zircons from Upper Ordovician

Las Vacas, La Cantera, and Empozada formations, NW Argentina. In: Albanesi GL, Beresi MS, Peralta SH (eds). Ordovician from the Andes. Serie Correlacin Geolgica 17. INSUGEO, Tucumn. pp. 191-196 Gnoli M, Serpagli E (1980) A sponge spicula assemblage from Lower Ordovician of Precordilleran Argentina. Riv Ital Paleontol 86: 267-272 Goldfuss A (1826-1833) Petrefacta Germaniae tam ea, quae in museum Universitatis Regiae Borussicae Fredericae Wilhelminae Rhenanae servantur quam ali quaecunque in Museis Hoeninghausiano Muensteriano aliisque extant. Iconibuset Descriptionibus Illustrata Erster Theil. Arnz & Comp, Dsseldorf Heredia SE (2001) Late Llanvirnian conodonts from the Ponn Trehu Formation, Mendoza, Argentina. GAIA 16: 101-117 Mehl D, Lehnert O (1997) Cambro-Ordovician sponge spicules assemblages in the Ordovician of the Argentine Precordillera and paleoenvironmental ties. Neues Jahrbuch Geol Palaontol Abhand 204: 204-246 Ott E, Volkheimer W (1972) Palaeospongilla chubutensis n.g et n.sp. ein Ssswasserschwamm der Kreide Patagoniens. Neues Jahrbuch Mineral Geol Palontol 140(1): 49-63 Pernas RD (1964) El gnero Protospongia en el Cmbrico de Mendoza. Miscelnea 1, Consejo Investigaciones Cientficas: 3-4. La Plata Quenstedt FA (1876-1878) Die Schwmme In: Petrefactenkunde Deutschlands. Der Ersten Abtheilung, Fnfter Band. Korallen (Schwmmen). Leipzig Rauff H (1893-1894) Palaeospongiologie. Palaeontographica 40: 1-346 Ramos VA, Jordan TA, Allmendinger R, Mpodozis C, Kay SM, Corts J, Palma M (1986) Paleozoic terranes of the Central Argentine-Chilean Andes. Tectonics 5(6): 855-880 Raymond PE, Okulitch VJ (1940) Some Chazyan sponges. Bull Mus Comp Zool 86(5): 197-214 Roemer FA (1864) Die Spongitarien des norddeutschen Kreigebirges. Palaeontographica 8: 1-64 Rusconi C (1955) Fsiles cmbricos y ordovcicos al oeste de San Isidro, Mendoza. Rev Mus Hist Nat Mendoza 8: 3-64 Sarmiento GN (1985) La biozona de Amorphognathus variabilisEoplacognathus pseudoplanus (Conodonta), Llanvirniano inferior, en el flanco oriental de la Sierra de Villicm. 1 Jornadas sobre Geologa de la Precordillera, Actas 1: 119-123 Schrammen A (1903) Zur Systematik der Kieselspongien. Mittelungen aus dem Roemer-Museum, Hildescheim 19: 1-21 Schrammen A (1937) Die Kieselspongien des Oberen Jura von Sddeutschland, B, Besonderer teil. Palaeontographica 85: 1-114 Thomas WA, Astini RA (2003) Ordovician accretion of the Argentine Precordillera terrane to Gondwana: a review: J S Am Earth Sci 16: 67-79 Volkmer-Ribeiro C, Reitner J (1991) Renewed study of the type material of Palaeospongilla chubutensis Ott and Volkheimer (1972). In: Reitner J, Keupp H (eds). Fossil and recent sponges. Springer, Berlin. pp.121-133 Walcott CD (1920) Middle Cambrian Spongiae. Cambrian geology and paleontology. Smithsonian Misc Collect 67(6): 261-364 Zittel KA (1877) Protachileum Kayseri: in Uber Primordiale und untersilurische fossilien aus der Argentine Republik. Beitrge Geologie und Palontologie der Argentine Republik, 2: 22-23

Porifera research: Biodiversity, innovation and sustainaBility - 2007

23

Morphological and cytological descriptions of a new Polymastia species (Hadromerida, Demospongiae) from the North-West Mediterranean Sea
Nicole Boury-Esnault(1*), Chantal Bzac(2)
Aix-Marseille Universit, CNRS UMR-6540 DIMAR, Centre dOcanologie de Marseille. Station Marine dEndoume. Rue Batterie des Lions, 13007. Marseille, France. nicole.boury_esnault@univmed.fr (2) Aix-Marseille Universit, CNRS UMS-2196, Centre dOcanologie de Marseille. Station Marine dEndoume. Rue Batterie des Lions, 13007. Marseille, France
(1)

Abstract: A new species of Polymastia (Hadromerida, Polymastiidae), P. harmelini is described from the coast of Provence (NW Mediterranean). Although this region has been intensively studied, new species are regularly found there. Its description includes morphological, anatomical and cytological features and the species is compared to the Polymastia species from the Atlanto-Mediterranean area. Keywords: Hadromerida, Polymastia, skeleton, anatomy, cytology, taxonomy

Introduction
Even in the new era of bar-coding precise morphological and anatomical descriptions of organisms are still necessary for an unassailable systematics. As Jenner (2006) stressed The study of morphology needs no excuse. It is the uncontested and irreplaceable documentation of lifes diversity. This is particularly essential in animals such as sponges. The framework of the existing classification for Porifera has been recently revisited in a collective book Systema Porifera (Hooper and van Soest 2002). In the last 20 years the taxonomy of the genus Polymastia Bowerbank, 1864 (Demospongiae, Hadromerida, Polymastiidae) has been improved by taking into account the precise organisation of the skeleton in the main body and in the papilla (BouryEsnault 1987, Kelly-Borges and Bergquist 1997, Morrow and Boury-Esnault 2000, Boury-Esnault 2002). It has been shown also the importance of cytological criteria as discriminating characters (Boury-Esnault 1974, Boury-Esnault et al. 1994). In a survey of the sponge fauna from the caves of the NW Mediterranean coast, Jean-Georges Harmelin has discovered at the entrance of the 3PP cave a new species belonging to the genus Polymastia (Fig. 1).

skeleton in HNO3 was done using routine procedures (BouryEsnault and Rtzler 1997), and then mounted on a slide in a drop of epoxy resin. For the skeleton thin sections were made after inclusion in epoxy resin of small pieces of the specimen following Boury-Esnault et al. (2002). Sections of about 1 mm were made with an 11-1180 Isomet low speed saw (Buehler). The sections were then adhered to a slide, ground and polished with a polisher (ESCIL 200 GTL) to a thickness of 15 m. The finishing touches were done by hand with abrasive papers n 600 and n 1200, and 8 and 3 m alumina powder. The thin section was then coloured with toluidine blue under heat for several seconds. A coverslip with a small drop of resin was placed on the thin slides for observation. For cytology in light and transmission electron microscopy (TEM), the specimens were fixed in glutaraldehyde 2.5% in a mixture of 0.4 M cacodylate buffer and seawater (4 vol: 5 vol) (Boury-Esnault et al. 1984). They were postfixed during 1h in 2% osmium tetroxide in seawater, dehydrated through an alcohol series, and embedded in Araldite. Semi-thin sections were stained with toluidine blue. Thin sections, contrasted with uranyl acetate and lead citrate, were observed under a ZEISS EM912 transmission electron microscope.

Materials and methods


The specimens were collected by SCUBA diving during a survey of La Ciotat 3PP cave (4309N, 536E). The 3PP cave (Vacelet et al. 1994) is a long term biodiversity research focal site of the NW Mediterranean (Warwick et al. 2003). The specimens were collected in 1999, 2002 and 2004 fixed in buffered formalin 4% and then transferred to alcohol 70%. To study the shape and size of spicules, dissociation of siliceous

Results Polymastia Bowerbank, 1864


Polymastia Bowerbank, 1864: p. 177; type species Halichondria (Spongia) mamillaris by original designation. Pencillaria Gray, 1867: p. 527; type species Spongia mamillaris by original designation

24

Fig. 1: A. Polymastia harmelini sp. nov. Living specimen photographed in situ. The specimen was covered by sediments, scale bar: 0.8 cm. B. Detail of an exhalant papilla of P. harmelini sp. nov. Note the dark ring below the oscule, scale bar: 0.4 cm. (photos Roland Graille). C. Type specimen in alcohol, scale bar: 0.5 cm.

Rinalda Schmidt, 1870: p. 51; type species Rinalda uberrima by original designation. Diagnosis: Polymastiidae, thickly encrusting, spherical or cushion-shaped, always with papillae. Skeleton composed of radial tracts of principal spicules with free spicules scattered in between. Cortex composed of at least two layers, the superficial layer is a palisade of small tylostyles, the lower layer is made of intermediary spicules, tangential, semi-tangential or perpendicular to the surface. Exotyles echinating the surface may be present. The principal spicules can be tylostyles, subtylostyles, styles, or strongyloxeas, intermediary spicules are most often tylostyles, and ectosomal spicules are always tylostyles.

Museum national dHistoire naturelle de Paris (MNHNDNBE.1562). Type locality: on the threshold of the 3PP cave (4309N/536E) at the basis of the west wall of the entrance at about 18 m deep.

Description
External characters (Fig. 1): The specimens are cushion shaped and cover a surface of about 100 cm2. The thickness is about 3-5 mm. In situ (Fig. 1A) the papillae only are visible. The body is covered by sediments and particles trapped by the hispid surface. The colour of the papillae is brown, as well as the surface. The choanosome has a deep yellow colour in life. The cortex is difficult to tear but it is easily detachable from the choanosome. There are about 28 inhalant papillae and 1 exhalant papilla bearing an oscule per specimen. A dark ring followed by a white one surrounds the oscule (Fig. 1B). The length of the inhalant papilla is 4-10 x 1.5-3 mm and that of the exhalant ones is 8-12 x 4 mm.

Polymastia harmelini sp. nov.


Material examined: 5.08.1999 type specimen (Fig. 1C); 13.09.1999; 6.11.2002; threshold of the 3PP cave in La Ciotat (Provence coast). Type specimen deposited in the

25

Fig. 2: Polymastia harmelini sp. nov. Organisation of the main body skeleton. A. General view, scale bar: 175 m. B. Detail of the cortex, scale bar: 115 m. C. Detail of the cortex, scale bar 115 m. D. detail of the base, scale bar: 115 m.

Skeleton (Fig. 2-3): The ectosomal skeleton is about 320370 m thick and composed of three layers: the upper one is a dense palisade (150-170 m) of tylostyles which lie on a layer of collagen (90-120 m) (Fig. 2A). The basal layer of the cortex is a tangential layer (50-80 m) made of intermediary spicules (Figs 2A-C). Right below the surface is a layer of cells with granular inclusions (25 m) which is responsible for the brown colour of the ectosome (Fig. 2B-C). The basal part in contact with the substratum is constituted by the tangential layer of intermediary spicules (Figs 2A and 2D). The palisade is absent and the sponge is fixed to the substratum by a collagen layer (Fig. 2D). Choanosomal tracts of principal spicules can reach 340 m in thickness at the basis. These tracts are divided into two or three smaller ones (170 m) below the ectosome (Fig. 2A). They cross the ectosome and echinate the surface at distances of approximately 400-500 m (Fig. 2C). Ectosomal and intermediary spicules are scattered between the choanosomal tracts (Fig. 2). The skeleton of the papilla consists of ascending multispicular tracts running through the length of the papillae (Fig. 3). About 25 to 35 tracts are present in a papilla and each tract has a diameter of 50-100 m. The central exhalant canal is about 160 m in diameter. It is surrounded in the exhalant papilla by about 10 inhalant canals 80 to 150 m in diameter. The septa between the canals are strengthened by intermediary spicules (Fig. 3). The ectosomal skeleton of the papilla is about 260-300 m thick and composed of two layers (Fig. 3). Towards the periphery there is a layer of tangentially arranged intermediary spicules (50 m) and followed by a palisade of ectosomal spicules (180-290 m). Towards the surface, the extremities of the ectosomal spicules form a regular hispidation of about 100 m in height. Below the cell surface there is a layer of spherulous cells of about 50 m thick. Spicules (Fig. 4): Ectosomal spicules are tylostyles with a well-marked head 122-239 x 1.7-5.2 m (mean = 193 x 2.8 m) straight or slightly bent (Fig. 4C). Intermediary spicules

Fig. 3: Polymastia harmelini sp. nov. Organisation of the papilla skeleton. A. Exhalant papilla, scale bar: 100 m. B. Detail of the inhalant part of a papilla. The arrow indicates inhalant opening, scale bar: 100 m. Abbreviations: C: cortex; E: exhalant canal; F: transversal section of fascicle of principal spicules. I: inhalant canal.

26

Fig. 4: Polymastia harmelini sp. nov. SEM views of spicules. A. Principal spicules, scale bar: 48 m. B. Intermediary spicules, scale bar: 38 m. C. Ectosomal spicules, scale bar: 15 m. Abbreviations: i: intermediary spicules; e: ectosomal spicules.

Fig. 5: A Polymastia harmelini sp. nov. anatomy of the main body, semithin sections. A. General view, scale bar: 100 m. B. Detail of the limit between ectosome and choanosome, scale bar: 50 m. C. Detail of the upper part of the ectosome, scale bar: 25 m. D. Detail of the choanosome, scale bar: 25 m. Abbreviations: C: cortex; Ch: choanosome; cc: choanocyte chamber; S: location of spicules.

are styles, subtylostyles or tylostyles straight 370-583 x 5.311 m (mean = 456 x 6.5 m) (Fig. 4B). Principal spicules are styles, subtylostyles or tylostyles straight 646-837 x 8-16 m (mean = 745 x 11 m) (Fig. 4A). Anatomy (Fig. 5-6): The cortex, 430-600 m thick, is collagenous with few cells present except close to the surface (Fig. 5). The choanosome has a higher density of cells. On semi-thin sections, choanocyte chambers have a diameter of about 15-25 m which correspond to an estimated volume of 1750-8120 m3. The number of choanocytes is 8-18 on a section of a choanocyte chamber. Using the indirect method of Rasmont and Rozenfeld (1981) the estimated number of choanocytes is 75-120 per chamber. The choanocytes have a periflagellar sleeve between the flagellum and the collar of microvillies. Oocytes are visible in the semithin sections in the specimen collected in August 1999. The oocytes are ovoid or spherical in shape. The size is about 32 x 12 m and the nucleus 8.5 x 5 m. They have a homogeneous content. The papillae have a higher cell density than the cortex especially close to the surface where cells with inclusions constitute a layer of about 30 m (Fig. 6A-B). The exhalant

canal is surrounded by a sphincter of contractile cells, absent around the inhalant canals (Fig. 6A and 6C). Cytology (Figs. 7-9): The most abundant cells are the cells with granular inclusions, which constitute a layer close to the surface (Figs. 2, 5A, 6A, 7) and which confer a brown colour to the cortex. These cells are dispersed in the mesohyl. They have an ovoid to spherical shape (Fig. 7A-B) and the size is about 6.3-11.6 x 2.8-7.9 m. The cytoplasm is reduced to small strands and the nucleus is distorted by the abundance of granular inclusions the diameter of which varies from 0.8 to 4.9 m (Fig. 7A-B). In some cells the inclusions seem to have completely fused and the cell has a granular appearance (Fig. 7B). The distorted nucleus has a diameter from 1.6 to 2.6 m and is often smaller than the inclusions. Cells with a cytoplasmic paracrystalline inclusion are present in the mesohyl (Fig. 8). The cells are ovoid in shape and are 5.6-6.9 m in length and 3.2-5.9 m in thickness. The cytoplasm is reduced to thin strands due to the presence of vacuoles (0.9-1.7 m in diameter) with a heterogenous content and a paracrystalline rod of 2.4-5.6 m in length to 1.6-2.4 m in thickness (Fig. 8A). The crystalline structure

27

has a mesh of 0.03 m in diameter (Fig. 8B). The nucleus is 1.7-1.9 m in diameter. Spiculocytes are present in the mesohyl of the choanosome. They are elongated cells which contain a vacuole with a triangular axial filament around which a spicule is secreted. The nucleus is often nucleolated and numerous mitochondria are present in the cytoplasm. The contractile cells present around the exhalant canals and the oscule have a length which can reach more than 20 m for a thickness of about 3 m at the level of the nucleus (Fig. 9A). The nucleus is ovoid (3 x 1.6 m). All along the length of the cell, contractile filaments of about 0.025 m thick are aligned (Fig. 9B). Archaeocytes are present in the mesohyl (Fig. 9C). They are ovoid cells about 6 x 3.5 m and a nucleus of 3.2 x 2.6 m. The nucleus is nucleolated and the diameter of the nucleolus is about 0.5 m. A very active Golgi apparatus is always present. A variable number of phagosomes (about 1 m in diameter) is observed in the cytoplasm. Rare glycocytes which possess small osmiophilic inclusions and rosettes of glycogen are also present in the mesohyl (Fig. 9D). They measure 5.4-8.6 x 2-4.6 m; the nucleus is about 2 m in diameter and the osmiophilic inclusions about 0.2-0.3 m.

Discussion
Fig. 6: Polymastia harmelini sp. nov. Anatomy of the papillae, semithin section. A. General view, scale bar: 70 m. B. Detail of the external part of the ectosome, scale bar: 25 m. C. Detail of the internal part and of the sphincter of contractile cells around the exhalant canal, scale bar: 25 m. Abbreviations: E: exhalant canal; Ec: ectosome; I: inhalant canal.

In the Atlanto-Mediterranean area three Polymastia species have a cortex made of three layers: an external palisade of tylostyles, an intermediary layer of collagen, and an internal layer of tangential intermediary spicules: P. mamillaris (Mller, 1806), P. arctica (Merejkowsky, 1878) and P. grimaldi (Topsent, 1913) and these species have been often mixed up

Table 1: Comparison of P. harmelini sp. nov. with the three species of the Atlantic area sharing a cortex of three layers as recently redescribed in Boury-Esnault (1987) for P. grimaldi, Morrow and Boury-Esnault (2000) for P. mamillaris and Plotkin and Boury-Esnault (2004) for P. arctica (measures in m). Characters Cortex Thickness Number of layers Palisade layer Collagenous layer Tangential layer Subcortical layer of free spicules Free spicule type Number/specimen Budding Ectosomal Size Intermediary Size Principal Size Exotyles Size P. harmelini sp. nov 350 3 170 100 80 absent ectosomal and intermediary >10 absent tylostyles 190 x 3 tylostyles 456 x 6.5 tylostyles 745 x 11 absent NW Mediterranean 18 m P. mamillaris 400 3 300 20 80 500 ectosomal >10 absent fusiform tylostyles 170 x 12 subtylostyles 445 x 13 fusiform strongyloxea 1052 x 24 absent Swedish west coast 76-225 m P. arctica 560 3 235 130 200 560 ectosomal >100 present fusiform tylostyles 170 x 5 styles 410 x 10 fusiform tylostyles 800 x 14 absent White and Barrents Sea 4-109 m P. grimaldi 650 3 250 150 250 absent ectosomal >100 absent tylostyle 220 x 7 fusiform tylostyles 440 x 14 fusiform strongyloxea 1800 x 23 present 4000 x 10 Boreal Atlantic 70-650 m

Choanosome

Papillae Spicules

Distribution Depth range

28

Fig. 7: Polymastia harmelini sp. nov. TEM micrographs of cells with inclusions. A. Cell with individualized granular inclusions, scale bar: 1.5 m. B. Cell with fused granular inclusions, scale bar: 1.5 m. Abbreviations: n: nucleus; g: granular inclusion.

Fig. 8: Polymastia harmelini sp. nov. TEM micrographs. A. Cell with a paracrystalline inclusion, cell with granular inclusion, scale bar: 1.6 m B. Detail of a paracrystalline inclusion, scale bar: 0.3 m. Abbreviations: c: paracrystalline inclusion; g: granular inclusion; n: nucleus.

Table 2: Comparison of the cytology of P. harmelini sp. n with the three species of Polymastia for which we have cytological data. Cell types Exopinacocytes Cells with intranuclear paracrystalline inclusions Cells with paracrystalline inclusions in the cytoplasm Spherulous cells Vacuolar cells Bacteriocyte Glycocytes Contractile cells around exhalant canals and oscule Periflagellar sleeve P. harmelini sp. nov T-shaped + + + + P. penicillus T-shaped endopinacocyte + + + + + P. robusta T-shaped several vacuoles + + + + P. janeirensis T-shaped collencytes, glycocytes 1 vacuole + and at the limit ectosome/ choanosome +

until the recent redescription of their type specimens (Table 1) (Boury-Esnault 1987, Morrow and Boury-Esnault 2000, Plotkin and Boury-Esnault 2004). Polymastia harmelini sp. nov. shares with these three species a cortex constituted by three layers. Polymastia grimaldi differs from the three other species by the presence of a fringe of exotyles at the

limit between the upper and the lower surface. Polymastia mamillaris (type species of the genus) differs from the other species by the shape and size of ectosomal spicules, and the thinness of the collagenous layer. Polymastia mamillaris and P. arctica share the presence of a layer of groups of ectosomal spicules at the limit of the choanosome (Morrow and Boury-

29

Fig. 9: Polymastia harmelini sp. nov. TEM micrographs. A. Elongated contractile cells in the sphincter around the exhalant canal, scale bar: 3 m. B. Detail of the contractile filaments of a contractile cell, scale bar: 0.3 m. C. Archaeocyte with a large nucleolated nucleus, scale bar: 0.7 m. D. Glycocyte with small osmiophilic inclusions and cell with a paracrystalline inclusion, scale bar: 1 m. Abbreviations: c: paracrystalline inclusion; co: collagen; f: contractile filaments; i: osmiophilic inclusion; n: nucleus.

Esnault 2000, Plotkin and Boury-Esnault 2004) absent in P. harmelini sp. nov. and P. grimaldi. Polymastia arctica is the only species of Polymastia known so far which show buds at the extremity of the papillae. The cytology is known only in three species: P. penicillus (Montagu, 1818) [under the name P. mamillaris], P. robusta (Bowerbank, 1861) [Boury-Esnault 1974, Boury-Esnault 1976] and P. janeirensis (Boury-Esnault, 1973) [BouryEsnault et al. 1994]. The four species show identical cytological characters such as T-shaped exopinacocytes as it is general in Demospongiae, the presence of contractile cells around exhalant canals and oscules and, at the limit of ectosome and choanosome in P. janeirensis, of a periflagellar sleeve around the flagella, a character of Hadromerida. The volume of the choanocyte chamber is in the same range as that known for P. janeirensis (3400-7800 m3) and more generally in Hadromerida (Boury-Esnault 2006). Glycocytes are present in the four species even if they are less abundant in P. harmelini sp. nov. The cells with inclusions are the most characteristic features of the four species. Spherulous cells are present in P. penicillus, cells with paracrystalline inclusions in the cytoplasm and cells with granular inclusions in P. harmelini sp. nov., endopinacocytes with intranuclear paracrystalline inclusion in P. penicillus and collencytes with

intranuclear paracrystalline inclusion in P. janeirensis and vacuolar cells in P. robusta and P. janeirensis.

Biogeography
In the Mediterranean Sea six Polymastia species have been recorded: P. mamillaris, P. robusta, P. inflata Cabioch, 1968, P. polytylota Vacelet, 1969, P. tissieri (Vacelet, 1961) [Uriz and Rosell 1990], and P. sola Pulitzer-Finali, 1983. The specimens under the name P. mamillaris are probably P. penicillus (Morrow and Boury-Esnault 2000). Thanks to the precise drawing it is possible to reassign the specimens collected by Uriz (1983) to P. penicillus but such a reassignment is difficult in many other cases (Sar 1958, Carballo and Gmez 1994). The Polymastia species collected in the Mediterranean Sea so far are bathyal or circalittoral species and are also present in the nearby Atlantic (Boury-Esnault et al. 1994). Polymastia harmelini sp. nov. has been collected on the threshold of a cave at 18 m. Sar (1958) has collected a P. mamillaris from littoral cave of the Italian coast. However the description of Sar is not sufficiently precise to understand to which species the specimens collected belong. Carballo and Garcia-Gmez (1994) have also collected specimens of Polymastia in a littoral cave of the Gibraltar strait. There is

30

no description in the paper and it is impossible to understand to which species the specimens belong. Polymastia sola is insufficiently described and the type specimen is not available. In conclusion with this new species six species have been found in NW Mediterranean: P. penicillus [under the name P. mammillaris], P. robusta, P. inflata, P. tissieri, P. polytylota and this new species P. harmelini sp. nov. which is for the time being the only Mediterranean endemic species.

Acknowledgements
We would like to thank our friend Jo Harmelin who discovered the first specimen of this sponge, Roland Graille for in situ photographs and Christian Marschal for his ability to make the sections of the skeleton. We thank also the service de microscopie lectronique of the IBDM and Jean-Paul Chauvin to have given access to the TEM.

References
Boury-Esnault N (1973) Campagnes de la Calypso au large des ctes atlantiques de lAmrique du Sud (1961-1962). 29. Spongiaires. Rs Sci Camp Calypso 10: 263-295 Boury-Esnault N (1974) Structure et ultrastructure des papilles dponges du genre Polymastia Bowerbank. Arch Zool exp gn 115: 141-165 Boury-Esnault N (1976) Morphognse exprimentale des papilles inhalantes de lponge Polymastia mamillaris (Mller). Arch Zool exp gn 117 : 181-196 Boury-Esnault N (1987) The Polymastia species (Demosponges, Hadromerida) of the Atlantic area. In: Vacelet J, Boury-Esnault N (eds). Taxonomy of Porifera from the N.E. Atlantic and Mediterranean Sea, vol. 13. Springer-Verlag, Berlin Heidelberg. pp. 29-66 Boury-Esnault N (2002) Family Polymastiidae Gray, 1867. In: Hooper JNA, van Soest RWM (eds). Systema Porifera: a guide to the classification of sponges, vol. 1. Kluwer Academic/Plenum Publishers, New York. pp. 201-219 Boury-Esnault N (2006) Systematics and evolution of Demospongiae. Can J Zool 84: 205-224 Boury-Esnault N, Rtzler K (1997) Thesaurus of sponge morphology. Smithsonian Press, Washington Boury-Esnault N, De Vos L, Donadey C, Vacelet J (1984) Comparative study of the choanosome of Porifera: I. The Homoscleromorpha. J Morphol 180: 3-17 Boury-Esnault N, Hajdu E, Klautau M, Custdio M, Borojevic R (1994) The value of cytological criteria in distinguishing sponges at the species level: the example of the genus Polymastia. Can J Zool 72: 795-804 Boury-Esnault N, Marschal C, Kornprobst J-M, Barnathan G (2002) A new species of Axinyssa Lendenfeld, 1897 (Porifera, Demospongiae, Halichondrida) from the Senegalese coast. Zootaxa 117: 1-8 Bowerbank JS (1861) List of British sponges. In: McAndrew R (ed). List of the British marine invertebrate fauna. Rep Br Ass Advmt Sci 30: 235-236 Bowerbank JS (1864) A monograph of the British Spongiadae. Robert Hardwicke, London Cabioch L (1968) Contribution la connaissance de la faune des Spongiaires de la Manche occidentale. Dmosponges de la rgion de Roscoff. Cah Biol mar 9: 211-246

Carballo JL, Garca-Gmez JC (1994) Esponjas del Estrecho de Gibraltar y reas prximas, con nuevas aportaciones para la fauna iberica. Cah Biol mar 35: 192-211 Gray JE (1867) Notes on the arrangement of sponges, with the description of some new genera. Proc Zool Soc London 1867(2): 492-558 Hooper JNA, van Soest RWM (2002) Systema Porifera: a guide to the classification of sponges, vol. 1. Kluwer Academic/Plenum Publishers, New York Jenner RA (2006) Challenging received wisdoms: Some contributions of the new microscopy to the new animal phylogeny. Integr Comp Biol 46: 93-103 Kelly-Borges M, Bergquist PR (1997) Revision of Southwest Pacific Polymastiidae (Porifera: Demospongiae: Hadromerida) with descriptions of new species of Polymastia Bowerbank, Tylexocladus Topsent, and Acanthopolymastia gen. nov. from New Zealand and the Norfolk Ridge, New Caledonia. N Z J Mar Freshwat Res 31: 367-402 Merejkowsky C de (1878) Les ponges de la mer Blanche. Mm Acad imp Sci, St Petersburg 26: 1-51 Montagu G (1818) An essay on sponges, with descriptions of all the species that have been discovered on the Coast of Great Britain. Mem Werner Nat Hist Soc 2: 67-121 Morrow CC, Boury-Esnault N (2000) Redescription of the type species of the genus Polymastia Bowerbank, 1864 (Porifera, Demospongiae, Hadromerida). Zoosystema 22: 327-335 Mller OF (1806) Zoologia danica. Havniae, Copenhagen Plotkin AS, Boury-Esnault N (2004) Alleged cosmopolitanism in sponges: the example of a common Arctic Polymastia (Porifera, Demospongiae, Hadromerida). Zoosystema 26: 13-20 Pulitzer-Finali G (1983) A collection of Mediterranean Demospongiae (Porifera) with, in appendix, a list of the Demospongiae hitherto recorded from the Mediterranean Sea. Ann Mus Civ Stor Nat Genova 84: 445-621 Rasmont R, Rozenfeld F (1981) Etude micro-cinmatographique de la formation des chambres choanocytaires chez une ponge deau douce. Ann Soc R Zool Belg 111: 33-44 Sar M (1958) Studio sui Poriferi di una grotta di marea del Golfo di Napoli. Arch Zool Ital 43: 203-280 Schmidt O (1870) Grundzge einer Spongien Fauna des Atlantischen Gebietes. Wilhelm Engelmann, Leipzig Topsent E (1913) Spongiaires provenant des campagnes scientifiques de la Princesse Alice dans les mers du Nord (1898-1899 - 19061907). Imprimerie de Monaco, Monaco Uriz MJ (1983) Contribucin a la fauna de esponjas (Demospongia) de Catalunya. An Sec Cienc Col Univ Gerona 7: 1-220 Uriz MJ, Rosell D (1990) Sponges from bathyal depths (10001750m) in the western Mediterranean Sea. J Nat Hist 24: 373-391 Vacelet J (1961) Quelques ponges remarquables de Mditerrane. Rec Trav Inst Pches maritimes 25: 351-354 Vacelet J (1969) Eponges de la Roche du Large et de ltage bathyal de Mditerrane (rcoltes de la Soucoupe plongeante Cousteau et dragages). Mm Mus natl Hist nat 59: 145-219 Vacelet J, Boury-Esnault N, Harmelin J-G (1994) Hexactinellid cave, a unique deep-sea habitat in the scuba zone. Deep-Sea Res 41: 965-973 Warwick RM, Emblow C, Fral J-P, Hummel H, Avesaath P van, Heip C (2003) European marine biodiversity research sites. NIOOCEME, Yerseke, the Netherlands

Porifera research: Biodiversity, innovation and sustainaBility - 2007

31

Two new haplosclerid sponges from Caribbean Panama with symbiotic filamentous cyanobacteria, and an overview of sponge-cyanobacteria associations
Maria Cristina Diaz(1,2*), Robert W. Thacker(3), Klaus Rtzler(1), Carla Piantoni(1)
Invertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington, D.C. 20560-0163, USA. ruetzler@si.edu (2) Museo Marino de Margarita, Blvd. El Paseo, Boca del Ro, Margarita, Edo. Nueva Esparta, Venezuela. crisdiaz@ix.netcom.com (3) Department of Biology, University of Alabama at Birmingham, Birmingham, AL 35294-1170, USA. thacker@uab.edu
(1)

Abstract: Two new species of the order Haplosclerida from open reef and mangrove habitats in the Bocas del Toro region (Panama) have an encrusting growth form (a few mm thick), grow copiously on shallow reef environments, and are of dark purple color from dense populations of the cyanobacterial symbiont Oscillatoria spongeliae. Haliclona (Soestella) walentinae sp. nov. (Chalinidae) is dark purple outside and tan inside, and can be distinguished by its small oscules with radial, transparent canals. The interior is tan, while the consistency is soft and elastic. The species thrives on some shallow reefs, profusely overgrowing fire corals (Millepora spp.), soft corals, scleractinians, and coral rubble. Xestospongia bocatorensis sp. nov. (Petrosiidae) is dark purple, inside and outside, and its oscules are on top of small, volcano-shaped mounds and lack radial canals. The sponge is crumbly and brittle. It is found on live coral and coral rubble on reefs, and occasionally on mangrove roots. The two species have three characteristics that make them unique among the families Chalinidae and Petrosiidae: filamentous, multicellular cyanobacterial symbionts rather than unicellular species; high propensity to overgrow other reef organisms and, because of their symbionts, high rate of photosynthetic production. These are the first descriptions of West Atlantic haplosclerid species associated with an Oscillatoria-type symbiont; all previous records of haploscleridcyanobacteria associations were of symbioses with unicellular cyanobacteria. High rates of photosynthetic production of Oscillatoria spongeliae could explain the abundance and overgrowth capability of the two host sponges in the regions reef environments. An overview of associations between sponges and cyanobacteria is presented. Keywords: Haplosclerida, new species, cyanobacteria, Panama

Introduction
The marine subtidal habitats of the Bocas del Toro region (coral reef, mangrove, and sea grasses) are abundantly colonized by marine sponges. A recent survey of sponges from non-cryptic habitats in this region reports 120 described species (Diaz 2005). The Haplosclerida Topsent, 1928 represent the most diverse sponge order at Bocas del Toro, with thirty five species spread across five sponge families: Chalinidae (12 spp.), Petrosiidae (8 spp.), Niphatidae (7 spp.), Callyspongiidae (4 spp.), and Phloeodictydae (4 spp.). Two undescribed species were encountered during this survey, one belonging to Haliclona Grant 1835, sub-genus Soestella de Weerdt, 2000, family Chalinidae Gray, 1867, and the second one to Xestospongia de Laubenfels, 1932, family Petrosiidae van Soest, 1980. Both are thin to thickly encrusting species copiously packed with filamentous cyanobacteria identified as Oscillatoria spongeliae (Thacker et al. 2007). The presence of filamentous cyanobacteria as symbionts in these sponges

constitutes a unique occurrence, both phylogenetically and geographically. To date, 100 sponge species in 29 families are known to harbor cyanobacteria (Table 1). The order Haplosclerida contains the highest number of species with this type of association (25, in 11 genera). Of these, 24 species support unicellular cyanobacteria, while only one undescribed Caribbean species in the family Niphatiidae van Soest, 1980 is reported to have filamentous symbionts (Diaz 1996). This unique association seems to have two striking ecological consequences: a competitive advantage over other reef organisms through overgrowth, including even aggressive reef species such as Millepora (Hydrozoa, Cnidaria) and Neofibularia Hechtel, 1965 (Demospongeae, Porifera), and high photosynthetic rates which characterize these two species as phototrophic sponges (Thacker et al. 2007). The present paper describes the morphology and ecological features of both new species and discusses their systematic affinities with close relatives in the Caribbean.

32

Table 1: Sponge species with cyanobacterial symbionts, modified from Diaz (1996). Families assigned to orders: 1. Homoclerophorida; 2. Astrophorida; 3. Halichondrida (sensu van Soest et al., 1989); 4. Poecilosclerida; 5. Lithistida; 6. Hadromerida; 7. Haplosclerida (sensu de Weerdt, 1985); 8. Dictyoceratida; 9. Dendroceratida; 10. Verongida; 11. Clathrinida; 12. Leucettida; 13. Sycettida; 14. Spirophorida. Symbionts (SYM) include the unicellular Aphanocapsa feldmanni-like (Af), A. raspaigella-like (Ar), Prochloron spp. (Pro), Synechococcus spongiarum (S.spo), Synechocystis trididemni-like (St), Synechocystis spp.-like (Sy), the filamentous Oscillatoria spp.-like (O.sp), Oscillatoria spongeliae-like (O.spo), ? = uncertain status and * = only cyanobacterial pigments detected with thin layer chromatography. Some species have more than one described symbiont; others may contain synonymous Aphanocapsa and Synechococcus symbionts. The regions surveyed: Australia (AUS), Bahamas (BAH), Belize (BEL), Great Barrier Reef (GBR), Guam (GU), Japan (JP) Mediterranean (MED), North and South Baja California (NBC, SBC), Palau (PAL), Papua New Guinea (PNG), Puerto Rico (PR), Red Sea (RS), Sulawesi (SUL) and Zanzibar (ZZ). Family Plakinidae1 Plakinidae1 Ancorinidae2 Ancorinidae2 Ancorinidae2 Ancorinidae2 Ancorinidae2 Geodiidae2 Geodiidae2 Geodiidae2 Axinellidae3 Axinellidae3 Halichondriidae3 Halichondriidae3 Halichondriidae3 Dictyonellidae3 Dictyonellidae3 Desmacellidae4 Desmacellidae4 Chondropsidae4 Crambeidae4 Hymedesmiidae4 Isodictyidae4 Microcionidae4 Mycalidae4 Rhabderemidae4 Rhabderemidae4 Theonellidae5 Theonellidae5 Theonellidae5 Theonellidae5 Theonellidae5 Theonellidae5 Theonellidae5 Siphonidiidae5 Alectonidae6 Chondrosiidae6 Chondrosiidae6 Clionaidae6 Clionaidae6 Clionaidae6 Tethyidae6 Spirastrellidae6 Latrunculiidae6 Callyspongiidae7 Callyspongiidae7 Taxa Oscarella sp. Placinolopha mirabilis Penares aff. schulzei Jaspis stellifera Stelletta clavosa Stelletta kallitetilla Stelletta pudica Geodia papyracea Geodia neptuni Geodia sp. 1 Cymbastela sp. Pseudaxinella tubulosa Axinyssa aplysinoides Halichondria sp. Pseudaxinyssa sp. Dictyonella funicularis Svenzea zeai Neofibularia irata Neofibularia notilangere Batzella melanos Crambe sp. Phorbas sp. Coelocarteria singaporense Clathria sp. Mycale hentscheli Rhabderemia sorokinae Rhabderemia sp. Discodermia dissoluta Theonella conica Theonella swinhoei Theonella swinhoei Theonella sp. 1 Theonella sp. 2 Theonella sp. 3 Leiodermatium sp. Neamphius huxleyi Chondrilla australiensis Chondrilla nucula Spheciospongia florida Spheciospongia sp. Cliona sp. Tethya sp. Spirastrella sp. Latrunculia sp. Callyspongia sp. Siphonochalina sp. Sym Ar O.spo Af Af Af S.spo S.spo Af Af Af ? S.spo ? Ar Sy Ar S.spo Af Af St Af Af, Ar ? Af Sy * Af Af Af, O.sp, S.spo Pro Af S.spo Af Af O.sp * Af S.spo Af, S.spo S.spo Af Af O.sp St Af Af Af Region MED SUL SUL GBR PNG BAH BAH BEL BEL BEL PNG BAH PNG MED GBR BEL BAH GBR BEL GBR MED MED PNG MED NZ PNG SUL BEL SUL ZZ JP RS SUL SUL SUL PNG PNG, SUL AUS MED ZZ BEL MED MED GBR SUL GBR RS Source Wilkinson 1980 Daz 1996 Daz 1996 Wilkinson 1979 Daz 1996 Steindler et al. 2005 Steindler et al. 2005 Rtzler 1990 Rtzler 1990 Rtzler 1990 Daz 1996 Steindler et al. 2005 Daz 1996 Wilkinson 1980 Larkum et al. 1988 Rtzler 1981 Steindler et al. 2005 Wilkinson 1980 Rtzler 1990 Larkum et al. 1988 Wilkinson 1980 Wilkinson 1980 Daz 1996 Wilkinson 1980 Webb and Maas 2002 Daz 1996 Daz 1996 Daz 1996 Daz 1996 Steindler et al. 2005 Hentschel et al. 2002 Wilkinson 1978 Steindler et al. 2005 Daz 1996 Daz 1996 Daz 1996 Daz 1996 Daz 1996 Usher et al. 2004a, 2004b Sar 1966 Steindler et al. 2005 Rtzler 1990 Sar 1966 Sar 1966 Cox et al. 1985 Daz 1996 Wilkinson 1980 Wilkinson 1978

33 Table 1 (cont.)

Chalinidae7 Chalinidae7 Niphatidae7 Niphatidae7 Niphatidae7 Niphatidae7 Niphatidae7 Petrosiidae7 Petrosiidae7 Petrosiidae7 Petrosiidae7 Petrosiidae7 Petrosiidae7 Petrosiidae7 Petrosiidae7 Petrosiidae7 Petrosiidae7 Petrosiidae7 Phloeodictyidae7 Phloeodictyidae7 Phloeodictyidae7 Phloeodictyidae7 Dysideidae8 Dysideidae8 Dysideidae8 Dysideidae8 Dysideidae8 Dysideidae8 Dysideidae8 Irciniidae8 Irciniidae8 Irciniidae8 Irciniidae8 Irciniidae8 Spongiidae8 Spongiidae8 Spongiidae8 Spongiidae8 Spongiidae8 Thorectidae8 Thorectidae8 Thorectidae8 Thorectidae8 Thorectidae8 Thorectidae8 Thorectidae8 Aplysillidae9 Darwinellidae9 Aplysinellidae10 Aplysinellidae10 Aplysinidae10 Aplysinidae10

Haliclona sp. Haliclona (Reniera) sp. Amphimedon sp. 1 Amphimedon sp. 2 Cribrochalina dura Cribrochalina vasculum Niphates sp. Neopetrosia exigua Neopetrosia subtriangularis Petrosia ficiformis Petrosia pellasarca Petrosia sp. Xestospongia muta Xestospongia proxima Xestospongia rosariensis Xestospongia sp. Xestospongia testudinaria Xestospongia wiedenmayeri Calyx podatypa Oceanapia sp. Oceanapia ambionensis Pellina semitubulosa Dysidea granulosa Dysidea sp. Dysidea sp. 1 Dysidea sp. 2 Dysidea sp. 3 Lamellodysidea chlorea Lamellodysidea herbacea Ircinia campana Ircinia felix Ircinia ramosa Ircinia variabilis Psammocinia sp. Coscinoderma sp. Phyllospongia alcicornis Phyllospongia foliacens Phyllospongia papyracea Spongia sp. Carteriospongia foliascens Carteriospongia sp. Carteriospongia sp. Dactylospongia elegans Hyrtios violaceus Lendenfeldia frondosa Lendenfeldia dendyi Aplysilla sp. Darwinella sp. 1 Suberea azteca Suberea mollis Aplysina aerophoba Aplysina archeri

* Ar Ar Af Af Af O.sp Af, S.spo Af, S.spo Af Af S.spo Af, S.spo S.spo Af Af Af Af Af * Ar Af O.spo O.spo O.spo O.spo O.sp O.spo O.spo Af, S.spo Af, S.spo * Af, Ar, S.spo * Af Af Af Af ? S.spo Af Af * O.spo Ar Pro, O.spo Ar Af Af O.sp Af, S.spo Af, S.spo

RS MED SUL SUL BEL BEL BAH, BEL PNG, SUL, PAL BEL MED PR ZZ BEL BAH PR SUL PNG, SUL BEL BEL PNG, SUL SUL MED GU GBR PNG, SUL PNG, SUL PNG, SUL PNG, SUL GBR BEL BEL GBR MED PNG GBR GBR GBR GBR MED ZZ SUL, PNG GBR PNG, SUL BEL SUL, PNG ZZ MED SUL SBC RS MED BEL

Wilkinson 1978 Wilkinson 1978 Daz 1996 Daz 1996 Rtzler 1990 Rtzler 1990 Daz 1996 Daz 1996 Thacker 2005 Rtzler 1990 Sar 1966 Vicente 1990 Steindler et al. 2005 Rtzler 1990 Steindler et al. 2005 Steindler et al. 2005 Vicente 1990 Daz 1996 Daz 1996 Rtzler 1990 Rtzler 1990 Daz 1996 Daz 1996 Sar 1966 Thacker and Starnes 2003 Larkum et al. 1987 Daz 1996 Daz 1996 Daz 1996 Daz 1996 Larkum et al. 1987 Rtzler 1990 Steindler et al. 2005 Rtzler 1990 Steindler et al. 2005 Wilkinson 1983 Sar 1971 Steindler et al. 2005 Daz 1996 Wilkinson 1980 Wilkinson 1992 Wilkinson 1978 Wilkinson 1992 Wilkinson 1980 Steindler et al. 2005 Daz 1996 Wilkinson 1992 Daz 1996 Rtzler 1990 Daz 1996 Steindler et al. 2005 Wilkinson 1980 Daz 1996 Daz 1996 Wilkinson 1978 Sar 1966 Hentschel et al. 2002 Rtzler 1990 Steindler et al. 2005

34 Table 1 (cont.)

Aplysinidae10 Aplysinidae10 Aplysinidae10 Aplysinidae10 Aplysinidae10 Aplysinidae10 Aplysinidae10 Aplysinidae10 Aplysinidae10 Clathrinidae11 Leucettidae12 Leucettidae12 Sycettidae13 Tetillidae14 Tetillidae14

Aplysina cauliformis Aplysina fistularis Aplysina fulva Aplysina gerardogreeni Aplysina lacunosa Aplysina sp. Verongula rigida Verongula gigantea Verongula reiswigi Clathrina sp. Pericharax heteroraphis Leucetta sp. Sycon sp. Cinachyrella australiensis Tetilla arb

Af, S.spo Af, S.spo Af, S.spo Af, S.spo Af, S.spo Af Af Af Af Ar Af ? Ar * Af

BEL BEL BEL SBC BEL BEL BEL BEL BEL MED GBR PNG MED PNG BCN

Rtzler 1990 Steindler et al. 2005 Rtzler 1990 Steindler et al. 2005 Rtzler 1990 Steindler et al. 2005 Daz 1996 Steindler et al. 2005 Rtzler 1990 Steindler et al. 2005 Daz 1996 Rtzler 1990 Rtzler 1990 Rtzler 1990 Wilkinson 1980 Wilkinson 1979 Daz 1996 Feldmann 1933 Daz 1996 Daz 1996

Materials and methods


Specimens were collected during field work in 2003 and 2005, using snorkel and SCUBA equipment while exploring two reefs (Swan Cay and Crawl Cay Canal) between 0-15 m deep in the Bocas del Toro region. Sponges were fixed in 10% formalin in seawater and preserved in 70% ethanol. Skeletal and histological preparations for light microscopy and scanning electron microsopy (SEM) followed standard methodology (Rtzler 1978). The skeletal arrangement was described, and the length and width of each spicule type were measured in each specimen. Type material is deposited in the Porifera collection of the Smithsonian Institutions National Museum of Natural History, Washington, DC (USNH), and in the Snithsonian Tropical Research Institute (STRI) laboratory at Bocas del Toro, Panama (BT).

Paratypes: USNM 1106221, Crawl Cay Canal (9o15050N, 82o07631W), 5 m, on top and along sides of Acropora cervicornis, collectors M.C. Daz and R. Thacker, 21-06-05. BT-045, Swan Cay (9o27198N, 82o18024W), 5 m deep, between fire coral (Millepora sp), and lettuce coral (Agaricia sp.) on a shallow reef with strong surge and currents, collector M.C. Daz, 08-2003.

Description
Shape and size: Thin encrusting sheets (1-2 mm thick) covering patches ranging from five to a few hundred cm2 (Fig. 1A, B). Surface: Smooth to irregularly rugose to the naked eye, porous under a microscope. Small oscules (1-2 mm in diameter) with transparent membranes, regularly distributed over the sponge surface. Radial canals converging toward oscules. Spicule tracts piercing through the skin (ectosome) create a microhispid appearance, only visible under a microscope (Fig. 1B). Colour: In live, deep dark- brown to purple outside, tan inside. Cream to white in alcohol. External color due to the photosynthetic cyanobacteria.
Fig. 1: In situ morphology and skeleton arrangement of the new species: A. Haliclona walentinae sp. nov. habit (scale: 6 cm); B. detail showing bumpy surface, radial canals, and oscules (1-2 mm) with white oscular membranes (scale: 5 mm); C. cross section through the choanosome with Soestella-type arrangement of subanisotropic choanosomal skeleton of ill defined paucispicular primary lines connected by paucispicular secondary ones (scale: 100 m); D. Xestospongia bocatorensis sp. nov. habit (scale: 2 cm); F. isotropic unispicular to paucispicular reticulation (2-3 spicules across) forming polygonal-shaped meshes (scale: 120 m).

Results Systematic descriptions


Class Demospongiae Sollas, 1885. Order Haplosclerida Topsent, 1928 Family Chalinidae Gray, 1867 Genus Haliclona Grant, 1835 Sub-Genus Soestella de Weerdt, 2000 Haliclona (Soestella) walentinae sp. nov. (Figs. 1-3; Table 2) Material examined. Holotype: USNM 1106220, Crawl Cay Canal (9o15050N, 82o07631W), 5-10 m deep, covering top and sides of Acropora cervicornis on a shallow reef where Millepora, and Porites were the dominant coral species, collectors M.C. Daz and R. Thacker, 21-06-05.

35

36

Table 2: Spicule measurements of specimens of Haliclona walentinae sp. nov. [max.-min. length (meanSD) x max.-min. width (meanSD)] in m. Material studied USNM 1106220 USNM 1106221 BT-045 Oxea 130161 (1409.3) x 69 (7.60.9) 130160 (1409.2) x 39 (4.81.6) 100180 (13219) x 38 (51)

Haliclona walentinae a very interesting subject for both ecological and evolutionary studies. Etymology: The species is named after Dr. Walentina de Weerdt (University of Amsterdam) whose work with the Haplosclerida has been essential in our understanding of the group. Family Petrosiidae van Soest, 1980 Genus Xestospongia de Laubenfels, 1932 Xestospongia bocatorensis sp. nov. (Figs. 1-3; Table 3) Material examined. Holotype: USNM 1106222, Crawl Cay Canal (9o15050N, 82o07631W), 12 m, top of Acropora cervicornis on a shallow reef where Millepora and Porites were the dominant coral species, collectors M.C. Daz and R. Thacker, 21-06-05. Paratypes: BT-019, Crawl Cay Canal (9o15050N, 82o07631W), 6 m, between fire coral, and Agaricia spp. colonies, on a shallow reef, collector: M.C. Daz, 08-2003; BT-163, same data as holotype.

Consistency: soft, compressible, and resilient, easy to peel off the substrate. Ectosomal skeleton: Poorly developed, some paucispicular spicule tracts and loosely strewn spicules (Fig. 1C). Ectosome not peelable. The ectosome on the underside of the sponge accumulates sand. Choanosomal skeleton: Paucispicular, loosely organized primary skeleton tracts (20-40 m in diameter), and mostly unispicular tracts or single spicules connecting them. Spicule tracts densely enveloped by filamentous cyanobacteria (Fig. 3A, B). Spongin scarce, barely discernable. Spicules: Hastate to fusiform oxea, straight or slightly curved (100-180 x 3-9 m). (Table 2, Fig. 2A). Ecology: The species was found thriving on a shallow reef, profusely overgrowing fire corals (Millepora spp.), soft corals, scleractinians, and other sponges, such a Neofibularia nolitangere (Duchassaing and Michelotti, 1864). It appeared to be a rather aggressive species, dominating all neighboring sessile invertebrates. Remarks: This species is here assigned to the subgenus Soestella, following the definition by de Weerdt (2000) where ill defined paucispicular primary lines, irregularlly connected by unispicular secondary lines characterize the skeletal architecture. Eight additional species in this subgenus occur in the Caribbean: H. (Soestella) caerulea (Hechtel, 1965), H. (S.) lehnerti de Weerdt (2000), H. (S.) luciencis de Weerdt (2000), H. (S.) melana Muricy and Ribeiro (1999), H. (S.) piscaderaensis (van Soest, 1980), H. (S.) smithsae de Weerdt (2000), H. (S.) twincayensis de Weerdt et al. (1991), and H. (S.) vermeuleni de Weerdt (2000). Four of these, H. (S). caerulea, H. (S.) piscaderaensis, H. (S.) twincayensis, and H. (S.) vermeuleni are among common species in the region of Bocas del Toro (Diaz 2005). None of these, nor any other species of Chalinidae, are known to be associated with cyanobacteria (Table 1). Two species in Soestella (melana, and luciencis) are black to dark brown color, but only darkly pigmented cells are reported, at least for the former (de Weerdt 2000). Distinct morphological and ecological differences separate H. (S.) walentinae from the other Haliclona (Soestella) Caribbean species. Among them a thinly encrusting growth habit, soft but resilient consistency, characteristic oscule morphology, and possession of cyanobacterial symbionts. The filamentous cyanobacteria turn out to be a branch of Oscillatoria spongeliae, with genetic affinities to certain Pacific sponge symbionts (Thacker et al. 2007), making

Description
Shape and size: Thinly encrusting species (2-5 mm thick), in patches from five to a few hundred cm2 . Surface: Smooth. Oscules (1-2 mm diameter) on top of low volcano-shaped mounds (1-2 mm of height). Consistency: Crumbly and brittle. Colour: In live, dark purple, inside and out (Fig. 1D). Cream to white in alcohol. Ectosomal skeleton: No organization, spicules strewn tangentially (Fig. 1E). Choanosomal skeleton: Isotropic unispicular to paucispicular reticulation forming polygonal meshes (100-320 m in diameter), and paucispicular primaries (2-3 spicules across) 200-300 m apart. Filamentous cyanobacteria densely packed around the skeleton (Fig. 3C, D). Spicules: Fusiform to slightly hastate stout oxeas in one size class (230-320 x 8-15 m) (Table 3), with pointed ends. Sigmas, c-shaped (10-26 x <1 m) (Fig. 2B). Ecology: The species was found growing in small patches on mangrove roots, empty shells, or coral rubble, and occasionally profusely overgrowing live corals. Also found on shallow reefs (0-15 m deep) growing over coral and bare rock substrates. Remarks: The predominance of unispicular over paucispicular reticulation, and the relatively light spicule density, compared to other Xestospongia, makes this species slightly atypical for the genus. However, two other Caribbean congeners, X. arenosa van Soest and de Weerdt, 2001, and X. wiedenmayeri van Soest, 1980 are precedents for similar skeleton structure. The assignment to Xestospongia is based on spicule size, skeleton structure (more petrosiid than chalinid), and petrosiid consistency (brittle and crumbly); it was first suggested by Dr. Walentina de Weerdt who examined our

37

Fig. 2: SEM photomicrographs of spicules: A. Haliclona walentinae sp. nov. (USNM 1106220) oxeas; B. Xestospongia bocatorensis sp. nov. (USNM 1106222), oxeas and sigmas.

Table 3: Spicule measurements of specimens of Xestospongia bocatorensis sp. nov. [max-min. length (meanSD-) x max.- min. width (mean SD)] in m. Material studied USNM 1106222 BT-019 BT-163 Oxea 280320 (30211.5) x 1215 (130.9) 230260 (24811.2) x 812 (11.80.7) 270305 (29312.4) x 812 (10.61.2) Sigma (length in m) 2025 (221.6) 1012 (11.80.7) 1026 (191.22)

material. Seven other Xestospongia species are recognized in the Caribbean: X. arenosa van Soest and de Weerdt (2001), X. caminata Pulitzer-Finali (1986), X. deweerdtae Lehnert and van Soest (1999), X. muta (Schmidt, 1870), X. portoricensis van Soest (1980), X. proxima (Duchassaing and Michelotti, 1864), X. rosariensis Zea and Rtzler (1983), none of these has the thinly encrusting morphology of X. bocatorensis sp. nov. Three are very common inhabitants of Bocas del Toro reefs: X. proxima, X. muta, X. rosariensis. Even though all of these species harbor symbiotic cyanobacteria, Xestospongia bocatorensis sp. nov. is unique for its possession of a hostspecific clade of filamentous Oscillatoria spongeliae, rather than the more typical unicellular symbionts, Candidatus Synechococcus spongiarum (Usher et al. 2004a, 2004b, Thacker et al. 2007). Etymology: The species is named after the Bocas del Toro region, an extensive system of islands with well developed

mangrove communities and patchy reefs in northeastern Panama where the new species was found.

Discussion and conclusions


To evaluate the relative frequency of associations between cyanobacterial symbionts and marine sponges, we compiled data from morphological and phylogenetic studies of sponges and their symbionts (Table 1, Diaz 1996, Steindler et al. 2005). Prior to genetic studies, many unicellular cyanobacterial symbionts were classified as Aphanocapsa feldmanni Fremy, 1933; some of these have subsequently been recognized as members of the genus Synechococcus (Usher et al. 2006), including a proposed species of sponge-specific unicellular cyanobacteria, Candidatus Synechococcus spongiarum Usher, 2004. Here, we present symbiont names as given by the authors of each study, and recognize that some of

38

Fig. 3: Filamentous cyanobacteria (Oscillatoria spongeliae) and choanocyte chambers shown in sections of the new species: A, B. Haliclona walentinae sp. nov.; C, D. Xestospongia bocatorensis sp. nov.

these names may be synonyms (Table 1). Clearly, combined morphological and genetic studies are needed to resolve some of these issues. Symbiosis of sponges and filamentous (Oscillatoriatype) cyanobacteria is a common occurrence in the IndoPacific region where at least 10 common species are known for this association. The families concerned are Plakinidae (Homosclerophorida), Theonellidae (Lithistida), Dysideidae and Spongiidae (Dictyoceratida), and Aplysinidae (Verongida). In the much better studied Mediterranean Sea, only one Tethya (Tethyidae, Hadromerida) is known with this kind of symbiont. Until our discovery of Haliclona walentinae sp. nov. and Xestospongia bocatorensis sp. nov., only two records of sponges with Oscillatoria-type symbionts existed in the tropical western Atlantic. One is the common bleeder sponge Hyrtios violaceus (Duchassaing and Michelotti, 1864) (Thorectidae, Dictyoceratida), of which the symbiont fine-structure was studied (Rtzler 1990). The other is an undescribed species of Niphates (Niphatidae, Haplosclerida),

which was recorded from the Bahamas and Belize (Diaz 1996). The phototrophic properties of the new species, the nature of the cyanobacterial symbionts, and the phylogenetic affinities of the symbionts to those hosted by Pacific sponges (Thacker et al. 2007) lends these biological assemblages a unique ecological and evolutionary significance. An unsolved issue remains about the origin of the two new species: are they systematic and ecological oddities among Caribbean sponges, or are they invasive species that originated in the tropical Pacific?

Acknowledgments
We thank Dr. Walentina (Wallie) de Weerdt (Amsterdam) who kindly examined fragments of the specimens and commented on their identification. This is Caribbean Coral Reef Ecosystems (CCRE) contribution number 798, supported in part by the Hunterdon Oceanographic Research Fund.

39

References
Cox GC, Hiller RG, Larkum AWD (1985) An unusual cyanophyte containing phycourobilin and symbiotic with ascidians and sponges. Mar Biol 89: 149-163 de Weerdt WH (2000) A monograph of the shallow-water Chalinidae (Porifera, Haplosclerida) of the Caribbean. Beaufortia 50: 1-67 de Weerdt WH, Ruetzler K, Smith KP (1991) The Chalinidae (Porifera) of Twin Cays, Belize, and adjacent waters. Proc Biol Soc Wash 104(1): 189-205 Diaz MC (1996) Molecular and ecological studies of spongemicrobial associations. PhD Thesis. University of California, Santa Cruz Diaz MC (2005) Common sponges from shallow marine habitats from Bocas del Toro region, Panama. Carib J Sci 41(3): 466-475 Duchassaing de Fonbressin P, Michelotti G (1864) Spongiaires de la mer Caraibe. Natuurk Verh Holl Mij Wetensch Haarlem (2) 21(3): 1-124 Feldmann J (1933) Sur quelques cyanophyces vivant dans le tissue des ponges. Arch Zool Exp Gn 75: 331-404 Hechtel GJ (1965) A systematic study of the Demospongiae of Port Royal, Jamaica. Bull Peobody Mus Nat Hist 20: 1-94 Hentschel U, Hopke J, Horn M, Friedrich AB, Wagner M, Hacker J, Moore BS (2002) Molecular evidence for a uniform microbial community in sponges from different oceans. Appl Environ Microbiol 68: 4431-4440 Larkum AWD, Cox GC, Hiller RG, Dibbayawan TP (1988) Prokaryotic algal symbionts of coral reef sponges. Proc 6th Int Coral Reef Symp, Townsville 3: 163-169 Larkum AWD, Cox GC, Hiller RG, Parry DL, Dibbayawan TP (1987) Filamentous cyanophytes containing phycouribilin and in symbiosis with sponges and an ascidian of coral reefs. Mar Biol 95: 1-13 Lehnert H, van Soest RWM (1999) More North Jamaican deep forereef sponges. Beaufortia 49(12): 141-169 Muricy G, Ribeiro SM (1999) Shallow water Haplosclerida (Porifera, Demospongiae) from Rio de Janeiro State, Brazil (southwestern Atlantic). Beaufortia 49(9): 83-108 Pullitzer-Finalli G (1986) A collection of West Indian Demospongiae (Porifera). In appendix, a list of Demospongiae hitherto recorded from the West Indies. Ann Mus Civ Storia Nat Genova 86: 65-216 Rtzler K (1978) Sponges in coral reefs. In: Stoddart DR, Johannes RF (eds). Coral reefs: research methods. Monographs on Oceanographic Methodologies (UNESCO) 5: 299-314 Rtzler K (1981) An unusual bluegreen alga symbiotic with two new species of Ulosa (Porifera:Hymeniacidonidae) from Carrie Bow Cay, Belize. Mar Ecol 2: 35-50 Rtzler K (1990) Associations between Caribbean sponges and photosynthetic organisms. In: Rtzler K (ed). New perspectives in sponge biology. Smithsonian Institution Press, Washington DC. pp. 455-466 Sar M (1966) Associazioni fra Porifera e alghe in acque superficiali del litorale marino. Ric Sci 36: 277-282 Sar M (1971) Ultrastructural aspects of the symbiosis between two species of the genus Aphanocapsa (Cyanophyceae) and Ircinia variabilis (Demospongiae). Mar Biol 11: 214-221 Schmidt O (1870) Grundzge einer Spongien-Fauna des Atlantischen Gebietes. Wilhelm Engelmann, Leipzig

Steindler L, Huchon D, Avni A, Ilan M (2005) 16S rRNA phylogeny of sponge-associated cyanobacteria. Appl Environ Microbiol 71(7): 4127-4131 Thacker RW (2005) Impacts of shading on sponge-cyanobacteria symbioses: a comparison between host-specific and generalist associations. Int Comp Biol 45: 69-376 Thacker RW, Diaz MC, Rtzler K, Erwin PM, Kimble SJA, Pierce MJ, Dillard SL (2007) Phylogenetic relationships among the filamentous cyanobacterial symbionts of Caribbean sponges and a comparison of photosynthetic production between sponges hosting filamentous and unicellular cyanobacteria. In: Custdio MR, Lbo-Hajdu G, Hajdu E, Muricy G (eds). Porifera research: biodiversity, innovation and sustainability. Srie Livros 28. Museu Nacional, Rio de Janeiro. pp. 621-626 Thacker RW, Starnes S (2003) Host specificity of the symbiotic cyanobacterium Oscillatoria spongeliae in marine sponges, Dysidea spp. Mar Biol 142: 643-648 Usher KM, Fromont J, Sutton DC, Toze S (2004a) The biogeography and phylogeny of unicellular cyanobacterial symbionts in sponges from Australia and the Mediterranean. Microb Ecol 48: 167-177 Usher KM, Kuo J, Fromont J, Toze S, Sutton DC (2006) Comparative morphology of five species of symbiotic and non-symbiotic coccoid cyanobacteria. Eur J Phycol 41(2): 179-188 Usher KM, Toze S, Fromont J, Kuo J, Sutton DC (2004b) A new species of cyanobacterial symbiont from the marine sponge Chondrilla nucula. Symbiosis 36: 183-192 van Soest RWM (1980) Marine sponges from Curaao and other Caribbean localities. Part II: Haplosclerida. Stud fauna Curacao Caribb Isl 62(191): 1-173 van Soest RWM, de Weerdt WH (2001) New records of Xestospongia species (Haplosclerida: Petrosiidae) from the Curaao reefs, with a description of a new species. Beaufortia 51: 109-117 Vicente V (1990) Response of sponges with autotrophic symbionts during the coral bleaching episode in Puerto Rico. Coral Reefs 8(1): 99-202 Webb VL, Maas EW (2002) Sequence analysis of 16S rRNA gene of cyanobacteria associated with the marine sponge Mycale (Carmia) hentscheli. FEMS Microbiol Lett 207(1): 43-47 Wilkinson CR (1978) Microbial associations in sponges. I. Ecology, physiology, and microbial populations of coral reefs sponges. Mar Biol 49: 161-167 Wilkinson CR (1979) Nutrient translocation from symbiotic cyanobacteria to coral reef sponges. In: Lvi C, Boury-Esnault N (eds). Biologie des spongiaires. Colloques Internationaux du CNRS, vol. 291. ditions du CNRS, Paris. pp. 373-380 Wilkinson CR (1980) Cyanobacteria symbiotic in marine sponges. In: Schwemmler W, Schenck HEA (eds). Endocytobiology, Endosymbiosis and Cell Biology. De Gruyter, Berlin. pp. 9931002 Wilkinson CR (1983) Net primary productivity in coral reef sponges. Science 219: 410-412 Wilkinson CR (1992) Symbiotic interactions between marine sponges and algae. In: Reiser W (ed.) Algae and symbioses. Biopress Ltd, Bristol. pp.111-151 Zea S and K Rtzler (1983) A new species of Xestospongia (Porifera, Demospongiae), from the Colombian Caribbean. Caldasia 13: 817-831

Porifera research: Biodiversity, innovation and sustainaBility - 2007

41

Sponge embryology: the past, the present and the future


Alexander V. Ereskovsky
Aix-Marseille Universit, CNRS UMR-6540 DIMAR, Centre dOcanologie de Marseille. Station Marine dEndoume. Rue Batterie des Lions, 13007. Marseille, France (corresponding address); and Department of Embryology, Biological Faculty, Saint-Petersburg State University, Universitetskaya nab. 7/9, 199034 Saint-Petersburg, Russia. aereskovsky@mail.ru Abstract: Developmental biology of sponges has a 140-year-old history. It made important contributions to spongiology in general: the creation of the subkingdom Enantiozoa, the separation of the Calcinea and the Calcaronea in the Calcarea, and the separation of the Tetractinomorpha and the Ceractinomorpha within the Demospongiae. Nevertheless, embryonic development has been studied in only 93 sponge species. This review must be restricted primarily to embryonic development and metamorphosis of sponges, because full modern information on its normal development is limited to only a few studies. Mechanisms of morphogenetic movements emerged in the course of evolution prior to the separation of the covering cell layer as ectoderm and the internal digestive cell mass as endoderm. Therefore, it is incorrect to apply the term gastrulation to sponge development. It is difficult to use comparative embryological data on sponges for phylogenetic interpretations because their development is highly polymorphic. The same cleavage pattern and blastula type may be characteristic of different larval types. On the other hand, the same larval types may develop from different cleavage patterns and types of morphogenesis. However, embryological data do indicate groups or types of sponge development. The observed variety of developmental patterns indicates that no linear ways of developmental evolution are common for all Porifera. It testifies to an early divergence of sponge macrogroups or, probably, paraphyly and their long parallel evolution. The basal phylogenetic position of the Porifera among the Metazoa and its suggested paraphyly make new investigations on embryology and larvae especially important. Relatively few homologues of developmental genes are known in the Porifera. Comparison of transcription factors that regulate genes expression during sponge morphogenesis will provide an evolutionary perspective to relationships among basal metazoan phyla. Keywords: development, evolution, morphogenesis, phylogeny, sponges

The past
Developmental studies of sponges have a 140-year-old history. Ever since the work of Ernst Haeckel (1866) (Fig. 1), they have inspired, enriched and modified evolutionary thought. Altogether, about 540 articles concerning sponge embryology have been published. They involve approximately 36 species of Calcarea, 140 species of Demospongiae, and as little as 3 Hexactinellida species. The first period of sponge development studies falls on the last third of the 19th century. It was the Golden Age of sponge embryology. About 110 articles on this topic were published (Fig. 2). Uncontested leadership in this research field belonged to German zoologists: Schulze, Maas, Schmidt, Keller (Fig. 3) and others. The basis of sponge comparative embryology was laid at that time. Haeckel (1874) admitted that embryological studies of calcareous sponges (Haeckel 1872) were the starting point for his ideas about the origin of Metazoa, later formalized as the Gastraea theory of ontogeny recapitulating phylogeny, in which the gastrula is viewed as the recapitulation of a gastraean ancestor that evolved via selection on a simple,

planktonic hollow ball of cells to develop the capacity to feed (Haeckel 1874). On the basis of comparative embryological data of some demosponges, Delage (1892, 1899) (Fig. 4) discovered that during a metamorphosis of parenchymella larvae external flagellated cells migrate inward to form the choanoderm of the adult sponge. These observations have allowed Delage to propose a hypothesis of inversion of the germ layers. Being based on this hypothesis, he separated sponges from Metazoa into a special group, Enantiozoa that signified inside out animals. Bidder (1898), following Minchin (1896), subdivided Calcarea into two subclasses, Calcinea and Calcaronea, distinguished deep embryological differences (e.g., coeloblastula in Calcinea, amphiblastula in Calcaronea), and the position of the nucleus in the choanocytes (with nucleus basal in choanocyte independent of flagellum in Calcinea and with nucleus apical in choanocyte linked to the flagellum in Calcaronea). The second period falls on the first half of the 20th century (1900-1960), when interest in sponge development declined (Fig. 2). Almost the only active researches were made in Belgium by Brien (Fig. 5), Meewis, Leveaux, and in France by Tuzet, Duboscq and Lvi (Fig. 6). Claude Lvi

42

Fig. 1: Ernst Haeckel (1834-1919) and Nicolas Miklucho-Maclay (1846-1888) during the expedition to the Red Sea in 1866 (From. I.I. Kanaev, 1966).

in his famous work (1956) was the first to use embryological characters of sponges in systematics. However, this period was marked by the emergence of a major branch in developmental biology of sponges. Wilson

(1907) pioneered the use of sponges as model animals for cell adhesion research. He described species-specific reaggregation of mechanically dissociated sponge cells. His works provided an impulse for studies of behaviour of separate cells and regeneration in sponges. The third period started with the application of electron microscopy and new optical and experimental methods to sponge studies (Fig. 2). Spermatogenesis and oogenesis, fertilization (in Calcaronea) and larval structure (in all poriferan classes) were investigated ultrastructurally. The results of these works were extensively applied to evolutionary and phylogenetic constructions concerning both Porifera and Metazoa in general. At the same time, complete development from egg to juvenile was investigated at the ultrastructural level only in some species, including some Spongillidae (see: Weissenfels 1989), Halisarca dujardini Johnston, 1842 (Demospongiae, Halisarcida) (Ereskovsky and Gonobobleva 2000, Ereskovsky 2002, Gonobobleva and Ereskovsky 2004a, 2004b, Ereskovsky et al. 2005, 2007a, Mukhina et al. 2006), some species of Oscarella (Ereskovsky and Boury-Esnault 2002, Boury-Esnault et al. 2003, Ereskovsky 2005, Ereskovsky et al. 2007b) and Amphimedon queenslandica Hooper and van Soest, 2006 (as Reniera sp.) (Leys and Degnan 2002, Degnan et al. 2005, Larroux et al. 2006). Looking back, we can see that out of the 540 articles on sponge embryology, only 93 are devoted to embryonic development in the strict sense. They deal with 21 species of Calcarea, 2 species of Hexactinellida and about 70 species of Demospongiae (Fig. 7). Strangely enough, there is only two publications (Hill et al. 2004, Laroux et al. 2006) on the development of sponges during sexual reproduction where molecular-biological methods were used. Researchers only start to decode complex embryonic morphogenesis at the ultrastructural level (Boury-Esnault et al. 1999, 2003, Ereskovsky and Gonobobleva 2000, Ereskovsky and Boury-

Fig. 2: The trend in general sponge development publications between 1870 and 2006.

43

Fig. 3: German spongiologs portraits. A. Frans Eilhard Schulze (1840-1921) ZM B IX-608; B. Otto Maas (1867-1916); C. Oscar Schmidt (1823-1886); D. Conrad Keller (1848-1930) ZM B I/1753. (The photos are kindly given by C. Eckert).

Esnault 2002, Leys and Degnan 2002, Gonobobleva and Ereskovsky 2004a, Usher and Ereskovsky 2005, Leys et al. 2006) and investigate developmental genes expression during embryonic development (Hill et al. 2004, Laroux et al. 2006).

The present Gastrulation: verbal or real problem in sponges?


Applicability of the term gastrulation to sponge development is one of the sore points in our discussion (see: Efremova 1997, Leys 2004, Ereskovsky and Dondua 2006). There are two principal definitions of this term. The first is used by most, but not all developmental biologists: Gastrulation is the process in embryonic development in the course of which three primary germ layers are formed and the gut is formed through complex

cell migrations (Technau and Scholz 2003, Stern 2004, Keller 2005, Martindale 2005). The second definition is rare: Gastrulation is the process that results in a multilayered organism during embryonic development (Efremova 1997, Leys and Degnan 2002, Maldonado 2004, Leys 2004). According to these authors, the formation of a multilayered embryo during embryogenesis in sponges should be considered as gastrulation, since mechanisms of cell reorganization in the blastula are similar with those recognized as gastrulation in cnidarians. This contradiction stems from the absence of a generally accepted point of view on the homology of embryonic processes and their derivatives in sponges and other animals. Despite recent impressive progress in morphogenetic research in general, works on sponge embryonic morphogenesis are very rare. Therefore, investigations of mechanisms of sponge embryonic development are currently

44

Fig. 5: Paul Brien (1894-1975), Brussels, 1968. (The photo is kindly given by Ph. Willenz). Fig. 4: Yves Delage (1854-1920) at the Roscoff Marine laboratory, 1905 (From: Beetscen and Fischer, 2004).

much more important than terminological discussions. Morphogenesis is the mechanism responsible for creation of body plan during embryonic development, metamorphosis, asexual reproduction and regeneration. Morphogenetic investigations are a promising branch in developmental biology of sponges. Formation of multilayer embryos in Metazoa is achieved either by the migration of individual cells, or, by movements of cell sheets (Keller et al. 2003, Keller 2005). The former morphogenetic movements are known as mesenchymal morphogenesis or epithelialmesenchymal transitions. One such example is multipolar ingression (Shook and Keller 2003). The latter is epithelial morphogenesis and invagination is such an example (Keller et al. 2003, Gilbert 2003). Morphogenetic cell movements are determined by complex and specific gene systems. Their origin and evolution resulted in the diversity of metazoan developmental types. Apparently, they are involved in multicellular embryos formation in all animals, including sponges. For instance, formation of sponge larvae is accompanied by almost all types of cell movements, characteristic of Eumetazoa (Efremova 1997, Leys 2004, Maldonado 2004, Ereskovsky 2005, Leys and Ereskovsky 2006, Ereskovsky and Dondua 2006): cell delamination (Hexactinellida Oopsacas minuta Topsent, 1927) (Fig. 8A), morula delamination (Demospongiae: Dendroceratida, Dictyoceratida, Halichondrida, Haplosclerida) (Fig. 8B), invagination,

unipolar and multipolar ingression (Demospongiae: Halisarcida Halisarca dujardini) (Fig. 8C, D). At the same time, some unique morphogeneses, not found in other multicellular animals, have been described in sponges. They are, for example, multipolar egression in Homoscleromorpha (Demospongiae) (Fig. 8E), polarized delamination (Demospongiae: Poecilosclerida and Halichondrida) (Fig. 8F), excurvation in Calcaronea (Calcarea) (Fig. 8G), formation of blastula (pseudoblastula) by means of ingression of maternal cells into the embryo in Chondrosia reniformis Nardo, 1833 (Demospongiae: Chondrosida) (Fig. 8H), and unipolar proliferation (Demospongiae: Verticillitida Vaceletia crypta (Vacelet, 1977) (Fig. 8I). According to comparative embryological data on Porifera and Cnidaria, ancestors of Metazoa must have been able to form epithelial layers and to disaggregate these layers into individual cells. They were capable of epithelial morphogenesis and also had regulatory mechanisms controlling cell ingression and ensuring directed movement of cell masses. It may be therefore concluded that mechanisms of morphogenetic movements emerged in the course of evolution prior to the separation of the covering cell layer as ectoderm and the internal digestive cell mass as endoderm. This testifies to the independence of processes of spatial distribution of cells and their specification in the forming embryo as ectodermal and endodermal tissues (Ereskovsky and Dondua 2006). So, it is incorrect to apply the term gastrulation to sponge development.

45

larvae have a strongly pronounced anterior-posterior polarity, distinct photoreception and other kinds of taxis (Maldonado 2004). Some demosponge larvae have desmosome-like cell junctions (Fig. 9). Finally, it has been shown that parenchymella of A. queenslandica possesses some of the transcription factor genes that appear to be characteristic of Metazoa. They are expressed during the development of this species (Larroux et al. 2006).

Cellular and molecular basis of embryonic morphogenesis in sponges Cellular basis of embryonic morphogenesis
During metazoan embryonic development, the cells can undergo changes either autonomously or in conjunction with their neighbors to form an embryo. Most of morphogenetic movements require that a subset of cells detach from their neighbors and acquire properties allowing them to migrate to new position. Obviously, the consequences of changes in cell shape and motility will be quite different if cells are joined in an epithelium or if they are unconstrained by neighbors. Cell motility is generated by contractile elements of the cytoskeleton. The following question requiring an answer arises: What is the cytoskeleton dynamics during embryonic morphogenesis in sponges?

Cell-extracellular matrix adhesion


One of the main molecules that mediate cell anchorage to the substratum during the morphogenesis is integrin, which are key molecules during early animal development (Darribere et al. 2000). (Integrins, members of the transmembrane linker proteins family, traverse the cell membrane, anchoring the actin microfilaments on the inside and may bind to the fibronectin and in other extracellular matrix proteins). Integrins were shown to be present in some adult demosponges: Ophlitaspongia tenuis and Microciona prolifera Ellis and Solander, 1786 (Brower et al. 1997, Kuhns et al. 2001, Sabella et al. 2004), Geodia cydonium (Jameson, 1811) (Pancer et al. 1997, Mller 1997) and in Suberites domuncula (Olivi, 1792) (Wimmer et al. 1999). The following questions arise: Are integrins involved in embryonic development of sponges? Is their morphogenetic role the same in sponges and in other animals?

Intercellular adhesion
Fig. 6: A. Odette Tuzet (1903-1976) and O. Duboscq (1868-1943), Banuls-sur-Mer Marine laboratory, 1937; B. Claude Lvi, Paris, 2000 (The photo is kindly given by J. Vacelet).

Evolutionary importance of larvae


Evolutionary importance has been attached to larvae of Bilateria since A. Kowalevskys studies on ascidia (1866). This idea has recently received molecular-biological support (Raff 1994, Peterson and Davidson 2000). Indeed, sponge

Cell-cell interactions are also important for tissue formation during development. A remarkable feature of sponges is that when dissociated to single cells they can undergo species-specific reaggregation (Wilson 1907). This is mediated by an extracellular proteoglycan complex, known as aggregation factor (AF) that acts as a bridge between receptor proteins on neighboring cells (Schutze et al. 2001). The AF receptor also possesses an RGD (Arg-GlyAsp attachment site) integrin-binding motif. RGD containing peptides will block AF-mediated aggregation. Both the RGD peptide and AF stimulate a range of intracellular responses (Wimmer et al. 1999). It was proposed that binding of AF promotes interaction between the RGD of the AF receptor

46

Fig. 7: The trend in sponge embryonic development publications between 1870 and 2006.

Fig. 8: Different types of morphogenesis in sponges resulting in larva formation: A. Cell delamination (Hexactinellida Oopsacas minuta); B. Morula delamination (Demospongiae: Dendroceratida, Dictyoceratida, Halichondrida, Haplosclerida); C. Invagination (Halisarca dujardini, Demospongiae); D. Multipolar ingression (H. dujardini, Demospongiae); E. Multipolar egression (Homoscleromorpha, Demospongiae); F. Polarized delamination (Demospongiae: Poecilosclerida and Halichondrida); G. Excurvation (Calcaronea, Calcarea); H. Formation of blastula (pseudoblastula) by means of ingression of maternal cells into the embryo (Chondrosia reniformis, Demospongiae: Chondrosida); I. Unipolar proliferation (Demospongiae: Verticillitida Vaceletia crypta). (From: Ereskovsky and Dondua 2006).

47

Fig. 9: Semi-thin micrographs of demosponges larvae with the desmosom-like cell junctions (insets). A. Parenchymella of Ircinia oros (Dictyoceratida); B. Disphaerula of Halisarca dujardini (Halisarcida); C. Cinctoblastula of Corticium candelabrum Schmidt, 1862 (Homoscleromorpha); D. Paren-chymella of Pleraplysilla spinifera (Schulze, 1879) (Dictyoceratida). Abbreviations: AP anterior pole, PP posterior pole. Scale bar, A 100 m; Inset 0,2 m; B - 50 m; Inset 25 nm; C - 50 m; Inset 0,2 m; D 50 m; Inset 0,2 m.

and sponge integrin proteins (Harwood and Coates 2004). Many excellent studies dealt with the fine mechanisms of cell-cell interactions in sponge cell cultures (see: FernandezBusquets et al. 2002, Misevic et al. 2004). Nevertheless, no cadherin, catenin or related proteins have been identified in sponges (Harwood and Coates 2004). However, there is not a single work demonstrating either specific or differential cellular adhesiveness in sponge embryonic development.

Developmental genes in sponges


The presence of metazoan developmental genes in demosponge genomes has been shown (e.g., Degnan et al. 1993, 1995, Coutinho et al. 1994, 2003, Seimiya et al. 1994, 1997, Hoshiyama et al. 1998, Richelle-Maurer et al. 1998, Manuel and Le Parco 2000, Adell et al. 2003, Perovic et al. 2003, Wiens et al. 2003a, b, Adell and Muller 2004, Hill et al. 2004, Manuel et al. 2004, Richelle-Maurer et al. 2006, Larroux et al. 2006, 2007). However, their roles in

embryogenesis and metamorphosis are unknown. To date, our understanding of sponge gene expression is restricted chiefly to asexual reproductive processes, such as gemmule germination, cell aggregation, and primorphs formation. Hill et al. (2004) followed the expression of non-Hox Antp-class Bar-/Bsh-like gene during larva releasing, larva swimming and metamorphosis. Laroux et al. (2006) demonstrated that an extensive range of metazoan transcription factor genes, including members of the ANTP class (outside Hox, ParaHox, and extended-Hox clades), Pax, POU, LIM-HD, Sox, nuclear receptor (NR), Fox (forkhead), T-box, Mef2, and Ets gene classes are expressed during A. queenslandica (Haplosclerida) development. These data combined with developmental gene expression patterns from other animals suggest that these genes may have played important regulatory roles in the embryos of the first metazoans. These works will probably now trigger an explosion of studies on the role of developmental genes in sponge development.

48

Fig. 10: The basal-apical and posterior-anterior axis of sponges on different stage of its life cycle. A. Haliclona sp. from White Sea. B. Diagram of demosponges organization. C, D. Semi-thin micrograph (C) and diagram (D) of Halisarca dujardini (Halisarcida) rhagon. E, F. SEM micrograph (E) and diagram (F) of Halisarca dujardini (Halisarcida) disphaerula larva. The arrows indicate the basal-apical (A D) and posterior-anterior (E, F) axis. Abbreviation: AP anterior pole, CC choanocyte chamber; Ep exopinacoderm; O osculum; PP posterior pole. A 2 cm; B 250 m; C - 50 m; D - 50 m; E - 50 m; F - 50 m.

Axis formation
An important characteristic distinguishing sponges from higher metazoans is the nature of body symmetry. Higher animals have two obvious body axes, anterior-posterior and dorsal-ventral, and are therefore bilaterally symmetrical (Bilateria). All young (rhagon or olynthus) and monooscular sponges, by contrast, have a single overt axis (apical-basal) defined by the presence of an osculum at one end (Fig. 10 AD). The question is: Does the apical-basal axis of an adult sponge correspond to the posterior-anterior axis of higher animals? Since the larvae of all the sponges investigated also possess an apical-basal axis, the answer to this question may be yes (Ereskovsky 2005) (Fig. 10E, F).

Development: phylogeny and evolution


The following question is very important in this respect: Can embryological data be applied to sponge phylogeny and evolution? According to the paraphyletic hypotheses, based on molecular data, Porifera consists of four groups: Calcarea, Hexactinellida, Demospongiae and Homoscleromorpha (Borchiellini et al. 2001, 2004). I proposed seven

developmental types in recent Porifera: I - trichimella (Hexactinellida); II - calciblastula (Calcinea); III - amphiblastula (Calcaronea); IV - cinctoblastula (Homoscleromorpha); V - disphaerula (Halisarcida); VI - direct development (Tetilla, Spirophorida); VII parenchymella (Ereskovsky 2004). There are four principal cleavage patterns in sponges: incurvational, polyaxial, radial and chaotic (Fig. 11: 1-4). They result in formation of three main types of blastula: stomoblastula, coeloblastula and stereoblastula (Fig. 11: 57). The latter two types are derived from different cleavage patterns. Different embryonic morphogeneses lead to 8 or 9 larval types (Fig. 11: 8-22). Difficulties of using embryological data for phylogenetic interpretation of Porifera are associated with a high degree of polymorphism of their development. The same cleavage pattern and blastula type may be characteristic of several different larval types. For example, radial cleavage, resulting in coeloblastula and stereoblastula, leads to parenchymella, trichimella, direct development, and coeloblastula (Fig. 11: 3-62-13, 14, 15; 71-16, 17, 18). On the other hand, the same larval type may be the result of different cleavage patterns and modes of morphogenesis.

49

Fig. 11: Diagram of sponges cleavage and morphogenesis, leading to the larvae. 14 Cleavage patterns in sponges: incurvational (1), polyaxial (2), radial (3), and chaotic (4). Three main form of sponges blastula: stomoblastula (5), coeloblastula (6), and stereoblastula (7). Different larval types of sponges: amphiblastula of Calcaronea (Calcarea) (8); calciblastula of Calcinea (Calcarea) (9); coeloblastula (10), parenchymella (11), and disphaerula (12) of Halisarca (Halisarcida); parenchymella of Vaceletia crypta (Verticillitida) (13); pseudoblastula of Chondrosia reniformis (Chondrosida) (14); trichimella of Oopsacas minuta (Hexactinellida) (15); juvenile of Tetilla under direct development (16); parenchymella of Tethya aurantium (Pallas, 1766; Hadromerida) (17); coeloblastula of Polymastia robusta Bowerbank, 1866 (Hadromerida) (18); parenchymella of Dictyoceratida (19); parenchymella of freshwater Haplosclerida (20); parenchymella of Poecilosclerida (21); cinctoblastula of Homoscleromorpha (22).

50

For example, parenchymella may originate from polyaxial, radial and chaotic cleavage, using different modes of morphogenesis (Fig. 11: 11, 13, 17, 19-21). Coeloblastula larva can result from polyaxial (Halisarcida and Calcinea) as well as radial cleavage (Hadromerida), through coeloblastula or stereoblastula stages (Fig. 11: 9, 10, 18). The fact that similar characters can result from different developmental pathways means that ontogenetically earlier stages can be evolutionarily altered. The opposite case showing early similarity with later occurring differences is more common. However, both aspects taken together reveal that in the course of evolution developmental stages may be altered at all levels, from the molecular to the morphogenetic, regardless of whether a stage occurs early or late during the ontogenetic process. Results of the comparative analysis of the cleavage and embryonic morphogenesis testify that these characters taken separately cannot form a basis for phylogenetic constructions within the Porifera. For example, Calcinea (Calcarea) embryogenesis (Fig. 11: 2-61-9) is much closer to the development of Halisarcida (Demospongiae) (Fig. 11: 2-6110) than to Calcaronea (Fig. 11: 1-5-8). Thus, a variety of cleavage patterns, types of blastulae and morphogenesis, leading to larvae formation in sponges, does not allow making a conclusion about certain linear ways of developmental evolution for all Porifera. It testifies to an early divergence of sponge macrogroups or, more likely, paraphyly and their long parallel evolution.

Acknowledgments
I thank Prof. Galina Korotkova, Prof. Archil Dondua (SaintPetersburg State University), Dr. Nicole Boury-Esnault, Dr. Jean Vacelet, Dr. Carole Borchiellini, Dr. Thierry Perez (Centre dOcanologie de Marseille, France) for helpful discussions, Carsten Eckert (Museum fr Naturkunde, Zentralinstitut der Humboldt, Universitt zu Berlin, Germany) and Dr. Philippe Willenz (Royal Belgian Institute of Natural Sciences, Brussels) and J. Vacelet for providing spongiologs portraits, Daria Tokina (Zoological Institute of RAS, Saint-Petersburg, Russia) for technical assistance, and Natalia Lentsman for improving the English. This work was funded by the program RFBR N 06-04-48660 and 06-04-58573.

References
Adell T, Grebenjuk VA, Wiens M, Mller WEG (2003) Isolation and characterization of two T-box genes from sponges, the phylogenetically oldest metazoan taxon. Dev Genes Evol 213: 421-434 Adell T, Mller WEG (2004) Cloning and characterization of T-box and forkhead transcription factors from Porifera. In: Pansini M, Pronzato R, Bavestrello G, Manconi R (eds). Sponge science in the new millennium. Bull Mus Ist Biol Univ Genova 68: 163-173 Bidder GP (1898) The skeleton and classification of calcareous sponge. Proc Roy Soc 64: 61-76 Borchiellini C, Manuel M, Alivon E, Boury-Esnault N, Vacelet J. Le Parco Y (2001) Sponge paraphyly and the origin of Metazoa. J Evol Biol 14(1): 171-179 Borchiellini C, Chombard C, Manuel M, Alivon E, Boury-Esnault N, Vacelet J (2004) Molecular phylogeny of Demospongiae: implications for classification and scenarios of character evolution. Mol Phyl Evol 32: 823-837 Boury-Esnault N, Efremova SM, Bzak C, Vacelet J (1999) Reproduction of a hexactinellid sponge: first description of gastrulation by cellular delamination in the Porifera. Invertebr Rep Dev 35: 187-201 Boury-Esnault N, Ereskovsky AV, Bezac C, Tokina D (2003) Larval development in Homoscleromorpha (Porifera, Demospongiae) first evidence of basal membrane in sponge larvae. Invertebr Biol 122: 187-202 Brower DL, Brower SM, Hayward DC, Ball EE (1997) Molecular evolution of integrins - genes encoding integrin beta subunits from a coral and a sponge. Proc Natl Acad Sci 94(17): 9182-9187 Coutinho CC, Vissers S, van de Vyver G (1994) Evidence of homeobox genes in the freshwater sponge Ephydatia fluviatilis. In: van Soest RWM, van Kempen TMG, Braekman JC (eds). Sponges in time and space: biology, chemistry, paleontology. Balkema, Rotterdam. pp. 385-388 Coutinho CC, Fonseca RN, Mansure JJ, Borojevic R (2003) Early steps in the evolution of multicellularity: deep structural and functional homologies among homeobox genes in sponges and higher metazoans. Mech Dev 120: 429-440 Darribere T, Skalski M, Cousin H, Gaultier A, Montmory C, Alfandari D (2000) Integrins: regulators of embryogenesis. Biol Cell 92: 5-25 Degnan BM, Degnan SM, Naganuma T, Morse DE (1993) The ets multigene family is conserved throughout the Metazoa. Nucl Acids Res 21(15): 3479-3484 Degnan BM, Degnan SM, Giusti A, Morse DE (1995) A Hox/Hom homeobox gene in sponges. Gene 155(2): 175-177

The future
We are now on the threshold of the fourth period of sponge developmental studies. To enhance our knowledge on this topic, the following steps are currently necessary: - To select some model sponge species with different types of development; - To investigate their development from egg to juvenile at ultrastructural level; - To decode morphogenetic mechanisms of development using ultrastructural and molecular methods. Studies of cellular and molecular basis of embryonic morphogenesis in sponges will provide answers to the following important questions: - What is the role of intercellular contacts and cytoskeleton dynamics in embryonic and postembryonic morphogenesis? - What is the role of cell-cell and cell-extacellular matrix interactions? - Are integrins, laminins, and signaling molecules involved in development the same in sponges and other metazoans? - Which developmental genes work during sponge embryonic development? - Does the apical-basal axis of adult sponge correspond to the anterior-posterior axis of higher animals?

51

Degnan BM, Leys SP, Larroux C (2005) Sponge development and antiquity of animal pattern formation. Integr Comp Biol 45: 335341 Delage Y (1892) Embryogenie des ponges. Arch Zool Expr Gn 10: 345-498 Delage Y (1899) On the position of sponges in the Animal Kingdom. In: Sedgwick A (ed). Proceedings of the 4th International Congress of Zoology. Clay & Sons, Cambridge. pp 57-62 Efremova SM (1997) Once more on the position among Metazoa - Gastrulation and germinal layers of sponges. In: Ereskovsky AV, Keupp H, Kohring R (eds). Modern problems of poriferan biology. Freie Universitat, Berlin. pp. 7-15 Ereskovsky AV, Gonobobleva EL (2000) New data on embryonic development of Halisarca dujardini Johnston, 1842 (Demospongiae: Halisarcida). Zoosystema 22: 355-368 Ereskovsky AV, Boury-Esnault N (2002) Cleavage pattern in Oscarella species (Porifera, Demospongiae, Homoscleromorpha), transmission of maternal cells and symbiotic bacteria. J Nat Hist 36: 1761-1775 Ereskovsky AV (2002) Polyaxial cleavage in sponges (Porifera): a new pattern of Metazoan cleavage. Dokl Biol Sci 386: 472-474 Ereskovsky AV (2004) Comparative embryology of sponges and its application for poriferan phylogeny. In: Pansini M, Pronzato R, Bavestrello G, Manconi R (eds). Sponge science in the new millennium. Bull Mus Ist Biol Univ Genova 68: 301-318 Ereskovsky AV (2005) Comparative embryology of sponges. St. Petersburg State Univ. Press. St. Petersburg Ereskovsky AV, Gonobobleva EL, Vishnyakov AE (2005) Morphological evidence for vertical transmission of symbiotic bacteria in the viviparous sponge Halisarca dujardini Johnston (Porifera, Demospongiae, Halisarcida). Mar Biol 146(5): 869-875 Ereskovsky AV, Dondua AK (2006) The problem of germ layers in sponges (Porifera) and some issues concerning early metazoan Evolution. Zool Anzeiger 245: 65-76 Ereskovsky AV, Konjukov P, Willenz P (2007a) Experimental metamorphosis of Halisarca dujardini larvae (Demospongiae, Halisarcida): evidence of flagellated cell totipotentiality. J Morphol 268(6): 529-536 Ereskovsky AV, Tokina DB, Bzac C, Boury-Esnault N (2007b) Metamorphosis of cinctoblastula larvae (Homoscleromorpha, Porifera). J Morphol 268(6): 518-528 Fernandez-Busquets XK, William J, Simpson TL, Ho M, Gerosa D, Grob M, Burger MM (2002) Cell adhesion-related proteins as specific markers of sponge cell types involved in allogeneic recognition. Dev Comp Immunol 26(4): 313-323 Gilbert SF (2003) Developmental biology. Sinauer Associates Inc. Sunderland Gonobobleva EL, Ereskovsky AV (2004a) Polymorphism in free-swimming larva of Halisarca dujardini (Demospongiae, Halisarcida). In: Pansini M, Pronzato R, Bavestrello G, Manconi R (eds). Sponge science in the new millennium. Bull Mus Ist Biol Univ Genova 68: 349-356 Gonobobleva EL, Ereskovsky AV (2004b) Metamorphosis of the larva of Halisarca dujardini (Demospongiae, Halisarcida). Bull Inst roy Sci Nat Belgique Biol 74: 101-115 Haeckel E (1866) Generelle Morphologie der Organismen. Berlin, George Reismer Haeckel E (1872) Die Kalksschwmme. Eine Monographie in zwei Bnden. Georg Reimer, Berlin

Haeckel E (1874) Die Gastrae Theorie, die phylogenetische Classification des Thierreichs und die Homologie der Keimblatter. Jena Ztschr Naturwiss 8: 1-55 Harwood A, Coates JC (2004) A prehistory of cell adhesion. Cur Opinion Cell Biol 16: 470-476 Hill A, Tetrault J, Hill M (2004) Isolation and expression analysis of a poriferan Antp-class Bar-/Bsh-like homeobox gene. Dev Genes Evol 214: 515-523 Hoshiyama D, Suga H, Iwabe N, Koyanagi M, Nikoh N, Kuma K, Matsuda F, Honjo T, Miyata T (1998) Sponge pax cDNA related to pax-2/5/8 and ancient gene duplications in the pax family. J Mol Evol 47(6): 640-648 Keller R, Davidson LA, Shook DR (2003) How we are shaped: the biomechanics of gastrulation. Differentiation 71: 171-205 Keller R (2005) Cell migration during gastrulation. Curr Opin Cell Biol 17: 533-541 Kowalevsky A.O. (1866) Entwiklungsgeschichte der einfachen Ascidien. Mm Acad Sci St Petersbourg 10(15): 1-16 Kuhns WJ, Rusciano D, Kaltenbach J, Ho M, Burger MM, FernandezBusquets X (2001) Up-regulation of integrins alpha(3) beta(1) in sulfate-starved marine sponge cells: functional correlates. Biol Bull 201(2): 238-239 Larroux C, Fahey B, Liubicich D, Hinman VF, Gauthier M, Gongora M, Green K, Wrheide G, Leys SP, Degnan BM (2006) Developmental expression of transcription factor genes in a demosponge: insights into the origin of metazoan multicellularity. Evol Dev 8: 150-173 Larroux C, Fahey B, Degnan SM, Adamski M, Rokhsar DS, Degnan BM (2007) The NK homeobox gene cluster predates the origin of Hox genes. Curr Biol 17: 1-5 Lvi C (1956) tude des Halisarca de Roscoff. Embryologie et systematique des Dmosponges. Trav Stat Biol Roscoff NS 7: 3181 Leys SP (2004) Gastrulation in sponges. In: Stern CD (ed). Gastrulation. From cells to embryo. Cold Spring Harbor Lab Press, New York. pp. 23-32 Leys SP, Degnan BM (2002) Embryogenesis and metamorphosis in a haplosclerid demosponge: gastrulation and transdifferentiation of larval ciliated cells to choanocytes. Invertebr Biol 121: 171-189 Leys SP, Cheung E, Boury-Esnault N (2006) Embryogenesis in the glass sponge Oopsacas minuta: formation of syncytia by fusion of blastomeres. Integr Compar Biol 46(2): 104-117 Leys SP, Ereskovsky AV (2006) Embryogenesis and larval differentiation in sponges. Can J Zool 84: 262-287 Maldonado M (2004) Choanoflagellates, choanocytes, and animal multicellularity. Invertebr Biol 123: 1-22 Manuel M, Le Parco Y (2000) Homeobox gene diversification in the Calcareous sponge, Sycon raphanus. Mol Phylog Evol 17: 97-107 Manuel M, Le Parco Y, Borchiellini C (2004) Comparative analysis of Brachyury T-domains, with the characterization of two new sponge sequences, from a hexactinellid and a calcisponge. Gene 340: 291-301 Martindale MQ (2005) The evolution of metazoan axial properties. Nature Rev Genetics 6: 917-927 Minchin EA (1896) Suggestions for a natural classification of the Asconidae. Ann Mag Nat Hist 18: 349-362 Misevic GN, Guerardel Y, Sumanovski LT, Slomianny MC, Demarty M, Karamanos Y, Maes E, Popescu O, Strecker G (2004) Molecular recognition between glyconectins as an adhesion self-assembly pathway to multicellularity. J Biol Chem 279: 15579-15590

52

Mukhina YI, Kumeiko VV, Podgornaya OI, Efremova SM (2006) The fate of larval flagellated cells during metamorphosis of the sponge Halisarca dujardini. Int J Dev Biol 50: 533-541 Mller WEG (1997) Origin of metazoan adhesion molecules and adhesion receptors as deduced from cDNA analyses in the marine sponge Geodia cydonium: a review. Cell Tissue Res 289(3): 383395 Pancer Z, Kruse M, Mller I, Mller WEG (1997) On the origin of metazoan adhesion receptors: cloning of integrin alpha subunit from the sponge Geodia cydonium. Mol Biol Evol 14: 391-398 Pavans de Ceccatty M (1986) Cytosceletal organisation and tissue patterns of epithelia in the sponge Ephydatia muelleri. J Morphol 189: 45-65 Perovic S, Schrder HC, Sudek S, Grebenjuk VA, Batel R, Mauro Tifani IMM, Mller WEG (2003) Expression of one sponge Iroquois homeobox gene in primmorphs from Suberites domuncula during canal formation. Evol Devel 5(3): 240-250 Peterson KJ, Davidson EH (2000) Regulatory evolution and the origin of the bilaterians. Proc Natl Acad Sci 97: 4430-4433 Raff R (1994) Developmental mechanisms in the evolution of animal form. In: Bengdson S (ed). Early life of earth. Nobel symposium. N 84. Columbia University Press, New York. pp. 489-500 Richelle-Maurer E, van de Vyver G, Vissers S, Coutinho CC (1998) Homeobox-containing genes in freshwater sponges: characterization, expression, and phylogeny. In: Mller WEG (ed). Molecular evolution: evidence for monophyly of Metazoa. Springer Verlag, vol. 19. Berlin. pp. 157-175 Richelle-Maurer E, Boury-Esnault N, Itskovich VB, Manuel M, Pomponi SA, van de Vyver G (2006) Conservation and phylogeny of a novel family of non-Hox genes of the Antp class in Demospongiae (Porifera). J Mol Evol 63: 222-230 Sabella CA, Faszewski EE, Kaltenbach JC, Kuhns WJ, Burger MM, Fernandez-Busquets X (2004) Immunocytochemical detection of integrins alpha(3) and beta(1) in allografts of the marine sponge, Microciona prolifera. Biol Bull 207: 162 Schutze J, Krasko A, Diehl-Seifert B, Mller WEG (2001) Cloning and expression of the putative aggregation factor from the marine sponge Geodia cydonium. J Cell Sci 114: 3189-3198

Seimiya M, Ishiguro H, Miura K, Watanabe Y, Kurosawa Y (1994) Homeobox-containing genes in the most primitive Metazoa, the sponges. Europ J Biochem 221: 219-225 Seimiya M, Watanabe Y, Kurosawa Y (1997) Identification of POUclass homeobox genes in a freshwater sponge and the specific expression of these genes during differentiation. Europ J Biochem 243: 27-31 Shook D, Keller R (2003) Mechanisms, mechanics and function of epithelialmesenchymal transitions in early development. Mech Devel 120: 1351-1383 Stern C (ed) (2004). Gastrulation: from cells to embryo. Cold Spring Harbor Press, Cold Spring Harbor Technau U, Scholz CB (2003) Origin and evolution of endoderm and mesoderm. Int J Dev Biol 47: 531-539 Usher K, Ereskovsky AV (2005) Larval development, ultrastructure and metamorphosis in Chondrilla australiensis Carter, 1873 (Demospongiae, Chondrosida, Chondrillidae). Invert Rep Dev 47: 51-62 Weissenfels N (1989) Biologie und Mikroskopische Anatomie der Swasserschwmme (Spongillidae). Gustav Fischer Verlag. Stuttgart, New York Wiens M, Mangoni A, DEsposito M, Fattorusso E, Korchagina N, Schroder HC, Grebenjuk VA, Krasko A, Batel R, Mller IM, Mller WEG (2003) The molecular basis for the evolution of the metazoan bodyplan: extracellular matrix-mediated morphogenesis in marine demosponges. J Mol Evol 57: S60-S75 Wiens M, Batel R, Korzhev M Mller WEG (2003) Retinoid X receptor and retinoic acid response in the marine sponge Suberites domuncula. J Exp Biol 206: 3261-3271 Wilson HV (1907) On some phenomena of coalescence and regeneration in sponges. J Exp Zool 5: 245-258 Wimmer W, Perovic S, Kruse M, Schroder HC, Krasko A, Batel R, Mller WEG (1999) Origin of the integrin-mediated signal transduction -functional studies with cell cultures from the sponge Suberites domuncula. Europ J Biochem 260: 156-165

Porifera research: Biodiversity, innovation and sustainaBility - 2007

53

Sponge coordination, tissues, and the evolution of gastrulation


Sally P. Leys
University of Alberta. CW 405, Biological Sciences. Edmonton, Alberta, T6G 2E9. Phone: (780) 492-6629. Fax: (780) 4929234. sleys@ualberta.ca Abstract: One of the unifying features of animals is that they carry out rapid, coordinated movement. This ability results from the early evolution of tissues that can both conduct signals and contract. The origin of tissues is thus intimately tied to the origin of nerves and muscle, which have long been considered the major innovations of cnidarians (anemones and jellyfish). However, the hypothesis that muscle may have conferred important selective advantages in preying and escape in the cnidarian ancestor suggests that this ancestor also had nerves, or at least the ability to coordinate contractions of its muscle. These ideas are supported by a considerable body of work showing that the genes which in triploblasts are involved in muscle specification and differentiation are expressed during the development of medusa and polyp in three cnidarian model species. Recent data suggest that sponges are paraphyletic, implying that cnidarians and sponges share a common ancestor that had a sponge-like body plan. Here I re-examine evidence for a coordinated contraction system in modern cellular sponges as evidence for functional tissues. I suggest that coordination of the animal is evidence that sponges do possess tissues which arise by gastrulation-like processes during embryogenesis as is the case in other metazoans. The sponge peristaltic contractile system may represent the foundations of coordinating tissues and have set the stage for the innovation of nerves and muscle in later animals. Keywords: evolution of nerves, coordination, evolution of tissues, Porifera

Introduction:
Although the Porifera have long been regarded as an evolutionary side-branch of metazoans (Parazoans), recent molecular phylogenies propose that sponges are paraphyletic that calcareous sponges (Calcispongia) and possibly Homoscleromorphs (Peterson, personal communication) are more closely related to cnidarians and other metazoans than they are to other sponges (Borchiellini et al. 2001, Medina et al. 2001). A paraphyletic Porifera suggests that the ancestral metazoan was a sponge-like organism, a suggestion that may not be as revolutionary as it first appears. The sponge body plan is only difficult to grasp in the light of work which suggests that sponges are colonies of a few types of cells organized around a system of water canals (Simpson 1984), animals that lack true epithelia (Mackie 1984, Tyler 2003), and the ability to coordinate activity at the level of the whole organism (Mackie 1979). In contrast, recent work shows that sponge epithelia are sealed units (Gonobobleva and Ereskovsky 2004) with tight junction proteins (Adell et al. 2004) and basement membranes (Boute et al. 1996, Boury-Esnault et al. 2003, Maldonado 2004). Sponges lack neurons (Pavans de Ceccatty 1989) but syncytial sponge tissues are nonetheless excitable and propagate electrical signals that control the feeding current (Leys and Mackie 1997). In cellular sponges external stimuli (such as contact by amphipods) trigger waves of contraction (Nickel 2004), and similar contractile waves are known to be widespread among

all cellular sponges. Though slow, the contractions in cellular sponges illustrate the components of peristaltic waves seen in animals with a nervous system. Peristalsis is an efficient mechanism for controlling fluid movement through a tube, and typically consists of a series of motor patterns that control relaxation in front of and contraction behind the object being moved by the fluid. The same pulsating movement occurs in the hearts of the invertebrate chordate Amphioxus (Holland et al. 2003) and ascidians, as well as in the gut and circulatory systems of insects and molluscs. In the sea pansy Renilla, a member of the most basal cnidarian group the Pennatulacea, peristaltic contractions of the gastrovascular cavity (GVC) a tube-like gut that pervades the entire animal control the movement of fluid for feeding and respiration as well as gamete release (Mechawar and Anctil 1997). I suggest that the sponge body represents the first elaboration of a peristaltic contractile system in Metazoa, a system that was later adapted for locomotion, digestive and circulatory activity, and which gave rise to the hydrostatic skeleton. It is likely that elements of signaling used in these activities in higher animals may be found in extant sponges. I further suggest that coordinated contractions in sponges is evidence that these animals do possess tissues, and that these must have arisen during embryogenesis via gastrulation-like processes as in other animals. It is our challenge to understand what elements of tissues known in higher animals are used in sponges.

54

Propagated contractions in sponges


Cellular sponges have long been known to contract inhalant and exhalent openings (ostia and oscula), and portions of their canal system (Mackie 1979). The slow rates of propagation (4-400 m s-1) and difficulty of observation in large animals have led to the conclusion that events are local and decremental. But at least three species show propagated contractions that are involved in expulsion of sediment (Nickel 2004) or gametes (Reiswig 1970), and rhythmic, diurnal pulses that may assist water flow through the animal (Weissenfels 1990, Nickel 2004). Given the slowness of the events, electrical signaling is not likely to be involved. Although glass sponges can propagate electrical signals, they do this through an uninterrupted giant syncytium (Leys and Mackie 1997). So far there is no convincing evidence of gap junctions (or other communicating junctions) in cellular sponges that would allow equivalent, rapid signalling (see Leys and Meech 2006). The slowness of the events suggests a slower mechanism of signaling via extracellular molecules is possible. Neurotransmitter molecules have been localized in tissues of calcareous sponges (Lentz 1966) and demosponge larvae (Weyrer et al. 1999), and many of these chemicals have been shown to affect the contraction of ostia and oscula (Parker 1910, Prosser et al. 1962, Emson 1966, Prosser 1967). Fascinating videos showing rhythmic contractions of the asconoid calcareous sponge Leucosolenia (C. Bond, personal communication) and time-lapse photographs of contractions in leuconoid calcareous sponges (Gaino et al. 1991) suggest the habit is widespread among cellular sponges.

Mechanism(s) of coordination of contractions


Video microscopy and image analysis of contractile behaviour show that both marine and freshwater sponges (de Vos and van de Vyver 1981, Weissenfels 1990, Nickel 2004) control the movement of water through a single aquiferous system (one osculum) in a manner similar to peristaltic contractions in the pennatulacean anthozoan Renilla. However, whereas most adult sponges are opaque to microscopy, juvenile demosponges are usually transparent so that individual cells crawling and cells forming epithelial linings to canals can be observed in vivo. Freshwater sponges have the added advantage that they can be readily grown in vitro from gemmules (overwintering cysts), thereby providing a relatively easy preparation for analysis of the mechanism of signaling. Experiments can be carried out on 7 day-old juvenile sponges hatched at room temperature using wellestablished protocols (de Vos 1971, de Vos and van de Vyver 1981, Francis and Poirrier 1986, Elliott 2004). Contractions begin after mechanical or chemical stimuli, and kinetics of contractions can be determined by computer assisted image analysis (1 image/10s) (Fig. 1, Elliott and Leys 2007). After each treatment the sponge carries out a stereotypical behaviour involving dilation and contraction, effectively expelling water (and any particulates) from the canal system. How are contractions propagated? The problem has been considered in depth by Jones (1962) who suggested the following possibilities: local changes in pressure that induce contractions some distance away; stretch receptors acting

sequentially in adjacent cells; release of an aqueous hormone into the water or of a chemical messenger into the mesohyl; and local (non-propagating) action potentials that function to excite adjacent cells. Although recent work has focused on the secreted hormone hypothesis (Ellwanger et al. 2004, Ellwanger and Nickel 2006), it is most likely that several mechanisms interact. For example, a local change in pressure could activate stretch receptors, which in turn could trigger the release of a locally acting messenger. In Ephydatia muelleri responses to mechanical stimuli involve a peristaltic-like wave of dilation and contraction (Leys and Meech 2006), yet spasms also occur simultaneously on either side of a single sponge (Ellwanger et al. 2004, Elliott and Leys 2007) how are these triggered? Perhaps minute changes in pressure stretch cell membranes at a distant location and trigger an apparently simultaneous contraction. Waves of contraction can also be seen to travel down (along) a canal and at the same time across canals. While a pressure wave could precede the contraction in both directions, evidence that amoeboid cells in the mesohyl cease crawling as the contraction passes (Fig. 2) point to a secreted messenger. Many molecules have been shown to trigger oscular and ostia closure, and to intiate contractions (reviewed by Jones 1962, Lawn 1982, Leys and Meech 2006). Widespread evidence for glutamate in signaling in plants (Demidchik et al. 2004), Paramecium (Yang et al. 1997) and astrocytes (Nedergaard et al. 2002), and evidence for metabotropic Glu/GABA receptors in sponges (Perovic et al. 1999), makes this molecule an especially good candidate for a signaling molecule. Indeed, recent experiments in both Tethya (Ellwanger and Nickel 2006) and Ephydatia (Elliott and Leys 2007) suggest that contractions can be triggered by application of glutamate in a concentration dependent manner. The working hypothesis is that contractions propagate via calcium waves as in mammalian astrocytes (Nedergaard 1994) and mast cells (Osipchuk and Cahalan 1992), either via the direct action of stretch receptors or by the release of locally acting chemicals, much as outlined by Jones (1962): a stimulus (pressure/mechanical) causes stretch receptors to trigger a rise in intracellular calcium, causing contraction of the cell, tension on adjacent cells, and stimulating release of a secreted messenger (such as glutamate), which in turn stimulates contraction of nearby cells, and so on. The slow rates of contraction around 20 m s-1 lend support to this hypothesis. However, some contractions, like that which travels up the osculum of E. muelleri at up to 375 m s-1 (McNair, 1923), are much faster. Since gap junction coupling is enhanced in the presence of glutamate (Enkvist and McCarthy 1994), it is possible that cells may be coupled by almost gap junctions, which connect cells as glutamate levels rise. During peristalsis in cnidarians and higher animals, nitric oxide signaling allows relaxation prior to contraction (Moroz et al. 2004, Anctil et al. 2005). Preliminary results show that nitric oxide (NO) synthase staining in E. muelleri tissues fixed for NADPH-diaphorase (Elliott and Leys 2007), and experiments by Ellewanger and Nickel (2006) suggest that NO modulates contractions in Tethya. Thus waves of contraction along the aquiferous canals in sponges may be modulated much in the same way they are in cnidarians

55

Fig. 1: Dilation and contraction of the aquiferous system of a 7-day old juvenile Ephydatia muelleri. White arrows point to the osculum, which increases in diameter by frame C as the canals dilate in A and B, and contract in C and D. The white regions between the dilated exhalent canals in (B) are the compressed inhalant canals (Elliott and Leys 2007).

Fig. 2: Cells crawling through the mesohyl stop when a wave of contraction passes by. A, B. India ink added to a dish with a sandwich preparation is taken into the chambers (black oblique lines). When all chambers are filled a wave of contraction propagates along the canals (white arrow pointing left) and across canals (white arrow pointing across). There is a slight delay before the contraction is seen moving along the second canal (2). C. Plot of the track (diamond) of a single cell crawling in the mesohyl of the first canal during the first contraction. As the canal diameter narrows during a contraction (10 minutes, 600 s after addition of dye), the cell ceases forward movement (long arrow and bar). Crawling commences once the canal is fully contracted (10 minutes later). Forward motion is slowed a second time (small arrows) when the incurrent canal (1) begins to dilate once more (Modified from Elliott and Leys, 2007).

even though the events occur at a much slower rate than allowed by nerves and true muscle.

Histology and ontogeny of contractile and signaling tissues


Coordination of contractions in cellular sponges remains a difficult problem because the concept of the sponge as an animal (Eumetazoa) is controversial. If sponges have a cellular level of organization (Parazoa) as is traditionally thought, there are no tissues: thus along what structure do contractions propagate and how does the animal maintain integrity among the cells during contractions? Most modern texts suggest that during the evolution of basal metazoans there was a graded acquisition of structured

tissues (Gilbert 2003), culminating in the invention of mesoderm by primitive bilaterians. Cnidarians are usually regarded as diploblastic animals with only endoderm and ectoderm; however genes whose bilaterian homologs are implicated in mesodermal specification and differentiation are expressed during development of the anthozoan cnidarian Nematostella (Martindale et al. 2004) as well as in a tissue that gives rise to striated muscle in the hydrozoan jellyfish Podocoryne (Spring et al. 2002). It has therefore been suggested that mesoderm in triploblasts may have arisen from the endoderm of diploblastic animals, or, alternatively, that cnidarians arose from a triploblastic ancestor and that diploblasty is a secondary simplification (Martindale et al. 2004, Siepel and Schmid 2005).

56

Tissues
Under the hypothesis of sponge paraphyly (see above), a sponge-like animal is presumed to have given rise to early cnidarians. The leap is large. Sponges are filter feeders that lack nerves and muscle. Furthermore, sponges are not generally considered to have tissues, yet to some extent this last point may be a problem of definition. For example, transport strands in the tropical sponge Aplysina function as a distinct tissue, carrying phagocytosed material to the tip of the sponge presumably for growth (Leys and Reiswig 1998); regional concentrations of symbiont-containing cells that provide nutrition to sponges may do the same (e.g., Yahel et al. 2003). Tissues arise from germ layers during ontogeny and are mesenchymal or epithelial in nature. While current theory holds that epithelia preceded mesenchyme both during evolution and in development (Hay 1968, Prez-Pomares and Muoz-Chpuli 2002, Price and Patel 2004), extant sponges are considered to be largely mesenchymal animals. The idea that their covering layer is not a true epithelium comes from the absence of a well-defined basement membrane (Woollacott and Pinto 1995). Strictly speaking, epithelia are considered to be sheets of cells with apical-basal polarity, cell-cell junctions and an extracellular matrix ECM, cuticle or equivalent apically, and basement membrane basally that maintains cell polarity (Tyler 2003, Cereijido et al. 2004). Other authors however, consider epithelia as physical barriers between two different extracellular environments, with or without the presence of a basal lamina (Prez-Pomares and Muoz-Chpuli 2002). Athough homoscleromorph sponges are considered to have a true basement membrane, the condensation of ECM below choanocyte chambers is extremely slight (Boury-Esnault et al. 2003), and readily can be confused with the mucous coat on the apical surface of cells. An image showing similar condensation of ECM under the epithelium of the larva of the demosponge Crambe crambe (Maldonado 2004), coupled with evidence that other sponges have a type of collagen functionally equivalent to Type IV collagen (a typical constituent of basement membranes) (Aouacheria et al., 2006) suggests that a comprehensive morphological study of sponge epithelia with a focus on seeking structures equivalent to a basal lamina is well-warranted. One practical tool for studying sponge epithelia is simple labeling of the actin cytoskeleton in epithelia (Pavans de Ceccatty 1986). Using modern fluorescent labels for actin microfilaments (Bodipy-phallacidin fluorescein, Molecular Probes, OR) the surface epithelium of 6-day old juvenile freshwater sponges shows remarkably extensive tracts of actin (Fig. 3). Bundles run from cell to cell forming continuous paths from the choanosome to the edge of the animal, a distance of several hundred microns. In fact, continuous tracts can traverse over 1 mm through the apical pinacoderm of these sponges. Where cells adjoin one-another there are dense plaques of actin reminiscent of adhesion plaques in smooth muscle, as shown in freeze fracture and thin section by Pavans de Ceccatty (1986) (Fig. 3C, D). The apical pinacoderm is a tri-layered structure formed by two epithelial sheets that sandwich a very thin collagenous (and

cellular) mesohyl (Elliott and Leys 2007). Like the transport pathways in Aplysina (Leys and Reiswig 1998), the apical pinacoderm of the juvenile sponge is designed to function as an epithelium.

Gastrulation
If sponges can be considered to possess functional tissues, how do these tissues arise? The problem of the origin of tissues and germ layers in sponges has been a longstanding debate with growing disagreement as to whether gastrulation-like processes occur (Efremova 1997, Leys 2004, Ereskovsky and Dondua 2006, reviewed in Leys and Ereskovsky 2006). Most studies of sponge development are largely descriptive. Given that the vast majority of sponges are ovoviviparous (brood their young), development cannot be readily followed in vitro as it can in many other phyla. Furthermore, mechanisms of development (cleavage patterns and segregation of cells to form layers) are diverse (reviewed in Leys and Ereskovsky 2006), and the lack of uniformity has led to a great disparity in views on comparative development. Experimental data is now needed to test the hypothesis that sponges undergo gastrulation-like processes during ontogeny (Leys and Degnan 2002, Leys 2004, Leys and EerkesMedrano 2005). Homologs of genes known to be involved in the formation of mesoderm in other animals have been sequenced from a calcareous sponge (Manuel et al. 2004) and from demosponges (Adell et al. 2003, Bebenek et al. 2004, Hill et al. 2004), but expression patterns in early gastrula-like stages are only just being examined (Larroux et al. 2006). Of specific interest should be the expression pattern in calcareous sponges, a group in which embryogenesis involves distinctly epithelial movements and invagination of the larva to form a blastopore-like structure (Leys and Eerkes-Medrano 2005).

Speculations and considerations


It is valuable to remember that all is possible with sponges (Boury-Esnault 2006). These animals are specialized for suspension feeding on bacteria and ultraplankton (Pile et al. 1996, 1997, Ribes et al. 1999), but this poses certain problems: the filter may occasionally become clogged, and the flow bringing in food may not be sufficient for gas exchange in certain habitats. The idea that peristaltic-like contractions (condensation contractions) may have arisen to assist the filtering mechanism in sponges is not new (Weissenfels 1990). However, with increasing knowledge of the physiology and development of other basal metazoans, it is now interesting to note how similar the rhythmic contractions in sponges are to those in pennatulaceans such as Renilla (Anctil 1989, 1991). Apparently the absence of nerves and true muscle in sponges is no handicap given the power of a hydrostatic skeleton. Seen in this light, the sponge body plan with its aquiferous system could be considered to contain the underpinnings for the evolution of peristaltic contractile systems found in later animals.

57

Fig. 3: A, B. The actin cytoskeleton in the apical pinacoderm (exopinacoderm) of a 6-day old juvenile sponge (E. muelleri). In A, arrows indicate actin tracts labeled with Bodipy phallacidin-fluorescein, and arrowheads mark points of contact of individual cells. In B, arrows indicate dense plaques of actin at points of contact of cells. C. Freeze fracture preparation showing a desmosome-like region on the basal epithelial membrane in E. muelleri. D. Thin section (TEM) showing actin microfilaments (mf) and a dense plaque at the point of contact between two cells (C and D from Pavans de Ceccatty, 1986).

Acknowledgements
I thank G. Elliott and G. Tompkins-MacDonald for interesting discussions and G. Elliott and I. Ijieke for contributions to figures 1 and 2. The foundations for these ideas derive from the research of M. Pavans de Cecatty and G.O. Mackie. Funding was kindly provided by NSERC.

References
Adell T, Gamulin V, Perovic-Ottstadt S, Wiens M, Korzhev M, Mller IM, Mller WEG (2004) Evolution of metazoan cell junction proteins: the scaffold protein MAGI and the transmembrane receptor tetraspanin in the demosponge Suberites domuncula. J Mol Evol 59(1): 41-50 Adell T, Grebenjuk VA, Wiens M, Mller WEG (2003) Isolation and characterization of two T-box genes from sponges, the phylogenetically oldest metazoan taxon. Dev Genes Evol 213: 421-434 Anctil M (1989) Modulation of a rhythmic activity by serotonin via cyclic AMP in the coelenterate Renilla koellikeri. J Comp Physiol B 159: 491-500

Anctil M (1991) Modulation of rhythmic contractions by melatonin via cyclic GMP in the coelenterate Renilla koellikeri. J Comp Physiol B 161: 569-575 Anctil M, Poulain I, Pelletier C (2005) Nitric oxide modulates peristaltic muscle activity associated with fluid circulation in the sea pansy Renilla koellikeri. J Exp Biol 208: 2005-2017 Aouacheria, A, Geourjon, C, Aghajari, N, Navratil, V, Delage, G, Lethias, C Exposito, J-Y (2006) Insights into early extracellular matrix evolution: spongin short chain collagen-related proteins are homologous to basement membrane Type IV collagens and form a novel family widely distributed in invertebrates. Mol Biol Evol 23: 2288-2302 Bebenek I, Gates R, Morris J, Hartenstein V, Jacobs D (2004) sine oculis in basal Metazoa. Dev Genes Evol 214: 342-351 Borchiellini C, Manuel M, Alivon E, Boury-Esnault N, Vacelet J, Le Parco Y (2001) Sponge paraphyly and the origin of Metazoa. J Evol Biol 14 (1): 171-179 Boury-Esnault N (2006) Systematics and evolution of Demospongiae. Can J Zool 84: 205-224

58

Boury-Esnault N, Ereskovsky A, Bezac C, Tokina D (2003) Larval development in the Homoscleromorpha (Porifera, Demospongiae). Invertebr Biol 122 (3): 187-202 Boute N, Exposito JY, Boury-Esnault N, Vacelet J, Nor N, Miyazaki K, Yoshizato K, Garrone R (1996) Type IV collagen in sponges, the missing link in basement membrane ubiquity. Biol Cell 88: 3744 Cereijido M, Contreras RG, Shoshani L (2004) Cell adhesion, polarity, and epithelia in the dawn of metazoans. Physiol Rev 84 (4): 1229-1262 de Vos L (1971) Etude ultrastructurale de la gemmulogense chez Ephydatia fluviatilis. J Microsc 20: 15-20 de Vos L, van de Vyver G (1981) Etude de la contraction spontane chez lponge deau douce Ephydatia fluviatilis cultive in vitro. Ann Soc R Zool Belg 111: 21-31 Demidchik V, Adobea Essah P, Tester M (2004) Glutamate activates cation currents in the plasma membrane of Arabidopsis root cells. Planta 219: 167-175 Efremova SM (1997) Once more on the position among Metazoa Gastrulation and germinal layers of sponges. Berl geowiss Abhand 20: 7-15 Elliott GRD (2004) The contractile apparatus of the sneezing freshwater sponge Ephydatia muelleri. Newsletter Am Microscr Soc November: 5-7 Elliott GRD, Leys SP (2007) Coordinated contractions effectively expel water from the aquiferous system of a freshwater sponge. J Exp Biol (in press) Ellwanger K, Brmmer F, Nickel M (2004) Glutamate, GABA and serotonin induce contractions in the sponge Tethya wilhelma (Porifera: Demospongiae). In: Kinzelbach R (ed). Jahrestagung der Deutschen Zoologischen Gesellschaft, Abstractband, Rostock. pp. 157 Ellwanger K, Nickel M (2006). Neuroactive substances specifically modulate rhythmic body contractions in the nerveless metazoon Tethya wilhelma (Demospongiae, Porifera). Front Zool 3: 7 Emson RH (1966) The reactions of the sponge Cliona celata to applied stimuli. Comp Biochem Physiol 18: 805-827 Enkvist MOK, McCarthy KD (1994) Astroglial gap junction communication is increased by treatment with either glutamate or high K+ concentration. J Neurochem 62: 489-495 Ereskovsky AV, Dondua AK (2006) The problem of germ layers in sponges (Porifera) and some issues concerning early metazoan evolution. Zoologischer Anzeiger 245: 65-76 Francis JC, Poirrier MA (1986) Particle uptake in two fresh-water sponge species, Ephydatia fluviatilis and Spongilla alba (Porifera: Spongillidae). Trans Am Microsc Soc 105(1): 11-20 Gaino E, Pansini M, Pronzato R, Cicogna F (1991) Morphological and structural variations in Clathrina clathrus (Porifera, Calcispongiae). In: Reitner J, Keupp H (eds). Fossil and recent sponges. Springer-Verlag, Berlin. pp. 360-371 Gilbert R (2003) Developmental Biology. 8th Edition. Sinauer Associates Inc. Sunderland Gonobobleva EL, Ereskovsky AV (2004) Metamorphosis of the larva of Halisarca dujardini (Demospongiae, Halisarcida). Bull Inst R Sci Nat Belg 74: 101-115 Hay ED (1968) Organization and fine structure of epithelium and mesenchyme in the developing chick embryo. In: Fleischmajer R, Billingham R (eds). Epithelial-mesenchyme interactions. Williams and Wilkins, Baltimore. pp. 31-55

Hill A, Tetrault J, Hill M (2004) Isolation and expression analysis of a poriferan Antp-class Bar-/Bsh-like homeobox gene. Dev Genes Evol 214: 515-523 Hille B (2002) Ionic channels of excitable membranes. Sinauer, Sunderland Holland ND, Venkatesh TV, Holland LZ, Jacobs DK, Bodmer R (2003) AmphiNK2-tin, an amphioxus homeobox gene expressed in myocardial progenitors: insights into evolution of the vertebrate heart. Dev Biol 255: 128-137 Jones WC (1962) Is there a nervous system in sponges? Biol Rev Camb Philos Soc 37: 1-50 Larroux C, Fahey B, Liubicich D, Hinman V, Gauthier M, Gongora M, Green CR, Woerheide G, Leys SP, Degnan BM (2006) Developmental expression of transcription factor genes in a demosponge: insights into the origin of metazoan multicellularity. Evol Dev 8(2): 150-173 Lawn ID (1982) Porifera. In: Shelton GAB (ed). Electrical conduction and behaviour in simple invertebrates. Clarendon Press, Oxford. pp. 49-72 Lentz TL (1966) Histochemical localization of neurohumors in a sponge. J Exp Zool 162: 171-180 Leys SP (2004) Gastrulation in sponges. In: Stern C (ed). Gastrulation: from cells to embryo. Cold Spring Harbor Press. pp. 23-31 Leys SP, Degnan BM (2002) Embryogenesis and metamorphosis in a haplosclerid demosponge: gastrulation and transdifferentiation of larval ciliated cells to choanocytes. Invertebr Biol 121 (3): 171189 Leys SP, Eerkes-Medrano DI (2005) Gastrulation in calcareous sponges: in search of Haeckels gastraea. Integr Comp Biol 45(2): 342-351 Leys SP, Ereskovsky A (2006) Embryogenesis and larval differentiation in sponges. Can J Zool 84: 262-287 Leys SP, Meech RW (2006) Physiology of coordination in sponges. Can J Zool 84: 288-306 Leys SP, Mackie GO (1997) Electrical recording from a glass sponge. Nature 387: 29-30 Leys SP, Reiswig HM (1998) Nutrient transport pathways in the neotropical sponge Aplysina. Biol Bull 195: 30-42 Mackie GO (1979) Is there a conduction system in sponges? In: Lvi C, Boury-Esnault N (eds). Biologie des spongiaires. Colloques Internationaux du CNRS, vol. 291. ditions du CNRS, Paris. pp 145-152 Mackie GO (1984) Introduction to the diploblastic level. In: Bereiter-Hahn J, Matoltsy JAG, Richards KS (eds). Biology of the integument. Springer-Verlag, Berlin. pp. 43-46 Mackie GO (1990) The elementary nervous system revisited. Amer Zool 30: 907-920 Maldonado M (2004) Choanoflagellates, choanocytes, and animal multicellularity. Invertebr Biol 123: 1-22 Manuel M, Le Parco Y, Borchiellini C (2004) Comparative analysis of Brachyury T-domains, with the characterization of two new sequences, from a hexactinellid and a calcisponge. Gene 340: 291301 Martindale MQ, Pang K, Finnerty JR (2004) Investigating the origins of triploblasty: mesodermal gene expression in a diploblastic animal, the sea anemone Nematostella vectensis (phylum, Cnidaria; class, Anthozoa). Development 131: 2463-2474 McNair GT (1923) Motor reactions of the fresh-water sponge Ephydatia fluviatilis. Biol Bull 44: 153-166

59

Mechawar N, Anctil M (1997) Melatonin in a primitive metazoan: seasonal changes in levels and immunohistochemical visualization in neurons. J Comp Neurol 387: 243-254 Medina M, Collins AG, Silberman J, Sogin ML (2001) Evaluating hypotheses of basal animal phylogeny using complete sequences of large and small subunit rRNA. Proc Natl Acad Sci USA 98: 9707-9712 Moroz LL, Meech RW, Sweedler JV, Mackie GO (2004) Nitric oxide regulates swimming in the jellyfish Aglantha digitale. J Comp Neurol 471: 26-36 Nedergaard M (1994) Direct signaling from astrocytes to neurons in cultures of mammalian brain cells. Science 263: 1768-1771 Nedergaard M, Takano T, Hansen AJ (2002) Beyond the role of glutamate as a neurotransmitter. Nat Rev Neurosci 3: 748-755 Nickel M (2004) Kinetics and rhythm of body contractions in the sponge Tethya wilhelma (Porifera: Demospongiae). J Exp Biol 207: 4515-4524 Osipchuk Y, Cahalan M (1992) Cell-to-cell spread of calcium signals mediated by ATP receptors in mast cells. Nature 359: 241-246 Parker GH (1910) The reactions of sponges with a consideration of the origin of the nervous system. J Exp Zool 8: 765-805 Pavans de Ceccatty M (1986) Cytoskeletal organization and tissue patterns of epithelia in the sponge Ephydatia mulleri. J Morphol 189: 45-65 Pavans de Ceccatty M (1989) Les ponges, laube des communications cellulaires. Science 142: 64-72 Prez-Pomares JM, Muoz-Chpuli R (2002) Epithelialmesenchymal transitions: a mesodermal cell strategy for evolutive innovation in metazoans. Anat Rec 268: 343-351 Perovic S, Krasko A, Prokic I, Mller IM, Mller WEG (1999) Origin of neuronal-like receptors in Metazoa: cloning of a metabotropic glutamate/GABA-like receptor from the marine sponge Geodia cydonium. Cell Tissue Res 296: 395-404 Pile AJ, Patterson MR, Savarese M, Chernykh VI, Fialkov VA (1997) Trophic effects of sponge feeding with lake Baikals littoral zone. 2. Sponge abundance, diet, feeding efficiency, and carbon flux. Limnol Oceanogr 42: 178-184 Pile AJ, Patterson MR, Witman JD (1996) In situ grazing on plankton <10 m by the boreal sponge Mycale lingua. Mar Ecol Prog Ser 141: 95-102

Price AL, Patel NH (2004) The evolution of gastrulation: cellular and molecular aspects. In: Stern C (ed). Gastrulation. From cells to embryos. Cold Spring Harbor Press, Cold Spring Harbor. pp. 695-701 Prosser CL (1967) Ionic analysis and effects of ions on contractions of sponge tissues. Z vergl Physiol 54: 109-120 Prosser CL, Nagai T, Nystrom RA (1962) Oscular contractions in sponges. Comp Biochem Physiol 6: 69-74 Reiswig HM (1970) Porifera: sudden sperm release by tropical demosponge. Science 170: 538-539 Ribes M, Coma R, Gili J-M (1999) Natural diet and grazing rate of the temperate sponge Dysidea avara (Demospongiae, Dendroceratida) throughout an annual cycle. Mar Ecol Progr Ser 176: 179-190 Siepel K, Schmid V (2005) Evolution of striated muscle: Jellyfish and the origin of triploblasty. Dev Biol 282: 14-26 Simpson TL (1984) The cell biology of sponges. Springer Verlag, New York Spring J, Yanze N, Josch C, Middel AM, Winninger B, Schmid V (2002) Conservation of Brachyury, Mef2, and Snail in the myogenic lineage of jellfish: a connection to the mesoderm of Bilateria. Dev Biol 244: 372-384 Tyler S (2003) Epithelium - The primary building block for metazoan complexity. Integr Comp Biol 43 (1): 55-63 Weissenfels N (1990) Condensation rhythm of fresh-water sponges (Spongillidae, Porifera). Eur J Cell Biol 53: 373-383 Weyrer S, Rtzler K, Rieger R (1999) Serotonin in Porifera? Evidence from developing Tedania ignis, the caribbean fire sponge (Demospongiae). Memoir Queensl Mus 44: 659-665 Woollacott RM, Pinto RL (1995) Flagellar basal apparatus and its utility in phylogenetic analyses of the porifera. J Morphol 226: 247-265 Yahel G, Sharp JH, Marie D, Hase C, Genin A (2003) In situ feeding and element removal in the symbiont-bearing sponge Theonella swinhoei: bulk DOC is the major source for carbon. Limnol Oceanogr 48: 141-149 Yang WQ, Braun C, Plattner H, Purvee J, Van Houten JL (1997) Cyclic nucleotides in glutamate chemosensory signal transduction of Paramecium. J Cell Sci 110: 2567-2572

Porifera research: Biodiversity, innovation and sustainaBility - 2007

61

Gemmules as a key structure for the adaptive radiation of freshwater sponges: a morphofunctional and biogeographical study
Renata Manconi(1), Roberto Pronzato(2)
Dipartimento di Zoologia e Genetica Evoluzionistica. Universit di Sassari. Via Muroni 25, I-07100 Sassari, Italy. r.manconi@uniss.it (2) Dipartimento per lo Studio del Territorio e delle sue Risorse. Universit di Genova. Corso Europa 26, I-16132 Genova, Italy. pronzato@dipteris.unige.it
(1)

Abstract: This paper concerns an excursus on morphology, diversity and biogeography of freshwater sponges to test if gemmules could be considered a strategic successful device in the natural history of Spongillina. Taxa richness of the six families belonging to the suborder Spongillina (Demospongiae, Haplosclerida) is notably high if compared to the other sessile filter feeder benthic taxa living in freshwater such as Cnidaria and Bryozoa. Although extremely conservative for some characters the plastic bauplan of Porifera favoured the adaptive radiation of Spongillina in inland waters worldwide and produced both structural and functional evolutionary novelties mainly at the level of resting bodies (gemmules). Clonation, modular architecture and cryptobiosis represent successful adaptive strategies to support survival and dispersal of freshwater sponges at all latitudes from the Arctic Circle to Patagonia in a wide variety of extremely discontinuous freshwater habitats. A comparative analysis on morpho-functional diversity of asexual resting bodies based on literature data vs. new investigations by scanning electron microscopy highlighted some trends in the evolution of the genera belonging to Metaniidae, Potamolepidae, and Spongillidae that display typical gemmular architectures. Other endemic sponges from ancient lakes, namely Lubomirskiidae, Malawispongiidae and Metschnikowiidae, are exclusively sexual and share this reproductive strategy by swimming larvae (parenchymellas) with the gemmule-producing families. The evolutionary success of Spongillina is not easy to be interpreted. In some cases a dispersalist model explains better the natural history of the taxon mainly for species till now considered cosmopolitan as in the case of Ephydatia fluviatilis. In other cases, e.g. the genus Corvospongilla, the high level of speciosity and endemicity seems to match the vicariance model. The distributive pattern of genera/families from ancient lakes represents a peculiar case; these taxa share both morphological (spiculation, skeletal architecture, absence of gemmules) and biological (perennial life cycle, absence of cryptobiosis) traits in spite of their extremely disjunct geographic distribution and high levels of endemism. Keywords: Biodiversity, Porifera, taxonomic richness, morpho-functional traits, cryptobiosis, distribution, ecology, discontinuous habitats

Introduction
The wide adaptive radiation of sponges has resulted in the colonization of continental waters at all latitudes from cold deserts to equatorial rainforests and hot deserts; from the coast line to high plains and mountains and subterranean environments. An extremely wide variety of lotic and lentic habitats have been colonised ranging from springs, water courses, and brackish waters in estuaries and enclosed seas, to thermal vents, caldera lakes, alpine lakes, and caves. Ephemeral pools, marshes, swamps, billabongs, oueds and pans, are also suitable habitats together with oceanic islands and man-made basins, such as pools in gardens and archaeological sites, reservoirs, water tanks and pipelines (see Manconi and Pronzato 2002, Pronzato and Manconi 2002). Recorded bathymetric distribution ranges from hundreds of meters in some lakes (Crane 1991, de Ronde et al. 2002)

to the surface exposed to sunlight during low-water levels (Manconi and Pronzato 1994, 2002). Freshwater sponges are able to survive extreme environmental conditions ranging from ice, hot waters, dry-up, anoxy, eutrophy, high levels of chemicals, hydrocarbons and heavy metals (Old 1932, Jewell 1935, 1939, Sar and Vacelet 1973, Harrison 1974, 1977, Rader 1984, Rader and Winget 1985, van Soest and Velikonja 1986, Willenz et al. 1986, Francis and Harrison 1988, Ricciardi and Reiswig 1993, Richelle-Maurer et al. 1994a, 1994b, Vacelet 1994, de Ronde et al. 2002, Rota and Manconi 2004). Body shape of sponges from inland waters range from thin whitish crusts to dark brown massive cushion, brilliant green branching or erect growth forms (Manconi and Pronzato 1991). In some seasons, according to the climate, most freshwater sponges are represented on the substratum only by small spherules or resting stages, known as gemmules

62

(Simpson and Fell 1974, Manconi and Pronzato 1991, 1994, 2002, Corriero et al. 1993, Pronzato et al. 1993, Pronzato and Manconi 1994, 1995, 2002). Sponges represent a natural resource for their functional role in auto-depurative processes of water bodies (Manconi and Desqueyroux-Faundez 1999, Manconi et al. 1999) playing a key-role in the re-cycling of organic matter. Their contribution to the energetic equilibrium of freshwater ecosystems by pumping rate is conspicuous and a fingersized Spongilla lacustris can filter more than 125 litres per day (Frost 1978, 1980, 1991). Sponges are also centres of biological association, representing a suitable but selective refuge microhabitat. They host a notably diverse assemblage of organisms ranging from animals and protists to bacteria and algae related by endocellular symbiosis, inquinilism, commensalisms, and highly specialized predation as in the case of Insecta belonging to the orders Neuroptera, Tricoptera, and Diptera. Most freshwater invertebrate taxa have been recorded in sponges, namely Hydrozoa, Nematoda, Oligochaeta, Polychaeta, Gastropoda, Bivalvia, Isopoda, Amphipoda, Ostracoda, Hydracarina, and Bryozoa, to several families of insects encompassing the typical and exclusive spongillaflies Sisyridae (Berg 1948, Brown 1952, Brnsted and Brnsted 1953, Brnsted and Loevtrup 1953, Parfin and Gurney 1956, Volkmer-Ribeiro and de Rosa-Barbosa 1974, Resh 1976, Resh et al. 1976, Steffen 1967, Frost and Williamson 1980, Kahl and Konopacha 1981, Konopacha and Socinski 1985, Kamaltynov et al. 1993, Weissmair and Mildner 1995, Gugel 1996, Oliveira Roque et al. 2004, Rota and Manconi 2004, Weinberg et al. 2004). Sponges host fishes and amphibians also to nest fertilized eggs (Kilian and Campos 1969, Manconi and Pronzato 2002), represent one of the food items for fishes, freshwater turtles and ducks (Dominey and Snyder 1988, McCauley and Longcore 1988, Seigel and Brauman 1994, Kennett and Tory 1996, Armstrong and Booth 2005), and are reported as a refuge for millipedes during inundation of Amazonian floodplains (Adis 1992). Freshwater sponges have been used since ancient times also by humans, and it is known that some African and Amazonian populations produce ceramics strengthened by sponge spicules (Linn 1925, Serrano 1933, Brissaud and Houdayer 1986, Adamson et al. 1987, McIntosh and MacDonald 1989). Other practical uses are in the field of cosmesis as in the case of dried spongillids used in the 19th century by Russian young ladies to scrub their faces to have rosy cheeks (Kuznetzow 1898), and at present some cosmetics are based on the action of siliceous spicule powder. In the 17th century Samuel Hahnemann enclosed freshwater sponges in his Materia Medica as a homeopathic remedy for psoriasis with the common pre-linnean Russian name of Badiaga (Allen 1986) although a notable confusion exists on the identification at the genus and species level of the used material, reported as Spongilla fluviatilis and Spongia palustris. Freshwater sponges are also useful indicators of palaeoenvironmental changes by the analysis of spicular remains in sediments (Harrison et al. 1979, Harrison and Warner 1986, Harrison 1988, Volkmer-Ribeiro and Turcq 1996, Candido et al. 2000).

No sponges are currently listed on the IUCN Red List, although official threatened species lists of few countries report on freshwater sponges (e.g. Brazil, Norway); in some cases they are indirectly protected being sympatric with umbrella species such as fishes and amphibians. Manconi and Desqueyroux-Faundez (1999) suggest that conservation of freshwater sponge fauna would represent a sustainable approach to maintain biodiversity and to improve the rational management of freshwater natural resources. It is proved also that freshwater sponges could control the presence of the bivalve Dreissena polymorpha (Ricciardi et al. 1995, Swierczynski 1996, Lauer and Spacie 2004).

Recent freshwater sponges: gemmular structure and biogeography


Recent freshwater sponges are ascribed to 45 genera in 6 families, namely Lubomirskiidae, Malawispongiidae, Metaniidae, Metschnikowiidae, Potamolepidae, and Spongillidae. Species richness, 217 species (Table 1), is high if compared to that of the other freshwater sessile invertebrates belonging to Cnidaria and Bryozoa (Manconi and Pronzato, in press a). Freshwater sponges occur worldwide, except for the Antarctic region and the North Pole. Geographic distribution is related of course to the geological and climatic history of the continents and to the long-term dynamics of hydrographic basins. The highest taxonomic diversity at the biogeographic scale is recorded from the Neotropical (63 species), Palaearctic (59 species), and Afrotropical (49 species) regions. Species richness is lower in the other zoogeographic regions: Oriental (37 species), Australasian (33 species), Nearctic (32 species), and Pacific Oceanic Islands (5 species) (Fig. 1; Table 1) (Manconi and Pronzato, in press a). The oldest fossils of sponges from inland water are very few and dated back to the Mesozoic Era (Ott and Volkheimer 1972, Dunagan 1999, Richter and Wuttke 1999). Eospongilla morrisonensis Dunagan, 1999 is known from the Colorado Upper Jurassic, while Palaeospongilla chubutensis Ott and Volkheimer, 1972 was recorded together with Spongilla patagonica Volkmer-Ribeiro and Reitner, 1991 from the Patagonian Lower Cretaceous in the Chubut Valley. More recent taxa are known from the European Eocene as in the case of Lutetiospongilla heily Richter and Wuttke, 1999. The presence of resistant bodies sharing most traits with recent Spongillidae in one of the best preserved fossil, Palaeospongilla chubutensis, indicates that gemmules have been extremely conservative structures since the Cretaceous (Manconi and Pronzato 2002). The process of freshwater colonization seems to be strictly related to the cryptobiosis phenomenon and to the evolutionary novelty represented by resistant bodies, the gemmules. Different approaches exist to the problem of inland water colonization and phylogeny of freshwater sponges (Brien 1966, 1969, Volkmer-Ribeiro 1979, 1986, 1990, Volkmer-Ribeiro and de Rosa Barbosa 1979, VolkmerRibeiro and Watanabe 1983, Pronzato and Manconi 2002). Our opinion is that only Haplosclerida, belonging to the suborder Spongillina, colonised inland waters. At present, we do not know, however, how many colonization processes

63

Table 1: Worldwide checklist of freshwater sponges (no fossil species). Zoogeographic Regions are reported as Nearctic (NA), Palaearctic (PA), Neotropical (NT), Afrotropical (AT), Oriental (OL), Australasian (AU), Pacific Oceanic Islands (PAC). Suborder SPONGILLINA Manconi and Pronzato, 2002 PA-NA-AT-OL-AU-NT-PAC (6 families, 45 genera, 217 species) Family Spongillidae Gray, 1867 PA-NA-AT-OL-AU-NT-PAC (21 genera, 146 species) Anheteromeyenia Schrder, 1927 (3 species) NA-NT A. argyrosperma (Potts, 1880) (Canada to Florida) type species NA A. ornata (Bonetto and Ezcurra de Drago, 1970) (Brazil, Argentina) NT A. cheguevarai Manconi and Pronzato, 2005 (Cuba) NT Corvoheteromeyenia Ezcurra de Drago, 1979 (2 species) NT C. australis (Bonetto and Ezcurra de Drago, 1966) (Neotropical) type species NT C. heterosclera (Bonetto and Ezcurra de Drago, 1966) (Argentina, Brazil, Venezuela, Curacao) NT Corvospongilla Annandale, 1911 (18 species) PA-NA-NT-AT-OL C. loricata (Weltner, 1895) (lower Nile basin) type species PA C. bhavnagarensis Soota, Pattanayak and Safena, 1984 (Gujarat, India) OL C. becki Poirrier, 1978 (Louisiana) NA C. boehmii (Hilgendorf, 1883) (Equatorial Africa, Brazil) AT-NT C. burmanica (Kirkpatrick, 1908) (Burma, India) OL C. caunteri Annandale, 1911 (India) OL C. lapidosa (Annandale, 1908) (India) OL C. mesopotamica Manconi and Pronzato, 2004 (Iraq) PA C. micramphidiscoides Weltner, 1913 (R. Congo) AT C. novaeterrae (Potts, 1886) (NE Nearctic) NA C. scabrispiculis Annandale, 1913 (Lower R. Nile) PA C. seckti Bonetto and Ezcurra de Drago, 1966 (Paran, R. Paraguay, R. Uruguay, Argentina, Brazil) NT C. sodenia Brien, 1969 (Cameroon) AT C. thysi Brien, 1968 (L. Kumba, Cameroon) AT C. ultima (Annandale, 1910) (S-India) OL C. zambesiana (Kirkpatrick, 1906) (R. Zambezi) AT C. victoriae Annandale, 1914 (Zambesi, Zambia) AT C. volkmeri (de Rosa-Barbosa, 1988) (Brazil) NT Dosilia Gray, 1867 (5 species) OL-AT-NA-NT D. plumosa (Carter, 1849) (India, Philippines) type species OL D. brouni (Kirkpatrick, 1906) (R. Nile, L. Baringo, E- Africa) AT D. palmeri (Potts, 1885) (Louisiana, Florida, Texas, Mexico, Central America) NA-NT D. pydanieli Volkmer-Ribeiro, 1992 (Brazil) NT D. radiospiculata (Mills, 1888) (Louisiana, Mexico) NA Duosclera Reiswig and Ricciardi, 1993 (1 species) NA D. mackayi (Carter, 1885) (NE-America) monotypic NA Ephydatia Lamouroux, 1816 (10 species) PA-NA-AT-OL-AU-NT-PAC E. fluviatilis (Linnaeus, 1759) (cosmopolitan) type species PA-NA-AT-OL-AU E. meyeni (Carter, 1849) (India, China) OL E. millsii (Potts, 1887) (Florida) NA E. muelleri (Lieberkuhn, 1855) (North Hemisphere) PA-NA E. facunda Weltner, 1895 (Central and South America) NT E. fortis Weltner, 1895 (Indonesia, Philippines, Japan, Vanuatu) OL-PA-PAC E. japonica (Hilgendorf, 1882) (USA, Manchuria, Japan) NA-PA E. ramsayi (Haswell, 1882) (Australia, New Zealand, New Guinea?) AU E. robusta (Potts, 1887) (E-USA, Mexico, California) NA E. syriaca Topsent, 1910 (Syria) PA Eunapius Gray, 1867 (16 species) PA-AT-OL-AU-NA-NT E. carteri (Bowerbank, 1863) (S-Asia, East Europe, Africa) type species OL-PA-AT E. aetheriae (Annandale, 1913) (Lower R. Nile) PA E. ambiguus (Annandale, 1909) (KwaZulu-Natal) AT E. calcuttanus (Annandale, 1911) (India) OL E. conifer (Annandale, 1916) (China, Korea) PA E. crassissimus (Annandale, 1907) (India, Australia, tropical SE Asia) OL-AU E. fragilis (Leidy, 1851) (Cosmopolitan) PA-NA-AT-NT-OL-AU E. geminus (Annandale, 1911) (India) OL E. geei (Annandale, 1918) (China) PA E. michaelseni (Annandale, 1914) (Central Africa) AT E. nitens (Carter, 1881) (Africa) AT E. potamolepis (Annandale, 1918) (Thailand) OL E. ryuensis (Sasaki, 1970) (Japan, Korea) PA E. sinensis (Annandale, 1910) (China, Manchuria, Korea, Russia, Australia) PA-AU E. subterraneus Sket and Velikonja, 1984 (Croatia) PA E. tinei (Gee, 1932) (Philippines) OL

64 Table 1 (cont.)

Heteromeyenia Potts, 1881 (7 species) NA-PA-NT-AU H. baileyi (Bowerbank, 1863) (North and Central America, Argentina, Europe) type species NA-PA-NT H. horsti Ezcurra de Drago, 1988 (Argentina) NT H. insignis Weltner, 1895 (Brazil) NT H. latitenta (Potts, 1881) (NE-USA, Mexico) NA H. stepanowii (Dybowsky, 1884) (Europe, Russia, China, Korea, Japan, Australia, Brazil, Argentina) PA-AU-NT H. tentasperma (Potts, 1880) (NE-USA) NA H. tubisperma (Potts, 1881) (NE-America) NA Heterorotula Penney and Racek, 1968 (7 species) AU-NT H. capewelli (Bowerbank, 1863) (Australia) type species AU H. contraversa (Racek, 1969) (E-Central Australia) AU H. fistula Volkmer and Motta 1995 (South America) NT H. kakauensis (Traxler, 1896) (New Zealand) AU H. multidentata (Weltner, 1895) (E-Australia, Tasmania) AU H. multiformis (Weltner, 1910) (W-Australia) AU H. nigra (von Lendenfeld, 1887) (E-Australia) AU Nudospongilla Annandale, 1918 (6 species) PA-AT-AU N. coggini (Annandale, 1910) (Yunnan Lakes, W-China ) type species PA N. cunningtoni (Kirkpatrick, 1906) (L. Tanganyika, Africa) AT N. ehraiensis Lizhen, 1998 (Yunnan, W-China) PA N. moorei (Evans, 1899) (L. Tanganyika) AT N. vasta (Weltner, 1901) (Sulawesi, Indonesia) AU N. yunnanensis (Annandale, 1910) (Yunnan, W-China) PA Pachyrotula Volkmer-Ribeiro and Rtzler, 1997 (1 species) PAC P. raceki (Rtzler, 1968) (New Caledonia) monotypic PAC Pectispongilla Annandale, 1909 (4 species) OL-AU-PA P. aurea Annandale, 1909 (India) type species OL P. botryoides Haswell, 1882 (Australia) AU P. stellifera Annandale, 1915 (S-India) OL P. subspinosa Annandale, 1911 (India, Japan, Korea) OL-PA Racekiela Bass and Volkmer-Ribeiro, 1998 (4 species) PA-NA-NT R. ryderi (Potts, 1882) (Amphiatlantic in the N-Hemisphere, Belize) type species PA-NA-NT R. biceps (Lindenschmidt, 1950) (Michigan, USA) NA R. pictovensis (Potts, 1885) (from E-Canada to New York) NA R. sheilae (Volkmer-Ribeiro, de Rosa Barbosa and Tavares, 1988) (South America) NT Radiospongilla Penney and Racek, 1968 (16 species) AU-PAC-NT-PA-AT-OL-NA R. sceptroides (Haswell, 1882) (E-Australia, New Zealand, New Guinea, New Caledonia) type species AU-PAC R. amazonensis Volkmer-Ribeiro and Maciel, 1983 (Brazil) NT R. cantonensis (Gee, 1929) (China) PA R. cerebellata (Bowerbank, 1863) (trop. Africa, Ind.-Pak., Indon., Philipp., Japan, N. Guinea, China, Russia?, SE-Eur.) AU-AT-OL-PA R. cinerea (Carter, 1849) (Bombay, Himalayas, Pakistan) OL R. crateriformis (Potts, 1882) (USA, Can., Mexico, W. Indies, Suriname, Brazil, China, Japan, S-Asia, Australia) NA-NT-PA-AU-OL R. hemephydatia (Annandale, 1909) (India, New Guinea, E-Australia) OL-AU R. hispidula (Racek, 1969) (Australia) AU R. hozawai (Sasaki, 1936) (Japan) PA R. indica (Annandale, 1907) (India, Indonesia, Philippines, New Guinea?) OL-AU R. multispinifera (Gee, 1933) (E-Australia) AU R. philippinensis (Annandale, 1909) (Philippines to N-Australia) OL-AU R. sansibarica (Weltner, 1895) (Zanzibar, L. Upemba, L. Sonfon, Sierra Leone, Zambia, Congo basin) AT R. sendai (Sasaki, 1936) (Japan, Korea) PA R. sinoica (Racek, 1969) (Australia) AU R. streptasteriformis Stanisic, 1978 (N. Territory, Australia) AU Sanidastra Volkmer-Ribeiro and Watanabe, 1983 (1 species) PA S. yokotonensis Volkmer-Ribeiro and Watanabe, 1983 (Japan, Sardinia and Corsica) monotypic PA Saturnospongilla Volkmer-Ribeiro, 1976 (1 species) NT S. carvalhoi Volkmer-Ribeiro, 1976 (R. Juru, Brazil, Venezuela) monotypic NT Spongilla Lamarck, 1816 (15 species) PA-NA-OL-AT-AU-NT S. lacustris (Linnaeus, 1759) (Palaearctic, Nearctic) type species PA-NA S. alba Carter, 1849 (Ind., SE-Asia, Turk., Afr., Madag., Australia, Papua-N. Guin., USA, S. Salvador, S. Am.) OL-AT-AU-NT-PA-NA S. aspinosa Potts, 1880 (E-Canada, USA, China) NA-PA S. cenota Penney and Racek, 1968 (Yucatan, Costa Rica, Florida) NT-NA S. chaohuensis Cheng, 1991 (Hebei, N-China) PA S. heterosclerifera Smith, 1918 (N-America) NA S. inarmata Annandale, 1918 (Japan) PA S. jiujiangensis Cheng, 1991 (Hebei, N-China) PA S. mucronata Topsent, 1932 ( Dienn-Timbuct, R. Niger) AT

65 Table 1 (cont.)

S. permixta Weltner, 1885 (Bibisande, central Africa) AT S. prespensis Hadzische, 1953 (L. Prespa, Macedonia) PA S. shikaribensis Sasaki, 1934 (Japan) PA S. spoliata Volkmer-Ribeiro and Maciel, 1983 (Venezuela, Brazil) NT S. stankovici Arndt, 1938 (L. Ohrid, Macedonia) PA S. wagneri Potts, 1889 (SE-USA, Florida, South Carolina) NA Stratospongilla Annandale, 1909 (9 species) OL-AT-PA-AU-NA S. bombayensis (Carter, 1882) (Mumbay, Natal) type species OL-AT S. africana Annandale, 1914 (Victoria Falls, Zambesi) AT S. akanensis (Sasaki, 1934) (Japan) PA S. clementis (Annandale, 1909) (Philippines, China, Japan, tropical W-Africa) OL-PA-AT S. gravelyi (Annandale, 1912) (Mumbay, India) OL S. indica (Annandale, 1908) (Thailand, India, Africa) OL-AT S. lanei Racek, 1969 (Australia) AU S. penney (Harrison, 1979) (Florida) NA S. sumatrana (Weber, 1890) (Indonesia, India, Africa) OL-AT Trochospongilla Vejdowsky, 1883 (14 species) PA-NA-NT-OL-AU-AT T. horrida (Weltner, 1893) (Holarctic) type species PA-NA T. delicata Bonetto and de Drago, 1967 (Argentina, Brazil) NT T. gregaria Bowerbank, 1863 (Venezuela) NT T. lanzamirandai Bonetto and Ezcurra de Drago, 1964 (Brazil) NT T. latouchiana Annandale, 1907 (India, Burma, China, SE-Australia, SW-Africa) OL-PA-AU-AT T. leidii (Bowerbank, 1863) (Florida, Panama) NA-NT T. minuta (Potts, 1881) (Argentina, Bolivia, Venezuela, E-Brazil) NT T. paulula (Bowerbank, 1863) (R. Amazon, Venezuela, Suriname, Argentina) NT T. pennsylvanica (Potts, 1882) (North America) NA T. petrophila Racek, 1969 (E-Australia) AU T. philottiana Annandale, 1907 (India, S-China, Philippines, Africa) OL-AT T. tanganyikae Evans, 1899 (L. Tanganyika) AT T. singpuensis Chen, 1991 ((Hebei, N-China) PA T. variabilis Bonetto and Ezcurra de Drago, 1973 (Argentina, Brazil) NT Umborotula Penney and Racek, 1968 (1 species) PA-OL-AU U. bogorensis (Weber, 1890) (N-China, Korea, SE-Asia, Andaman Islands, E-Australia) monotypic PA-OL-AU Uruguayella Bonetto and Ezcurra de Drago, 1969 (5 species) NT U. repens (Hinde, 1888) (R. Uruguay, upper R. Paran, Argentina) type species NT U. amazonica (Weltner, 1895) (R. Amazon) NT U. macandrewi (Hinde, 1888) (R. Paraguay, R. Paran) NT U. pygmea (Hinde, 1888) (R. Paraguay, R. Uruguay) NT U. ringueleti (Bonetto and Ezcurra de Drago, 1969) (upper R. Paran, R. Uruguay) NT Family lubomirSkiidae Rezvoi, 1936 (4 genera, 10 species) PA Baikalospongia Annandale, 1914 (4 species) PA B. bacillifera Dybowsky, 1880 (L. Baikal) type species PA B. intermedia Dybowsky, 1880 (L. Baikal) PA B. dzhegatajensis Rezvoi, 1927 (L. Djegataj Kul, Urianhajskaja Region) PA B. erecta Efremova, 2004 (L. Baikal) PA Lubomirskia Dybowsky, 1880 (4 species) PA L. baikalensis (Pallas, 1776) (L. Baikal) type species PA L. abietina Swartschewsky 1901 (L. Baikal) PA L. fusifera Soukatschoff, 1895 (L. Baikal) PA L. incrustans Efremova, 2004 (L. Baikal) PA Rezinkovia Efremova, 2004 (1 species) PA R. echinata Efremova, 2004 (L. Baikal) monotypic PA Swartschewskia Makushok, 1927 (1 species) PA S. papiracea (Dybowsky, 1880) (L. Baikal) monotypic PA Family malawiSpongiidae Manconi and Pronzato, 2002 (5 genera, 6 species) PA-AT-AU Cortispongilla (Annandale, 1918 (1 species) PA C. barroisi (Topsent, 1892) (L. Tiberiade, R. Jordan) monotypic PA Malawispongia Brien, 1972 (1 species) AT M. echinoides Brien, 1972 (L. Malawi) monotypic AT Ochridaspongia Arndt, 1937 (2 species) PA O. rotunda Arndt, 1937 (L. Ohrid, Macedonia) type species PA O. interlithonis Gilbert and Hadzische, 1984 (L. Ohrid) PA

66 Table 1 (cont.)

Pachydictyum Weltner, 1901 (1 species) AU P. globosum Weltner, 1901 (L. Posso, Sulawesi) monotypic AU Spinospongilla Brien, 1974 (1 species) AT S. polli Brien, 1974 (L. Tanganyika) monotypic AT Family metaniidae Volkmer-Ribeiro, 1986 (5 genera, 22 species) NA-NT-AT-OL-AU Acalle Gray, 1867 (1 species) NT A. recurvata (Bowerbank, 1863) (Brazil) monotypic NT Corvomeyenia Weltner, 1913 (4 species) NA-NT C. everetti (Mills, 1884) (NE-USA, S-Canada) type species NA C. carolinensis Harrison 1971 (South Carolina, NE-USA) NA C. epilithosa Volkmer-Ribeiro, de Rosa Barbosa and Machado, 2005 (Brazil) NT C. thumi (Traxler, 1895) (Brazil) NT Drulia Gray, 1867 (5 species) NT D. browni (Bowerbank, 1863) (R. Amazon, Rio Negro in Brazil, Rio Beni in Bolivia, Venezuela) Type species NT D. conifera Bonetto and Ezcurra de Drago, 1973 (Rio Orinoco, Venezuela) NT D. cristata (Weltner, 1895) (R. Amazon, Tapajos) NT D. ctenosclera Volkmer-Ribeiro and Mothes de Moraes, 1981 (Rio Negro, Amazonia) NT D. uruguayensis Bonetto and Ezcurra de Drago, 1969 (R. Uruguay, R. Paran, Argentina, Suriname) NT Houssayella Bonetto and Ezcurra de Drago, 1966 (1 species) NT H. iguazuensis Bonetto and Ezcurra de Drago, 1966 (R. Iguaz) monotypic NT Metania Gray, 1867 (11 species) NT-AT-AU-OL M. reticulata (Bowerbank, 1863) (Brazilian and Venezuelan Amazonian basin) type species NT M. fittkaui Volkmer-Ribeiro, 1979 (Amazonian Basin) NT M. godeauxi Brien, 1968 (Central Africa) AT M. kiliani Volkmer-Ribeiro and Costa, 1992 (Brazil) NT M. ovogemata Stanisic, 1979 (N-Australia) AU M. pottsi (Weltner, 1895) (Congo basin, Angola, Borneo, Indonesia) AT-OL M. rhodesiana Burton, 1938 (SE-Africa, Congo basin) AT M. spinata (Carter, 1881) (Amazonian basin) NT M. subtilis Volkmer-Ribeiro, 1979 (Amazonian basin) NT M. vesparia (von Martens, 1868) (Borneo, Indonesia, Australia) OL-AU M. vesparioides (Annandale, 1908) (Tenasserim, Burma, Australia) OL-AU Family metSchnikowiidae Czerniawsky, 1880 (1 genus, 1 species) PA Metschnikowia Grimm, 1876 (1 species) PA M. tuberculata Grimm, 1876 (Caspian Sea) monotypic PA Family potamolepidae Brien, 1967 (6 genera, 29 species) NT-AT-PAC Echinospongilla Manconi and Pronzato, 2002 (1 species) AT E. brichardi Brien, 1974 (L. Tanganyika) monotypic AT Oncosclera Volkmer-Ribeiro, 1970 (14 species) NT-PAC-AT O. jewelli Volkmer-Ribeiro, 1963 (Rio Grande do Sul, Brazil) type species NT O. atrata (Bonetto and Ezcurra de Drago, 1973) (R. Apur, Argentina) NT O. diahoti (Rtzler, 1968) (New Caledonia) PAC O. gilsoni (Topsent, 1912) (Fiji) PAC O. intermedia (Bonetto and Ezcurra de Drago, 1973) (R. Orinoco, Venezuela) NT O. navicella (Carter, 1881) (Brazil, Argentina) NT O. petricola (Bonetto and Ezcurra de Drago, 1973) (R. Uruguay, Argentina) NT O. ponsi (Bonetto and Ezcurra de Drago, 1973) (R. Uruguay, Argentina) NT O. rousseletii (Kirkpatrick, 1906) (R. Zambezi, Africa) AT O. schubarti (Bonetto and Ezcurra de Drago, 1973) (R. Uruguay, Argentina, Brazil) NT O. schubotzi Weltner, 1913 (Aruwimi, Congo basin) AT O. spinifera (Bonetto and Ezcurra de Drago, 1973) (R. Orinoco, Venezuela) NT O. stolonifera (Bonetto and Ezcurra de Drago, 1973) (R. Pir, Argentina) NT O. tonolli (Bonetto and Ezcurra de Drago, 1968) (Parami, Brazil) NT Potamolepis Marshall, 1883 (7 specie) AT P. leubnitziae Marshall, 1883 (Congo basin, L. Mweru, R. Niger, L. Tanganyika) type species AT P. belingana Lvi, 1965 (Cameroon, R. Ivindo Gabon) AT P. chartaria Marshall, 1883 (Isangila Congo basin, R. Luapula, L. Tanganyika, R. Niger) AT P. marshalli Burton, 1938 (Matadi Congo basin) AT P. micropora Burton, 1938 (Matadi Congo basin) AT P. pechuelii Marshall, 1883 (Matadi-Matemba Congo basin, L. Tanganyika) AT P. weltneri Moore, 1903 (L. Tanganyika, Zimbabwe) AT

67

Table 1 (cont.) Potamophloios Brien, 1970 (5 species) AT P. stendelli (Jaffe, 1916) (L. Mweru, L. Luapula, L. Tanganyika) type species AT P. gilberti Brien, 1969 (L. Mweru, R. Luapula) AT P. hispida Brien, 1969 (L. Mweru, R. Luapula) AT P. songoloensis Brien, 1969 (L. Mweru, R. Luapula) AT P. symoensi (Brien, 1967) (Luapula basin) AT Sterrastrolepis Volkmer- Ribeiro and de Rosa Barbosa, 1978 (1 species) NT S. brasiliensis Volkmer-Ribeiro and de Rosa Barbosa, 1978 (R. Turvo, Brazil) monotypic NT Uruguaya Carter, 1881 (1 species) NT U. corallioides (Bowerbank, 1863) (Brazil, Uruguay, Venezuela) monotypic NT incertae SediS (3 genera, 3 species) NT-AT-PA Balliviaspongia Boury-Esnault and Volkmer-Ribeiro, 1992 (1 species) NT B. wirrmanni Boury-Esnault andVolkmer-Ribeiro, 1992 (L. Titicaca, Peru-Bolivia) monotypic NT Makedia Manconi, Cubeddu and Pronzato, 1999 (1 species) AT M. tanensis Manconi, Cubeddu and Pronzato, 1999 (L. Tana, Ethiopia) monotypic AT Ohridospongilla Gilbert and Hadzische, 1984 (1 species) PA O. stankovici Gilbert and Hadzische, 1984 (L. Ohrid, Macedonia) monotypic PA

Fig. 1: Taxonomic diversity of freshwater sponges at the zoogeographic scale recorded from the Neotropical (63 species, 22 genera, 3 families), Palaearctic (59 species, 21 genera, 4 families), Afrotropical (49 species, 18 genera, 4 families), Oriental (37 species, 11 genera, 2 families), Australasian (33 species, 13 genera, 3 families), Nearctic (32 species, 13 genera, 2 families), and Oceanic Pacific Islands (5 species, 4 genera, 2 families).

occurred in continental waters. It is proved that Spongillina share the general structure of both eggs and sperms, and the larval stage parenchymella type III sensu Ereskovsky (1999, 2004) with the other Haplosclerida suborders; the parenchymella of Spongillina shows however exclusive traits such as choanocyte chambers and canals. The key evolutionary novelty, represented by peculiar resistant bodies is known among marine Haplosclerina in the genus Haliclona (de Weert 2002), while resistant bodies are absent in the suborder Petrosina (Desqueyroux-Faundez and Valentine 2002). Among the recent Spongillina, the trait resistant body is shared only by the families Metaniidae, Potamolepidae, and Spongillidae, but it is absent in the other families Lubomirskiidae, Malawispongiidae, and Metschnikowiidae (Manconi and Pronzato 2002) (Fig. 2).

Freshwater sponges producing gemmules display a pluriannual life cycle characterised by four steps: vegetative growth phase, gemmulation/sexual reproduction, cryptobiosis, hatching of gemmules and regeneration (Fig. 3). The low metabolism of gemmules allows sponges to survive extreme environmental conditions and to re-establish an active sponge by the rapid proliferation of totipotent cells (Weissenfels 1989, Pronzato and Manconi 1995). The production of gemmules is a trait shared by most freshwater sponges but gemmules display different levels of morphological complexity. The functional role of gemmules is double as propagules and as resting bodies. These evidences suggest testing the working hypothesis the evolutionary success of freshwater sponges at the level of geographic range, species richness, and abundance is related to the efficiency of gemmules as dispersal devices. Moreover the

68

Fig. 2: Schematic reconstruction of the morpho-functional evolutionary trend of dormant bodies in sponges: a. Totipotent cell (several Porifera); b. Gemmule with a simple structure (few Haplosclerina* and few Spongillina**); c. Gemmule with tri-layered pneumatic theca (several Spongillina**); d. Spiny gemmulosclere radially arranged (several Spongillina**). All sponge species are characterized by the presence of totipotent cells able to regenerate or recover the sponge body. Only few species of Haplosclerina*, belonging to the genus Haliclona, are able to produce over wintering gemmules. Metaniidae and Spongillidae, among Spongillina**, bear complex gemmules with multilayered theca, pneumatic layer and spiny gemmuloscleres frequently radially arranged.

Fig. 3: Life cycle phases of Ephydatia fluviatilis recorded in a Sardinian stream by means of time lapse photography: a. Hatching of gemmules and growth; b. Vegetative phase; c. Sexual reproduction, gemmulation and degeneration; d. Cryptobiosis. The presence of the active sponge and gemmules are indicated by dotted and solid strips respectively.

structure of gemmules and their morphological traits are, at present, diagnostic at the family, genus and species level. The families Lubomirskiidae and Metschnikowiidae show an extremely restricted geographic range in the Baikal Lake and the Caspian Sea, respectively (Fig. 4). A peculiar case is represented by Malawispongiidae with a discontinuous distribution in ancient lakes from the Great African Rift Valley (Tanganyika and Malawi) to the Syrian-Palestinian Jordan Rift Valley (Lake Tiberias), and from the Balkanian

area (Ohrid Lake), to the extremely distant Wallacea in the Sulawesi Island (Poso Lake) (Fig. 4). These three families share the trait absence of gemmules being their reproductive mode exclusively sexual or by fragmentation (Manconi and Pronzato 2002). Potamolepidae show a circumtropical range of Gondwanian origin, in rainforests with 29 species (Fig. 4). Their reproductive mode is both sexual and asexual and resistant bodies are gemmules with a simple architecture (Fig. 5).

69

Fig. 4: Geographic distribution of the seven recent families belonging to the suborder Spongillina. The total number of species and genera are reported for each family on maps.

Spongillidae are cosmopolitan with 146 species, while the Gondwanian Metaniidae are Circumtropical with 22 species (Fig. 4). The two families share the traits sexual and asexual reproductive mode and gemmules with a complex architecture (Fig. 5). These complex gemmules with an armed gemmular theca to protect totipotent cells, seems to be perfect dispersal propagules (Fig. 5). Several species of Spongillidae are characterised by a gemmule with a well developed pneumatic layer to float and to perform dispersal downstream, spicules from tangentially to radially arranged to strengthen the gemmular theca, and spiny spicules are able to hook efficiently onto the carrier. In some genera, as Stratospongilla, a double spicular layer protects the gemmule more efficiently (Fig. 5),

and the genus Saturnospongilla shows the displacement of the pneumatic layer with the result of a ring-shaped pneumatic layer possibly to increase the performances for anemophilous-hydrochorous dispersal (Fig. 5). A comparative analysis of the gemmular architecture clearly shows that several species of the two families Metaniidae and Spongillidae share most gemmular diagnostic traits. Spicules are radially arranged in the gemmular theca, the pneumatic layer is well developed, gemmuloscleres are spiny, and a cage of megascleres protects the gemmule (Fig. 5). A first evidence of this analysis is that the working hypothesis freshwater sponge success is strictly related to the efficiency of gemmules as dispersal devices does not work. In fragmented-discontinuous habitats it appears that

70

Fig. 5: Gemmular structure in different families of freshwater sponges. a-b. Gemmules of Potamolepidae (a, gemmular theca of Potamophloios stendelli; b, Gemmular cage of Uruguaya corallioides). df. Gemmules of Metaniidae are more complex, with a pneumatic layer almost always present, and radially arranged gemmuloscleres bearing hooks in most species (c-e, Metania reticulata); in some speciess an external cage adds protection to the gemmular theca (f, Drulia browni). g-j. The general structure of gemmules is shared by Spongillidae and Metaniidae: developed pneumatic layer, spiny gemmuloscleres radially arranged (g-i, Anheteromeyenia argyrosperma), outer spicular cage (j, Stratospongilla bombayensis). k. Saturnospongilla carvalhoi, among Spongillidae, shows a gemmular structure with a peculiar ring-shaped pneumatic layer (arrows).

71

Fig. 6: The gemmule structure of Spongillidae. Umborotula bogorensis shows spiny gemmuloscleres radially arranged (a-c) within the pneumatic layer (c). Species belonging to the genus Eunapius display a peculiar pneumatic layer (d, E. carteri) with spiny gemmuloscleres not radially arranged (e, E. nitens). Some gemmules of Spongilla lacustris lack gemmuloscleres (f) and pneumatic layer (g).

the absence of gemmules determines the absence of dispersal at a large-scale and therefore a condition of endemism as in sponges from ancient lakes (Fig. 4). On the other hand, dispersal power would be low for simple gemmules (absence of spiny gemmuloscleres, absence of pneumatic layer) with consequently a relatively restricted geographic range as in the case of Potamolepidae (Fig. 4-5). Finally, taxa bearing complex gemmules would show a high dispersal power with a tendency to cosmopolitism. This might happen for Metaniidae and Spongillidae.

Biogeographic data on Metaniidae show however that a highly complex gemmular architecture does not support their relatively restricted distribution and low species richness. Although the gemmular complexity in Spongillidae and Metaniidae is comparable, a notable divergence exists between the worldwide distribution of the former and the Circumtropical pattern of the latter. The geographic range and the species richness of Metaniidae are, on the other hand, similar to that of Potamolepidae characterised by a simple gemmule (Figs 4-5).

72

pneumatic layer is absent (Figs 6-7) suggesting that the trait complexity of gemmular architecture does not support the relatively restricted distribution of the genus Umborotula vs. Eunapius and Spongilla. At the species level within the genus Spongilla, different biogeographic patterns are evident, some species being very common and widespread - as in the case of the Holarctic Spongilla lacustris - while other species are rare and monotopic - as for Spongilla prespensis Hadzische, 1953 and Spongilla stankovici Arndt, 1938 from the Balkanian area, respectively endemic to Lake Prespa and Lake Ochrid (Pronzato and Manconi 2002) (Fig. 8). It is consequently evident that it is not true that taxa with complex gemmules as perfect dispersal devices show a wider geographic range; this statement is valid at the family, the genus and the species level. We can consider a further condition among Spongillidae. Spongilla lacustris displays two gemmular types (Manconi and Desqueyroux-Faundez 1999) and its widespread distribution, with a Holarctic range, could be related to this peculiar condition. Also, species belonging to the genus Corvospongilla, one of the most speciose within Spongillidae, display two gemmular types diverging in their morpho-functional roles. A first type, the sessile gemmule, is strictly adherent to the substratum, with absence of pneumatic layer and well developed spicular cage; the supposed functional role of this heavy gemmule is to persist in situ. A second type is a free gemmule in the sponge skeleton, with a well developed pneumatic layer and without spicular cage; the supposed functional role of this light gemmule is dispersal (Fig. 9) (Manconi et al. 2004). The genus Corvospongilla shows at the biogeographic scale a notably disjunct range with 17 species extremely rare and known only from restricted areas. This biogeographic pattern matches better the vicariance model versus the dispersal one. The evolutionary novelty of two diverging gemmular architectures seems not to favour the wide spreading of the genus Corvospongilla, being most species endemic and rare (Manconi and Pronzato 2004).

Approaching conclusions
Different possible problems could affect our working hypothesis; they could be: i) errors in the systematics; ii) a high richness in species complexes with the result of misleading geographic ranges; iii) a diversified physiological control of gemmular dormancy. Another approach to explain the relationships between the structure of gemmules and the distribution of freshwater sponge taxa could be to consider the old axiom of Biogeography: species distribution and richness is frequently strictly related to the distribution and number of taxonomists. If we focus on the species richness of African freshwater sponges, since the first record by Carter in 1881, the temporal trend shows clearly that taxonomic richness increased slowly in more than one century, with a present maximum of 56 species at the continental scale (other 6 taxa are reported at the genus, family and suborder level) out of 175 findings (Manconi and Pronzato in press b, unpublished). Moreover endemicity s.s. is notably high (78.5%), 23 species are known

Fig. 7: Geographic distribution of the genera Umborotula, Eunapius and Spongilla. The latter two genera are cosmopolitan although their gemmules are simple (see Fig. 6), while the former is restricted to the Oriental Region.

The analysis of gemmular complexity at the genus level within Spongillidae indicates that the genus Umborotula, with a highly complex gemmule (Fig. 6), is monotypic (only 1 species) and restricted to E-Asia and Australia (Manconi and Pronzato 2002) (Fig. 7). On the other hand the genus Eunapius is cosmopolitan with a moderately complex gemmular architecture (spicules not radially arranged, spicules without hooks) (Figs 6-7). This condition is also shared by the genus Spongilla, where sometimes the

73

Fig. 8: Pattern of geographic distribution in the genus Spongilla. Holarctic range of S. lacustris vs. S. prespensis and S. stankovici each strictly endemic to two different Balkanian lakes.

only from the holotype, and the knowledge on some areas is scattered and fragmentary, if not lacking, as for Madagascar. All these data strongly suggest that taxonomic richness of African freshwater sponges is underestimated and species distribution is probably mislead (Manconi and Pronzato in press b). A historical analysis of the whole knowledge on freshwater sponges in the field of taxonomy and biogeography - based on the number of papers per year - shows that only 3 species have been described before the erection of the genus Spongilla by Lamarck (1816). The following 60 years are characterised by long-lasting periods of inactivity (no papers) or very low activity (1-2 paper/year). Successively a positive trend is evident, with peaks corresponding both to the most productive authors, and to the periodical maxima of taxonomic richness increase (Fig. 10). Annandale, for example, described 29 new species in 10 years (1907-1918), Potts described 16 species in 10 years (1880-1889), Weltner described 15 species in 21 years (1893-1913), Bonetto and Ezcurra de Drago described 17 species in 8 years (1966-1973) always together, VolkmerRibeiro described 13 species in 43 years (1963-2005) with

different co-authors, Brien described 11 species in 8 years (1967-1974) while Bowerbank described 11 species in a single year (1863). We can generalize that species richness of freshwater sponges seems, at present, to be underestimated and destined to increase with further researches based on a critical analysis and a synthesis on all materials in collections and on all taxonomic data from the literature, to have a strong morphological basis to compare and support the results of molecular biology. New sampling campaigns are also needed, as suggested by the recent new findings on the freshwaters sponge fauna. For example, one out of five samplings in the Australian Kakadu park resulted in the discovery of new morpho-traits for the genus Pectispongilla, and additional ones for the whole Spongillidae (Manconi et al. 2006). If we move to the Caribbean area, 12 samplings in the West Indies resulted in the discovery of a notably rich Spongillino-fauna in the island of Cuba (Manconi and Pronzato 2005). Finally, from a total of 209 contributions reported in the VIth Sponge Conference abstract book, ten concern freshwater sponges but only two focus on the field of systematics

74

Fig. 9: Corvospongilla mesopotamica is characterized by the presence of two gemmular morpha. The sessile heavier gemmule, strictly adherent to the substratum, lacks pneumatic layer and is protected by a reinforced spicular cage (a); the free lighter gemmule shows a well developed pneumatic layer (b) and few gemmuloscleres; c. Schematic reconstruction of the life cycle of C. mesopotamica to evidence the double role of gemmules diverging in their morpho-functional role. Circles indicate dispersal (light gemmules), squares indicate persistence (heavy gemmules).

Fig. 10: Yearly production of taxonomic and biogeographic papers on freshwater sponges after their actual separation from other sponge higher taxa. Data source: Zoological Record.

75

(Pansini and Pronzato 2002). This trend is confirmed by contents of the 7th International Sponge Symposium abstract book: only 16 contributions, out of 308, concern Spongillina (10 on systematics, faunistic and distribution) (Custdio et al. 2006). At present the number of taxonomists on freshwater sponges is dramatically low (less than ten) and without absolutely necessary new recruitments they are on the brink of extinction.

Acknowledgements
This paper is dedicated to the memory of the Brazilian specialist of freshwater sponges Rosaria de Rosa-Barbosa. R. Manconi is grateful to the organizers of the 7th International Sponge Symposium for their kind invitation and financial support. Research supported by the Italian Ministero dellIstruzione, dellUniversit e della Ricerca Scientifica e Tecnologica (MIUR-PRIN 2004057217 Zoogeography of Mediterranean-Southern African disjunct distributions by a multimethod approach), the European program INTERREG Sardinia-Corsica-Tuscany on Biodiversity, the Universit di Sassari and Universit di Genova.

References
Adamson DA, Clark JD, Williams MAJ (1987) Pottery tempered with sponge from the White Nile, Sudan. Afr Arch Rev 5: 115-127 Adis J (1992) On the survival strategy of Mesostoma hylaeicum Jeekel, a millipede from Central Amazonian floodplains. Ber Natmed Ver Innsbruck, suppl. 10: 183-187 Allen TF (1986) Encyclopaedia of pure materia medica, Vol. II, B. Jain Publishers, New Delhi Armstrong G, Booth DT (2005) Dietary ecology of the Australian freshwater turtle (Elseya sp.: Chelonia: Chelidae) in the Burnett River, Queensland. Wildlife Res 32: 349-353 Berg K (1948) Biological studies on the river Susaa. Faunistic and biological investigations. Porifera. Folia Limnol Scand 4: 10-23 Brien P (1966) Le poliphiletisme des ponges deau douce. Lembryognse et la larve chez Potamolepis stendelli (Jaff). C R Acad Sci Paris 263: 649-652 Brien P (1969) Les Potamolepides africaines. Polyphyletisme des ponges deau douce. Arch Zool Exp Gen 110: 527-561 Brissaud I, Houdayer A (1986) Sponge spicules as a characteristic of ancient African pottery from Mali. J Fld Arch 13: 357-358 Brnsted A, Brnsted HV (1953) The effect of symbiotic zoochlorellae on the germination rate of gemmules of Spongilla lacustris (L.). Vidensk Medd Dan Naturhist Foren 115: 133-144 Brnsted HV, Louvtrup E (1953) The respiration of sponge gemmules without and with symbiotic unicellular algae. Vidensk Medd Dan Naturhist Foren 115: 145-157 Brown HP (1952) The life history of Climacia areolaris (Hagen), neuropterous parasite of freshwater sponges. Amer Mid Nat 47: 130-160 Candido JL, Volkmer-Ribeiro C, Filho FLS, Turcq BJ, Desjardins T, Chauvel A (2000) Microsclere variations of Dosilia pydanieli (Porifera, Spongillidae) in Caracaran lake (Roraima - Brazil). Palaeoenvironmental implication. Biociencias 8(2): 77-92 Carter HJ (1881) History and classification of the known species of Spongilla. Ann Mag Nat Hist 7: 77-107 Corriero G, Vaccaro P, Manconi R, Pronzato R (1993) Life strategies of Ephydatia fluviatilis (L., 1759) in two different environments.

In: van Soest RWM, van Kempen TMG, Braekman JC (eds). Sponges in time and space. Balkema, Rotterdam. pp. 321-326 Crane K (1991) Hydrothermal vents in Lake Baikal. Nature 350(6316): 281 Custdio MR, Lbo-Hajdu G, Hajdu E, Muricy G (eds.) (2006) 7th International Sponge Symposium - Book of Abstracts (Armao dos Bzios, Brazil). Museu Nacional, Srie Livros 16, Rio de Janeiro Desqueyroux-Faundez R, Valentine C (2002) Phloeodictyidae, Petrosiidae. In: Hooper JNA, van Soest RWM (eds). Systema Porifera: a guide to the classification of sponges. Kluwer Academic/Plenum Publishers, New York. pp. 893-917 Dominey J, Snyder AM (1988) Kleptoparasitism of freshwater crabs by cichlid fishes endemic to Lake Barombi Mbo, Cameroon, West Africa. Env Biol Fish 22(2): 155-160 Dunagan SP (1999) A North American freshwater sponge (Eospongilla morrisonensis new genus and species) from the Morrison Formation (Upper Jurassic), Colorado. J Paleontol 73(3): 389-393 Ereskovsky AV (1999) Development of sponges of the order Haplosclerida. Russian J Mar Biol 25(5): 361-371 Ereskovsky AV (2004) Comparative embryology of sponges and its application for poriferan phylogeny. In: Pansini M, Pronzato R, Bavestrello G, Manconi R (eds). Sponge science in the new millennium. Boll Mus Ist Biol Univ Genova 68: 301-318 Francis JC, Harrison FW (1988) Copper and zinc toxicity in Ephydatia fluviatilis (Porifera: Spongillidae). Trans Am Microsc Soc 107(1): 67-78 Frost TM (1978) In situ measurements of clearance rates for the freshwater sponge Spongilla lacustris. Limnol Oceanogr 23(5): 1034-1039 Frost TM (1980) Clearance rate determinations for the freshwater sponge Spongilla lacustris: effect of temperature, particle type and concentration, and sponge size. Arch Hydrobiol 90(3): 330-356 Frost TM (1991) Porifera. In: Thorp JH, Covich AP (eds). Ecology and classification of North American freshwater invertebrates. Academic Press, New York. pp. 95-124 Frost TM, Williamson CE (1980) In situ determination of the effect of symbiotic algae on the growth of the fresh-water sponge Spongilla lacustris. Ecology 61: 1361-1370 Gugel J (1996) The sponge mite Unionicola minor (Acari, Hydrachnellae, Unionicolidae) in a pond at the spring of the Darmbach near Darmstadt. Hessische Faunistische Briefe 15(1): 11-16 Harrison FW (1974) Porifera. In: Hart CW, Fullers SLH (eds). Pollution ecology of freshwater invertebrates. Academic Press, New York. pp. 29-66 Harrison FW (1977) The taxonomic and ecological states of the environmentally restricted spongillid species of North America. III Corvomeyenia carolinensis Harrison, 1971. Hydrobiologia 56(2): 187-190 Harrison FW (1988) Utilization of freshwater sponges in paleolimnological studies. In: Gray J (ed). Paleolimnology: aspects of freshwater paleoecology and biogeography. Elsevier, Amsterdam. pp. 387-397 Harrison FW, Warner BG (1986) Fossil freshwater sponges (Porifera: Spongillidae) from Western Canada: an overlooked group of Quaternary paleoecological indicators. Trans Am Microsc Soc 105(2): 110-120

76

Harrison FW, Gleason PJ, Stone PA (1979) Paleolimnology of Lake Okeechobee, Florida: an analysis utilizing spicular components of freshwater sponges (Porifera: Spongillidae). Not Nat Acad Nat Sci Philadelphia 454: 1-6 Jewell ME (1935) An ecological study of the fresh-water sponges of northeastern Wisconsin. Ecol Monogr 5: 461-504 Jewell ME (1939) An ecological study of the freshwater sponges of Wisconsin. II. The influence of calcium. Ecology 20: 11-28 Kahl K, Konopacka A (1981) Oligochaeta inhabiting the colonies of the sponge Spongilla lacustris (L.) in the River Gac. Acta Hydrobiol 23(3): 243-249 Kamaltynov RM, Chernykh VI, Slugina ZV, Karabanov EB (1993) The consortium of the sponge Lubomirskia baikalensis in Lake Baikal, east Siberia. Hydrobiologia 271(3): 179-189 Kennett R, Tory O (1996) Diet of two Freshwater turtles, Chelodina rugosa and Elseya dentata (Testudines: Chelidae) from the wetdry tropics of Northern Australia. Copeia 2: 409-419 Kilian EF, Campos H (1969) Freshwater sponge as sites for hatching of the brood of a fish. Naturwissenschaften 5(6): 333 Konopacka A, Socinski J (1985) Macrofauna inhabiting the colonies of the sponge Spongilla lacustris (L.) in the River Gac. XIII. Ecology of aquatic organisms. 3. Animals. Verh Int Verein Limnol 22: 2968-2973 Kuznetzow J (1898) Fischerei und thierebentung in den Gewassern Russlands. Ministerium fur landwirthschaft und reichsolomanen. Department fur landwirthschaft, St. Petersburg Lamarck JBPA (1816) Histoire naturelle des animaux sans vertbres. Verdire, Paris Lauer TE, Spacie A (2004) Space as a limiting resource in freshwater systems: competition between zebra mussels (Dreissena polymorpha) and freshwater sponges (Porifera). Hydrobiologia 517(1): 137-145 Linn S (1925) The technique of the South American ceramics. Gteb K Vetensk Vitter Hets-Samh-Handl Sjatte Foljden Ser. B 29(5): 1-199 Manconi R, Desqueyroux-Faundez R (1999) Freshwater sponges (Porifera, Spongillidae) from the Lake of Geneva, Switzerland. Rev Suisse Zool 106(3): 571-580 Manconi R, Pronzato R (1991) Life cycle of Spongilla lacustris (Porifera, Spongillidae): a cue for environmental dependent phenotype. Hydrobiologia 220: 155-160 Manconi R, Pronzato R (1994) Spongillids of Mediterranean islands. In: van Soest RWM, van Kempen TMG, Braekman JC (eds). Sponges in time and space. Balkema, Rotterdam. pp. 333-340 Manconi R, Pronzato R (2002) Spongillina subord. nov. Lubomirskiidae, Malawispongiidae n. fam., Metaniidae, Metschnikowiidae, Potamolepidae, Spongillidae. In: Hooper JNA, van Soest RWM (eds). Systema Porifera: a guide to the classification of sponges. Kluwer Academic/Plenum Publishers, New York. pp. 921-1019 Manconi R, Pronzato R (2004) The genus Corvospongilla Annandale (Haplosclerida, Spongillina, Spongillidae) with description of a new species from eastern Mesopotamia, Iraq. Arch Hydrobiol suppl Mon Stud 151(1-2): 161-189 Manconi R, Pronzato R (2005) Freshwater sponges of the West Indies: discovery of Spongillidae (Haplosclerida, Spongillina) from Cuba with biogeographic notes and a checklist for the Caribbean area. J Nat Hist 39(36): 3235-3253 Manconi R, Pronzato R (in press a) Global diversity of sponges (Porifera: Spongillina) in freshwater. In: Balian E, Martens

K, Leveque C (eds). Freshwater animal diversity assessment. Hydrobiologia Manconi R, Pronzato R (in press b) Sponges from African inland waters: the genus Eunapius (Haplosclerida, Spongillina, Spongillidae). Arch Hydrobiol suppl Mon Stud Manconi R, Cubeddu T, Corriero G, Pronzato R (1999) Commercial sponges farming as natural control of floating cages pollution. In: Enne G, Greppi F (eds). New species for Mediterranean aquaculture. Biofutur Elsevier, Paris. pp 269-274 Manconi R, Saadalla HAA, Cubeddu T, Ferretti C, Pronzato R (2004) Resting stages of Corvospongilla sp. (Demospongiae, Spongillidae) from Mesopotamia, Iraq. In: Pansini M, Pronzato R, Bavestrello G, Manconi R (eds) Sponge science in the new millennium. Boll Mus Ist Biol Univ Genova 68: 441-448 Manconi R, Cubeddu T, Ferretti C, Pronzato R (2006) A new morphological trait for northern Australian pectispongillas. In: Custdio MR, Lbo-Hajdu G, Hajdu E, Muricy G (eds). 7th International Sponge Symposium - Book of Abstracts (Armao dos Bzios, Brazil). Museu Nacional, Srie Livros 16, Rio de Janeiro. pp. 225 McCauley DG, Longcore JR (1988) Foods of juvenile ring-necked ducks: relationship to wetland pH. J Wildl Manage 52(2): 177185 McIntosh SK, MacDonald KC (1989) Sponge spicules in pottery: new data from Mali. J Fld Arch 16: 489-494 Old MC (1932) Environmental selection of the fresh-water sponges (Spongillidae) of Michigan. Trans Amer Microsc Soc 51: 129-136 Oliveira Roque de F, Trivinho-Strixino S, Marques Couceiro SR, Hamada N, Volkmer-Ribeiro C, Messias MC (2004) Species of Oukuriella Epler (Diptera, Chironomidae) inside freshwater sponges in Brazil. Rev Bras Entomol 48(2): 291-292 Ott E, Volkheimer W (1972) Palaeospongilla chubutensis n.g. et n.sp. ein susswasserschwamm aus der Kreide Patagoniens. N Jb Geol Palaont Abh 140: 49-63 Pansini M, Pronzato R (eds) (2002) VIth International Sponge Conference, Book of Abstracts. Boll Mus Ist Biol Univ Genova 66-67: 1-706 Parfin SI, Gurney AB (1956) The spongillaflies with special reference to those of the Western Hemisphere (Sisyridae, Neuroptera). Proc Unit States Natl Mus 105: 421-529 Pronzato R, Manconi R (1994) Life history of Ephydatia fluviatilis: a model for adaptive strategies in discontinuous habitats. In: van Soest RWM, van Kempen TMG, Braekman JC (eds). Sponges in time and space. Balkema, Rotterdam. pp. 327-331 Pronzato R, Manconi R (1995) Long term dynamics of a freshwater sponge population. Freshwat Biol 33: 485-495 Pronzato R., Manconi R (2002) Atlas of European freshwater sponges. Ann Mus Civ St Nat Ferrara 4: 3-64 Pronzato R, Manconi R, Corriero G (1993) Biorhythm and environmental control in the life history of Ephydatia fluviatilis (Demospongiae Spongillidae). Boll Zool 60: 63-67 Rader RB (1984) Factors affecting the distribution of a freshwater sponge. Freshwat Invertebr Biol 3: 86-97 Rader RB, Winget RN (1985) Seasonal growth rate and population dynamics of a freshwater sponge. Hydrobiologia 123: 171-176 Resh MA (1976) Life cycles of invertebrate predators of fresh-water sponge. In: Harrison FW, Cowden RR (eds). Aspects of sponge biology. Academic Press, New York. pp. 299-354

77

Resh VH, Morse JC, Wallace JD (1976) The evolution of the sponge feeding habit in the caddis fly genus Ceraclea (Trichoptera: Leptoceridae). Ann Entomol Soc Amer 69(5): 937-941 Ricciardi A, Reiswig HM (1993) Freshwater sponges (Porifera, Spongillidae) of eastern Canada: Taxonomy, distribution, and ecology. Can J Zool 71(4): 665-682 Ricciardi A, FL Snyder, DO Kelch, HM Reiswig (1995) Lethal and sublethal effects of sponge overgrowth on introduced dreissenid mussels in the Great Lakes - St. Lawrence River system. Can J Fish Aquat Sci 52: 2695-2703 Richelle-Maurer E, Degoudenne Y, van de Vyver G, Dejinghe L (1994a) Some aspects of the ecology of Belgian freshwater sponges. In: van Soest RWM, van Kempen TMG, Braekman JC (eds). Sponges in time and space. Balkema, Rotterdam. pp. 341350 Richelle-Maurer E, Degoudenne Y, van de Vyver G, Dejinghe L (1994b) Some aspects of heavy metal tolerance in freshwater sponges. In: van Soest RWM, van Kempen TMG, Braekman JC (eds). Sponges in time and space. Balkema, Rotterdam. pp. 351354 Richter G, Wuttke M (1999) Lutetiospongilla heili n. gen. n. sp. und die eozne Spongillidenfauna von Messel. Cour Forschungsinst Senckenb 216: 183-195 Ronde De CEJ, Stoffers P, Garbe-Schonberg D, Jones B, Manconi R, Christenson BW, Browne PRL., Hissman K, Botz R, Davy BW, Schmitt M, Battershill CN (2002) Discovery of active hydrothermal venting in Lake Taupo, New Zealand. J Volcan Geotherm Res 115: 257-275 Rota E, Manconi R (2004) Taxonomy and ecology of spongeassociate Marionina spp. (Clitellata, Enchytraeidae) from the Horomatangi Geothermal System of Lake Taupo, New Zealand. Int Rev Hydrobiol 89: 58-67 Sar M, Vacelet J (1973) cologie des Dmosponges: influence des facteur physicochimiques. In: Grass PP (ed). Trait de Zoologie, Anatomie, Systematique, Biologie. 3(l) Masson et Cie, Paris. pp. 462-576 Serrano A (1933) Observacines sobre la lfareria de los-medanos de Coln. Mem Mus Parana 6: 1-2 Seigel RA, Brauman RJ (1994) Food habits of the yellow-blotched map turtle (Graptemys flavimaculata). Mus Tech Rep No. 28, Mississippi Museum of Natural Science, Jackson Simpson TL, Fell PE (1974) Dormancy among the Porifera: gemmule formation and germination in fresh-water and marine sponges. Trans Am Microsc Soc 93(4): 544-577 Steffen AW (1967) Ectosymbiosis in aquatic insects. Symbiosis 2: 207-209 Swierczynski M (1996) A cooccurrence of sponges (Porifera) and Dreissena polymorpha (Pall.) Przegl Zool 40(1-2): 119-121

Vacelet J (1994) Porifera. In: Juberthie C, Decu V (eds). Encyclopaedia Biospeologica, Socit Internationale de Biospologie. Pub. 1. Moulis, Bucarest. pp. 35-38 van Soest RWM, Velikonja M (1986) Porifera. In: Botosaneanu L (ed). Stygofauna Mundi. Brill, Leiden Volkmer-Ribeiro C (1979) Evolutionary study of the freshwater sponge genus Metania Gray, 1867: I. The new species. Amazoniana 6(4): 639-649 Volkmer-Ribeiro C (1986) Evolutionary study of the freshwater sponge genus Metania Gray, 1867: III. Metaniidae, new family. Amazoniana 9(4): 493-509 Volkmer-Ribeiro C (1990) A new insight into the systematics, evolution and taxonomy of freshwater sponges. In: Rtzler K (ed). New perspectives in sponge biology. Smithsonian Institution Press, Washington DC. pp. 323-33 l Volkmer-Ribeiro C, Turcq BJ (1996) SEM analysis of siliceous spicules of a freshwater sponge indicates paleoenvironmental changes. Acta Microsc 5B: 186-187 Volkmer-Ribeiro C, de Rosa-Barbosa R (1974) A freshwater spongemollusk association in Amazonian waters. Amazoniana 5(2): 285291 Volkmer-Ribeiro C, de Rosa-Barbosa R (1979) Neotropical freshwater sponges of the family Potamolepidae Brien, 1967. In: Lvi C, Boury-Esnault N (eds). Biologie des spongiaires. Coll Int CNRS 291. pp. 503-511 Volkmer-Ribeiro C, Watanabe Y (1983) Sanidastra yokotonensis, n. gen. and n. sp. of freshwater sponge from Japan. Bull Nat Sci Mus Tokyo Ser A 9(4): 151-159 Weinberg I, Glyzina O, Weinberg E, Kravtsova L, Rozhkova N, Sheveleva N, Natyaganova A, Bonse D, Janussen D (2004) Types of interactions in consortia of Baikalian sponges. In: Pansini M, Pronzato R, Bavestrello G, Manconi R (eds) Sponge science in the new millennium. Boll Mus Ist Biol Univ Genova 68: 655-663 Weert de WH (2002) Chalinidae. In: Hooper JNA, van Soest RWM (eds). Systema Porifera: a guide to the classification of sponges. Kluwer Academic/Plenum Publishers, New York. pp. 852-873 Wells A (1995) New Caledonian Hydroptilidae (Trichoptera), with new records, descriptions of larvae and a new species. Aquat Insects 17(4): 223-239 Weissenfels N (1989) Biologie und mikroskopische anatomie der susswasserschwamme (Spongillidae). Fischer, Stuttgart, New York Weissmair W, Mildner P (1995) On the study of Sisyridae (Neuroptera), their hosts and their aquatic habitat in Carinthia. Carinthia II 105(2): 535-552 Willenz P, Vray B, Maillard MP, van de Vyver G (1986) A quantitative study of the retention of radioactively labeled E. coli by the freshwater sponge Ephydatia fluviatilis. Physiol Zool 59(5): 495-504

Porifera research: Biodiversity, innovation and sustainaBility - 2007

79

Evolution of multicellularity in Porifera via selfassembly of glyconectin carbohydrates


Gradimir N. Misevic(1*), Camille Ripoll(1), Jonathan Norris(1), Vic Norris(1), Yann Guerardel(2), Emmanuel Maes(2), Gerard Strecker(2), Pascal Ballet(3), Yannis Karamanos(4), Lazar T. Sumanovski(5), Octavian Popescu(6), Nikola Misevic(7)
Laboratoire Assemblages Molculaires: Modlisation Imagerie et SIMS, FRE CNRS 2829, Facult des Sciences de lUniversit de Rouen, 76 821 Mont Saint Aignan Cedex, France, gradimir@gradimir.com (2) Unit de Glycobiologie Structurale et Fonctionnelle, Universit des Sciences et Technologies de Lille, UMR 8576 CNRS, 59655 Villeneuve DAscq, France (3) University of Brest, Brest, France (4) Laboratoire de Biochimie Molculaire et Cellulaire, Universit dArtois, Facult J Perrin, rue J. Souvraz, SP18, 62307 Lens, France (5) Department of Research, University Hospital of Basel, CH-4058 Basel, Switzerland, (6) Molecular Biology Center and Institute for Interdisciplinary Experimental Research, Babes-Bolyai University, 400006 Cluj-Napoca, Rumania (7) University of Bremen, 28359 Bremen, Germany
(1)

This work is dedicated to the memory of Maurice Demarty Abstract: Research done in the last century on Porifera has provided insights into the molecular mechanism of the biological processes of cell adhesion, innate immunity, and self-recognition. Evidence that this mechanism is based on glyconectin selfassembly is shown by the structure to function relationships deduced from studies of carbohydrates isolated from three different sponge species. The structural studies were performed on purified glyconectin carbohydrates from Microciona prolifera, Halichondria panicea and Cliona celata using nuclear magnetic resonance and mass spectrometry. Seventeen novel, speciesspecific carbohydrate sequences were revealed that belong to the Porifera glyconectin family. The functional, cell recognition analyses of carbohydrate self-association were performed by measuring binding forces between individual glycan molecules under physiological conditions; the results show that the association strength between homotypic pairs of glycans (400 pN) are higher than those between heterotypic pairs (20 pN). This difference is sufficient to explain the species-specific separation of glycan-coated beads in vitro and the sorting of cells in nature. We propose that the glyconectin carbohydrates, which are the constituents on the cell surface that are the most exposed to the environment, were responsible for the molecular recognition processes that underpinned the emergence of multi-cellularity. Keywords: evolution of multicellularity, Porifera, cell recognition and adhesion, glyconectin carbohydrates and proteoglycans, atomic force microscopy

An approach to study the evolution of multicellularity Why?


Changing patterns of matter and energy achieved in multicellular life their most sophisticated form so far known. During biological evolution, two essential steps occurred: the first was the emergence of cells and the second subsequent one was the development of multicellular assemblies. In an effort to understand the emergence and evolution of the complex and versatile multicellular life forms present today on earth, as well as to satisfy our curiosity of whether other kinds of similarly hierarchically organized life could exist in

the universe, whether it be in the past, present or future, two common, related questions may be formulated in the following way: what were the molecular mechanisms that enabled the creation and persistence of multicellular (or multi unit) life? And, can the similar or alternative mechanisms be predictable in both time and space? In order to devise experiments that could generate data to provide, in part, the answer to these questions, we begin with the following logical statement: the evolution of multicellular forms of life required the emergence of cellular self-non-self discrimination and adhesion, where the sensor molecules guiding such recognition and adhesion should be present at the outermost cell surface (although many processes based on novel as yet imaginary physico-

80

chemical principles might be invoked in extraterrestrial life forms). By gaining recognition and adhesion properties, the primordial multicellular organisms could preserve functional and morphological identity throughout their life cycles.

Structural analyses of glyconectin glycan self-nonself recognition molecules


Structural analyses of glyconectins (GNs) isolated from three sponge species Microciona prolifera (GN1), Halichondria panicea (GN2) and Cliona celata (GN3), used as a experimental model system to study evolution of the multicellularity, will be reviewed here. General approach, depicted in Fig. 1, consists of four steps ranging from the release of glycans of their protein core towards glycan fragmentation, fingerprinting and sequencing using nuclear magnetic resonance (NMR) and mass spectrometry (MS). In the first step protein free polysaccharide chains from purified GNs were prepared by extensive pronase digestion of the protein part (Misevic et al. 1982, Misevic and Burger 1993). This was followed by separation of intact glycans from free amino acids by gel filtration and ionexchange chromatography. In the second step glycans were separated and isolated by column chromatography and/or gel electrophoresis (Fig 1). GN1 was found to have two major glycans with molar masses 200 x 103 and 6 x 103, as previously reported (Misevic et al. 1982, Misevic and Burger 1986, 1990a, 1990b, 1993, Spillmann et al. 1993, Spillmann et al. 1995), GN2 had one major glycan with molar mass of 180 x 103 kD, representing more then 60% of the total carbohydrate content, and GN3 contained also one major glycan species (50% of total carbohydrates) with molar mass of 110 x 103 kD (Fig. 1). The third step in our analyses was chemical and enzymatic fragmentation of GN glycans. The results obtained revealed that each species has its own fingerprint signature. The last step was sequencing of each GN glycan fragment by combination of two dimensional COSY90 high resolution NMR and three types of MS: EI-MS - Electronic Impact Mass Spectrometry, CI-MS - Chemical Ionization Mass Spectrometry and MALDI-TOF MS - Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (Fig 1). Complex analyzes of NMR and MS fingerprinting data revealed that four large glycans g200 and g6, g180, and g110 of GN1, GN2 and GN3 respectively, are built by novel repetitive units (Misevic et al. 1982, 1987, Misevic 1989, Spillmann et al. 1993, 1995, Misevic and Burger 1986, 1990a, 1990b, 1993, Popescu and Misevic 1997) (Fig. 2). As shown in Table 1 four short sulfated and one pyruvilated unit in GN1, eight larger and branched pyruvilated oligosaccharides in GN2 which represent heterogeneous but related family of structures, and four sulfated units in GN3 were sequenced (Guerardel et al. 2004, Misevic et al. 2004). We propose two possible models of organization for GN1 and GN3 carbohydrate moieties within the glycan chain (Fig. 2A and B). The first model represents a high molecular weight, linear, acid sensitive polysaccharide connected to an acid resistant domain. This polysaccharide may be composed of either heterogeneous short repetitive units or a large homogeneous repetitive unit comprising acidic labile glycosidic bonds (Fuc/Ara). The actual size of a homogeneous repetitive unit is difficult to assess since most Fuc- and Araglycosidic linkages are cleaved in mild acidic conditions. The second model represents a mixed ramified polysaccharide composed of an acid resistant core connected through Fuc/

How?
In our experimental study of the molecular bases guiding the evolution of multicellularity, it was imperative to select the most appropriate model systems and organisms. We have chosen xenogeneic cell recognition and adhesion of several sponge species from the phylum Porifera because they represent today the simplest multicellular life forms, closest in terms of evolution to primordial multicellular life. In consequence, the molecular mechanism of self-non-self discrimination and adhesion in sponges should be most similar to the mechanism that operated during the emergence of multicellularity. At the beginning of the last century, fundamental phenomenological experiments showed that the xenogeneic re-aggregation of dissociated sponge cells is promoted by extracts made from the surfaces of their cells (Wilson 1907, Galstoff 1925, Curtis 1962, Moscona 1968). Numerous repetitions and variations of these experiments and characterization of aggregationpromoting extracts, called at the time aggregation factors, have been done (Humphreys 1963, Cauldwell et al. 1973, Muller and Zahn 1973, Jumblatt et al. 1980). More sophisticated purification and characterization of aggregation-promoting extracts have shown that they contain a new class of large cell surface proteoglycan-like molecules, heavily covered by long glycan chains, named by G.N. Misevic glyconectins (GNs, derived from glyco connecting, connecting cells via glycans; Papakonstantinou and Misevic 1993, Dammer et al. 1995, Misevic and Popescu 1995, Guerardel et al. 2004, Misevic et al. 2004). Large GN glycans are the outermost macromolecules on the cell surface and display an enormous variability despite their similar structures (Fig. 1). These glycans should therefore be considered as the primary candidates for sensing the environment and performing the self/non-self recognition and adhesion essential for the evolution of the multicellularity. In the first part of this report we describe the key experiments performed to identify, isolate and sequence functional glyconectin glycan molecules. In the second part we explain our novel experimental approach and show quantitative measurements of recognition and adhesion phenomenon on the molecular and cellular level. Finally, in the last part we present a few thoughts and facts about the value of the Porifera model system to basic research.

Emergence of complexity
The emergence of more complex multicellular organisms was based on the appearance of higher degrees of complexity and the multistep nature of cell recognition and adhesion systems. These can be related to 1) allogeneic self-non-self discrimination in the divergence of species and 2) syngeneic organ and tissue specificity during morphogenesis.

81

Fig. 1: The fist panel shows EM, AFM and X-ray images of glycans dimensions at cellular, molecular and atomic level. EM; the Electron Microscope image of cells stained for acidic polysaccharides. These glycans are the most peripheral molecules (over 200 nm) from the cell surface with very high density and abundance. AFM; Atomic Force microscope image of GN1 with g200 glycan arms of 180 nm. X-ray; model of protein on plasma membrane in blue with small glycans in yellow and large glycan in green which is an order of magnitude longer then presented if the real length of g200 glycan would be taken in account. In the second panel as the example of the second step of structural analyses, a polyacrylamide gel electrophoresis of purified glyconectin glycan fraction is presented. Electrophoresis of glyconectin glycans was performed on a polyacrylamide gradient gel (7.5-15%). Gels were stained with 0.3% alcian blue in 3% acetic acid in aqueous 25% isopropanol. Lane a, 20 g of GN1 glycans; lane b, 20 g of GN2 glycans; lane c, 20 g of GN3 glycans. The third panel shows the third step of structural analyses of glycans by fingerprinting with trifluoroacetic acid hydrolyses. TLC analysis of hydrolyzed fractions of GN1 and GN2 stained by sulfuric orcinol. Lane 1, standard Glc degrees of polymerization (DP); lane 2, 0.1 M trifluoroacetic acid hydrolysis of GN1; lane 3, 0.1 M trifluoroacetic acid hydrolysis of GN2; lane 4, 1 M hydrolysis of GN2. The forth step in structural analyses using NMR and MS sequencing are shown in the forth panel. Complex sequencing procedure in combined NMR and MS complementary approach requires sophisticated instrumentation and high skills.

Fig. 2: A and B. Two putative models of ultrastructural organization of GN1 and GN3 glycans with linear and/or ramified repetitive units (blue circles symbolize Hexose and/or GlcNAc, blue striped squares Fucose). C. Model of GN2 highly ramified repetitive glycan structure (blue circle symbolize Hexose and/or GlcNAc, yellow triangles Py(4,6)Gal), blue striped squares Fucose.

Ara residues to small oligosaccharides that are released by mild acidic hydrolysis. In contrast with GN1 and GN3, mild hydrolysis of GN2 released large oligosaccharides that were further fragmented in smaller units using stronger acidic conditions. Analysis

of both fractions revealed that the acid labile carbohydrate moiety of GN2 comprised a highly ramified polysaccharide backbone. It is constituted by an extremely heterogeneous mixture of hexose (mannose and galactose) oligomeres all terminated by Py(4,6)Gal residues and randomly interrupted

82

Table 1: Glycan structures obtained by NMR and MS after mild hydrolyses of isolated GN1, GN2 and GN3 polysaccharides.

GN1 GlcNAc-Fuc-(SO3)GlcNAc-Fuc GlcNAc-(SO3)Gal-Fuc Gal-(SO3)Gal-GlcNAc-Fuc Py(4,6)Gal-GlcNAc-Fuc (SO3)GlcNAc-[Fuc]Fuc

GN2 Py(4,6)Gal-(Hex)0-1-Fuc Py(4,6)Gal-(Hex)0-3-GlcNAc Py(4,6)Gal-(Hex)1-5 Py(4,6)Gal-(Hex)0-2[Hex]Hex Py(4,6)Gal-[Hex]Hex-Hex Py(4,6)Gal-Hex-[Py(4,6)Gal]Hex (Hex)4-[Py(4,6)Gal]Hex PyGal-Hex-[Hex]HexNAc

GN3 HexNAc-(SO3)Ara/Fuc-Fuc Hex-HexNAc-(SO3)Ara/Fuc-HexNAc-Fuc

by Fuc and GlcNAc residues. The observed heterogeneity of released oligosaccharides did not permit definitive conclusions about the ultrastructural organization of repetitive motifs. In conclusion, structural analyses of GN1, GN 2 and GN3 isolated from three different sponge species revealed that their carbohydrate content ranges between 4060% of their total mass thus characterized them as a heavily glycosilated macromolecules. The physico-chemical properties of each of four major GN glycan (GM1 g200 and g6, GN2 g189 and GN3 g110) such as size, composition, high content of anionic groups (carboxyl and/or pyruvate and/or sulfate), resistance to most glycosaminoglycan degrading enzymes, monoclonal antibodies mapping and their highly repetitive new type of sequences characterized them as novel class of acidic glyconectin type of glycans. Using the above interdisciplinary approach and technologies we have found that also higher invertebrates like sea urchins as well as vertebrates like mammals (rodents and humans) have similar type of glyconectin glycan structures (Papakonstantinou and Misevic 1993, Misevic and Popescu 1995). Therefore, glyconectin carbohydrates can be considered as a new family of species-specific glycans containing different classes of molecules present in Metazoans.

AFM measurements of intermolecular binding strength


Intermolecular binding forces between cell surface molecules are keeping cells together in multicellular organisms. To provide direct and quantitative evidence that glyconectin carbohydrates can indeed support cell adhesion, in 1993 we have developed a novel technology based on AFM measurements of binding strength between glyconectin carbohydrates under the physiological conditions (Dammer et al. 1995). Interactions between individual adhesion molecules (immunoglobulin, selectin, cadherin, integrins and extracellular matrix adhesions) were usually investigated by kinetic binding studies, calorimetric methods, x-ray diffraction, nuclear magnetic resonance and other spectroscopic analyses. These methods do not provide direct measurement of the intermolecular binding forces, which are fundamental for ligan-receptor association related to cell adhesion and recognition. To measure glyconectin to glyconectin interaction forces, we covalently attached glyconectins via their protein part to an AFM sensor tip and a flat mica surface (Fig. 3). The attachment process involved only glyconectin proteins but did not modify functional carbohydrate adhesion sites. As shown in the schematic presentation in Fig. 3 the cantilever tip having attached glyconectin molecules was carefully moved toward the substrate surface and a series of approach-andretract cycles were collected in physiological liquid medium. GN-GN binding was characterized by measuring both the force necessary to separate the GN-functionalized sensor tip from the GN surface (final jump-off) and the percentage of interaction events under different ionic conditions (Dammer et al. 1995). These two indicators of GN activity varied reversibly with the Ca2+ concentration, in agreement with GN-promoted cell adhesion and GN-coated bead aggregation (shown in the following section of functional analyses). At a Ca2+ concentration of 10 mM, the average force between GNs was 125 pN, ranging up to 400 pN, with high probability of binding (60 10%). At a Ca2+ concentration of 2 mM, cell adhesion and GN bead aggregation were sharply reduced, and the force (40 15 pN) and probability of binding (12 5%) were also reduced (Dammer et al. 1995). The interaction between GNs is Ca2+-selective, as reported with a cell aggregation assay. Indeed, 10 mM Mg2+ could not replace Ca2+ in AFM experiments or in adhesion of glyconectincoated beads (Dammer et al. 1995).

Functional measurements of glyconectin glycans self-non-self recognition properties by Atomic Force Microscopy and color coded cell-bead adhesion
For complete understanding of molecular mechanisms guiding evolution of multicellularity, it is necessary to complement structural studies on glyconectin glycans with quantitative functional measurements of cell adhesion and recognition. Such structure to function relationship in Porifera experimental model system was established by taking two complementary approaches. The first one was Atomic Force Microscopy (AFM) measurements of intermolecular binding strength between individual glyconectin glycans under physiological conditions (Dammer et al. 1995). The second one was quantification of color coded cell-cell, bead-bead and bead-cell recognition and adhesion mediated by glyconectin glycans (Popescu and Misevic 1997, Misevic et al. 2004).

83

Fig. 3: Schematic presentation of AFM measurements of intermolecular binding strength between glyconectin 1 carbohydrates.

Use of the monoclonal antibody (mAb) block 2 provided a third line of evidence that the AFM-measured interactions originate from binding between glyconectin glycans. This antibody recognizes a carbohydrate epitope of GN1 (see Table 1) and specifically inhibits GN1-promoted cell adhesion and GN1-coated bead aggregation (Dammer et al. 1995). In AFM experiments, block 2 Fab fragments in 10 mM Ca2+ SWT (Sea Water buffered with 20 mM Tris pH 7.4) reduced the interactive force to approximately the level measured at 2 mM Ca2+. A control mAb did not prevent GN1-GN1 binding under equivalent conditions. Thus, during AFM measurements in all tested experimental conditions, GN1-GN1 interactions resemble cell-cell adhesion events observed in vivo. The shape of the approach-and-retract cycles shows that string-like structures were responsible for GN1-GN1 interactions. These strings are likely to be the GN1 arms composed of glycans with a relative molecular weight of 200 x 103 (g200), which have been shown to mediate polyvalent GN1-GN1 binding (Fig. 3). This possibility is further supported by the fact that the length (180 nm) and the number (20 copies) of the g200 glycan per GN1 molecule are similar to the length and number of GN1 arms as measured by AFM and electron microscopy (Dammer et al. 1995). Finally, the inhibitory mAb block 2 is directed against a selfassociation epitope located on the g200 glycan. The shape of the approach-and-retract curves between glyconectins

suggested the presence of long-range interactions, interpreted as the lifting and extension of string-like glyconectin glycans, followed by further stretching until the elastic force of the cantilever equals the strength of the binding and the lever jumps off. At a physiological Ca2+ concentration of 10 mM in seawater multiple jump-offs were frequently observed, indicating polyvalent binding with an average adhesive force of 40 15 pN per release (Dammer et al. 1995). AFM measurements of intermolecular binding between homotypic pairs of GN2 and GN3 showed that intermolecular binding forces per pair of molecules are, as in GN1, in the range of 400 pN. Heterotypic combination of GNs revealed intermolecular binding strength of 20 pN (detailed results to be published). Similar results were obtained with purified GN glycans confirming the results of intact GNs where only carbohydrate moieties were available for interaction while the protein part was used for immobilization to surfaces (see Fig. 3). Therefore, carbohydrate to carbohydrate interaction is responsible for GN-GN self-non-self recognition and adhesion. In conclusion, measurement of binding forces intrinsic to adhesion molecules is necessary to assess their contribution to the maintenance of the anatomical integrity of multicellular organisms. Our atomic force microscopy results of the binding strength between cell adhesion glyconectin glycans under physiological conditions showed that homotypic adhesive force of 400 piconewtons per molecular

84

Fig. 4: Glyconectin glycoconjugates are cell adhesion and recognition molecules. Ca2+-dependent glyconectin to glyconectin interactions mediate species-specific cellcell recognition and adhesion. A. M. prolifera, B. H. panicea, and C. C. celata living sponges. Shown are self- and non-self-discrimination and adhesion in the suspension of mixed M. prolifera (orange), H. panicea (white), and C. celata (brown) live cells bearing glyconectins. D and E) ACMFSW (Artificial Calcium and Magnesium Free Sea Water) without 10 mM Ca2+ (D) and ACMFSW with 10 mM Ca2+ at 0C after 20 min of rotation (E). The microscopically observed color of the cells is somewhat different from that of the whole sponge. Early cell sorting experiments were usually done with binary sponge combinations at room temperature without rotation. The sorting is thus dependent on the presence of recognition molecules at the cell surface, cell motility, and speed of new synthesis and/or secretion of additional recognition molecules. Our rotary assays using either metabolically attenuated or fixed cells reduce the number of variable parameters.

85

pair could hold the weight of 1600 cells assuring the integrity of the multicellular sponge organism (Dammer et al. 1995). Interaction forces between heterotypic molecules were 20 times lower and are thus not sufficient to sustain existence of heterotypic aggregates under physiological hydrodynamic conditions of natural sea environment. Furthermore, this data also explain why small and loose unspecific aggregation was sometimes observed during the initial stage of heterotypic mixing under mild agitation.

Glyconectin glycans mediate color coded cell and bead adhesion


The cell adhesive function of three sponge glyconectins purified from Microciona prolifera (GN1), Halichondria panicea (GN2) and Cliona celata (GN3) was tested in a rotary reaggregation assay with live metabolically attenuated and/or fixed cells depleted of endogenous GNs. All three glyconectins, at concentrations mimicking in vivo conditions, mediated cell adhesion in the presence of physiological sea water with 10 mM CaCl2, and not below 1 mM CaCl2 (Guerardel et al. 2004, Misevic et al. 2004). In the absence of GNs, independently of CaCl2 concentration, no aggregation could be observed. Magnesium ions could not replace Ca2 titration experiments of Ca2+ concentration dependence of sponge glyconectin self-interactions revealed a transition at 5mM and 100% interactions at physiological 10 mM CaCl2, identical to that of Ca2+ dependent glyconectin promoted cell adhesion (Jumblatt et al. 1980, Dammer et al. 1995). These experiments indicated that a Ca2+ dependent glyconectin to glyconectin interactions play a pivotal role in cell adhesion of the three selected marine sponge species. The specificity of adhesion of GNs bearing cells was tested in a trinary species combination (Microciona prolifera, Halichondria panicea and Cliona celata) with living dissociated and metabolically attenuated cells in artificial sea water at 0C in the presence, and absence of 10 mM Ca2+ (physiological concentration in seawater). In a rotary assay, species-specific recognition and adhesion occurred only with 10 mM Ca2+ within 5-15 min. (Fig. 4). Upon removal of GNs from cell surface by repetitive washing none of the three species displayed aggregation in the presence of 10 mM Ca2+. Adding back the purified GNs to the same live cells at 0C completely restored species-selective cohesion. Similar results were obtained with non-living fixed cells. These experiments indicated that glyconectins and Ca2+ mediate the initial steps of xenogeneic cell recognition and adhesion of the three selected sponge species and were extending previously reported phenomenological and biochemical studies about the role of proteoglycan-like glycoconjugates in binary assays of dissociated sponge cells (Wilson 1907, Galstoff 1925 , Curtis 1962, Humphreys 1963, Moscona 1968, Cauldwell et al. 1973 , Mller and Zahn 1973, Jumblatt et al. 1980). In the second type of recognition assay, we reconstituted the observed cell recognition by using artificial system of glyconectin color coated beads. Glyconectin 1 was attached via its protein part to fluorescent pink, glyconectin 2 to fluorescent green, and glyconectin 3 to fluorescent blue latexamidine beads leaving glycan molecules free for interactions. Unlabeled glyconectins were immobilized on a nitrocellulose

Fig. 5: Simultaneous species-specific glyconectin to glyconectin recognition in suspension and blotting assay. Letters were drawn using 4 l of 1.5 mg/ml glyconectins on a Hybond-C extra nitrocellulose membrane (Amersham Biosciences) and probed in SWT with pink, green, and blue fluorescent beads coated with glyconectin 1, 2, and 3, respectively. A. SWT without 10 mM Ca2+. B. SWT with 10 mM Ca2+. All photographs were taken after 30 min of mixing.

membrane in such a manner that the three molecules were used to draw the subsequent letters of the words GLYCONECTIN RECOGNITION. The three bead types were mixed and added to the coated membrane in the presence of 10 mM CaCl2 or absence of calcium ions. As shown in Fig. 5, within 5-15 min of constant rotation species-specific bead-bead aggregation and homophilic recognition between membrane-bound and beadbound glyconectins were observed through three separate colored aggregates and selective staining of each letter only with 10 mM CaCl2. Both processes occurred at apparently similar rates for each of the three glyconectins. In control experiments with glyconectin 1 separately attached to pink, yellow and white beads, as expected, mixed color aggregates were formed upon addition of 10 mM CaCl2. In the absence of 10 mM CaCl2, bead aggregation did not occur either in the mixture of three glyconectins or of one glyconectin coated to three color beads. Ex vivo color coded cell-bead experiments

86

Fig. 6: Species-specific glyconectin to glyconectin interactions mediate bead-cell recognition and adhesion. Xenogeneic glyconectin selfrecognition in a mixture of glutaraldehyde-fixed cells and glyconectin-coated beads in SWT in the presence of 10 mM Ca2+. M. prolifera cells bearing glyconectin 1 were incubated with: glyconectin 1 (pink beads) (A), glyconectin 2 (yellow beads) (D), and glyconectin 3 (white beads) (G). H. panicea cells bearing glyconectin 2 were incubated with: glyconectin 1 (B), glyconectin 2 (E), and glyconectin 3 (H) color-coded beads. C. celata cells bearing glyconectin 3 were incubated with: glyconectin 1 (C), glyconectin 2 (F), and glyconectin 3 (I) color-coded beads (glutaraldehyde fixation changes cell colors, i.e. M. prolifera, orange to yellowish white; H. panicea, white to yellowish brown; and C. celata, brown to brownish orange. We did not observe differences in adhesion properties between fixed and live metabolically attenuated cells in a rotary assay.

showed that artificial beads covered with glyconectin glycans will co-aggregating in the species-specific manner only with homotypic cells having same glyconectin glycans (Fig. 6). Similar types of experiments were also done with purified glycans from all three species. Results obtained confirmed that self-non-self discrimination of GNs is based on selective carbohydrate to carbohydrate self-assembly (Misevic et al. 1987, Misevic and Burger 1993) which represents a novel mechanism complementary to well studied protein to protein and protein to carbohydrate interactions of adhesion and recognition molecules. Color coded bead experiment was also performed by overlaying agarose gel containing electrophoretically separated three glyconectins with color coated glyconectin beads. As shown in Fig. 7, after overnight incubation at room temperature, in the presence of 10 mM CaCl2 under gentle agitation, species specific staining of gel glyconectin bands identical to ones stained with Toluidine blue and Amido black showed that glyconectin to glyconectin interactions are highly species-specific (Guerardel et al. 2004, Misevic et al. 2004).

The combinations of the above described experiments demonstrate species-specific molecular self-recognition of glyconectins in an elementary reconstituted bead adhesion system which fully resembles glyconectin mediated cellcell recognition and adhesion. Thus, glyconectin glycans mediate self and non-self discrimination via selective glycan to glycan assembly in the initial step of sponge cell adhesion and xenogeneic recognition.

Evolution of the Porifera model system in research


In the second part of the past century, zoology and ecology research on Porifera was highly considered. Unfortunately, the same sponge model system was often neglected in the field of biochemistry and molecular biology. This research was put to the bottom of the list of priory and was classified as risky, marginal and not serious (e.g. comments that if possible this research should be avoided for the sake of the scientists and institutions involved). Fortunately, and in

87

References
Cauldwell CB, Henkart P, Humphreys T (1973) Physical properties of sponge aggregation factor. A unique proteoglycan complex. Biochemistry 12: 3051-3055 Curtis ASG (1962) Pattern and mechanism in the reaggregation of sponges. Nature 196: 245-248 Dammer U, Popescu O, Wagner P, Anselmetti D, Guntherodt HJ, Misevic GN (1995) Binding strength between cell adhesion proteoglycans measured by atomic force microscopy. Science 267: 1173-1175 Galstoff PS (1925) Regeneration after dissociation (an experimental study on sponges) I. Behavior of dissociated cells of Microciona prolifera under normal and altered conditions. J Exp Zool 42: 183221 Guerardel Y, Czeszak X, Sumanovski LT, Karamanos Y, Popescu O, Strecker G, Misevic GN (2004) Molecular fingerprinting of carbohydrate structure phenotypes of three porifera proteoglycanlike glyconectins, J Biol Chem 279: 15591-15603 Humphreys T (1963) Cell aggregation: chemical dissolution and in vitro reconstruction of sponge cell adhesions: I. Isolation and functional demonstration of the components involved. Dev Biol 8: 27-47 Jumblatt JE, Schlup V, Burger MM (1980) Cell-cell recognition: specific binding of Microciona sponge aggregation factor to homotypic cells and the role of calcium ions. Biochemistry 19: 1038-1042 Misevic GN (1989) Immunoblotting and immunobinding of acidic polysaccharides separated by gel electrophoresis. Methods Enzymol 179: 95-104 Misevic GN, Burger, MM (1986) Reconstitution of high cell binding affinity of a marine sponge aggregation factor by cross-linking of small low affinity fragments into a large polyvalent polymer. J Biol Chem 261: 2853-2859 Misevic GN, Burger MM (1990a) Involvement of a highly polyvalent glycan in the cell-binding of the aggregation factor from the marine sponge Microciona prolifera. J Cell Biochem 43: 307-314 Misevic GN, Burger MM (1990b) The species-specific cell-binding site of the aggregation factor from the sponge Microciona prolifera is a highly repetitive novel glycan containing glucuronic acid, fucose, and mannose. J Biol Chem 265: 20577-20584 Misevic GN, Burger, MM (1993) Carbohydrate-carbohydrate interactions of a novel acidic glycan can mediate sponge cell adhesion, J Biol Chem 268: 4922-4929 Misevic GN, Popescu, O (1995) A novel class of embryonic cell adhesion glycan epitopes is expressed in human colon carcinomas. J Mol Recognit. 8: 100-105 Misevic GN, Finne J, Burger MM (1987) Involvement of carbohydrates as multiple low affinity interaction sites in the self-association of the aggregation factor from the marine sponge Microciona prolifera. J Biol Chem 262: 5870-5877 Misevic GN, Guerardel Y, Sumanovski LT, Slomianny MC, Demarty M, Ripoll C, Karamanos Y, Maes E, Popescu O, Strecker G (2004) Molecular recognition between glyconectins as an adhesion selfassembly pathway to multicellularity. J Biol Chem 279: 1557915590 Misevic GN, Jumblatt JE, Burger MM (1982) Cell binding fragments from a sponge proteoglycan-like aggregation factor. J Biol Chem 257: 6931-6936

Fig. 7: Electrophoretic separation of sponge glyconectins. A. 0.75% agarose gel stained with 0.02% toluidine blue followed by 0.1% Amido Black 10B. a-c, GNs from M. prolifera GN1, H. panicea GN2, and C. celata GN3, respectively (10 g each). B. 0.75% agarose gel stained with color-coded fluorescent beads coated with GN1 (pink) (a), GN2 (green) (b), and GN3 (blue) (c) in the presence of SWT with 10 mM CaCl2.

contrast to the expectations of the official representatives of the scientific community, molecular- and cellular-oriented fundamental research on sponges - exemplified in this article by evolution of multicellularity, as well as by other reports in this book - have generated a vast body of knowledge of new structures, novel molecular mechanisms and new nanotechnologies. Consequently this interdisciplinary research on sponges, which integrates biology, physics, chemistry and mathematics, starts to gain deserved respect as measured by the appearance of publications in journals with high impact factors and the citations of these papers, and the level of attendance at the international conferences in the now clearly established interdisciplinary sponge field. In conclusion we are arguing that any model system is valuable if competent scientists can use it to develop and test original ideas to help solve fundamental scientific questions.

Acknowledgments
This work is supported by private funds of Gradimir Misevic, Swiss National Science Foundation, European Union 6th Framework Network of Excellence Nanobeams, CNRS France, University of Rouen France, Rgion Normandy France, University of Lille France, and region Nord pas de Calais.

88

Moscona AA (1968) Cell aggregation: properties of specific cellligands and their role in the formation of multicellular systems. Dev Biol 18: 250-277 Muller WEG, Zahn RK (1973) Purification and characterization of a species-specific aggregation factor in sponges. Exp Cell Res 80: 95-104 Papakonstantinou E, Misevic GN (1993) Isolation and characterization of a new class of acidic glycans implicated in sea urchin embryonal cell adhesion. J Cell Biochem 53: 98-113 Popescu O, Misevic GN (1997) Self-recognition by proteoglycans. Nature 386: 231-232

Spillmann D, Hard K, Thomas-Oates J, Vliegenthart JF, Misevic G, Burger MM, Finne J (1993) Characterization of a novel pyruvylated carbohydrate unit implicated in the cell aggregation of the marine sponge Microciona prolifera. J Biol Chem 268: 13378-13387 Spillmann D, Thomas-Oates JE, van Kuik JA, Vliegenthart JF, Misevic G, Burger MM, Finne J (1995) Characterization of a novel sulfated carbohydrate unit implicated in the carbohydratecarbohydrate-mediated cell aggregation of the marine sponge Microciona prolifera. J Biol Chem 270: 5089-5097 Wilson HV (1907) On some phenomena of coalescence and regeneration in sponges. J Exp Zool 5: 245-258

Porifera research: Biodiversity, innovation and sustainaBility - 2007

89

Porifera: an enigmatic taxon disclosed by molecular biology/cell biology


Werner E.G. Mller(*), Isabel M. Mller
Institut fr Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universitt, Duesbergweg 6, D-55099 Mainz; Germany. tel.: +49-6131-39-25910; fax.: +49-6131-39-25243. wmueller@uni-mainz.de Abstract: It was a long and painful scientific process to position the most enigmatic metazoan phylum, the Porifera, into the correct phylogenetic place among the eukaryotes in general and the multicellular animals in particular. This well studied taxon, the sponges, was first grouped to the animal-plants or plant-animals then to the Zoophyta or Mesozoa and finally to the Parazoa. Only after molecular biological techniques became available, was it possible to place the Porifera monophyletically with the other metazoan phyla, justifying a unification of all multicellular animals to only one kingdom, the Metazoa. The first strong support came from the discovery that cell-cell and cell-matrix adhesion molecules cloned from sponges (the main work was performed with the demosponges Suberites domuncula and Geodia cydonium) and subsequently expressed, share high sequence similarity/functional identity with the corresponding molecules of other metazoans. Besides these evolutionary novelties for Metazoa, the sponges possess also morphogens and transcription factors in common with the other metazoan phyla. Surprising was the fact that even those elements exist in Porifera which are characteristic for pattern and axis formation (e.g. Frizzled receptor). The cell culture system from sponges, the primmorphs, provided a suitable tool for the understanding of morphogenetic events. Furthermore, stem cell marker genes could be isolated, underscoring that sponge cells have the capacity to differentiate. Hence, we can conclude that the body plan of the Urmetazoa must have been constructed by similar genomic regulatory systems as found now in higher Metazoa. In a relatively short period, within approximately 200 million years, from 700 to 500 million years ago, the basic pathways are established which allowed the transition to complex, individual metazoan systems through the formation of adhesion molecules; based on the development of a complex immune system and of the apoptotic machinery an integrated system, the Urmetazoa, could be reached. Hence, from the insights into the genome repertoire of some representative species within the Porifera it became overt that the ancestor of all multicellular animals, the Urmetazoa, was provided with a surprisingly high complexity of structural and regulatory molecules. These data caused a paradigmatic change; the Porifera are complex and simple but by far not primitive. The different hurdles which had to be taken to state that the Porifera are not Parazoa [alongside animals] or Mesozoa [middle animals], but Metazoa are reviewed here with special emphasis on the contributions coming from molecular biological approaches. Keywords: Evolution; Metazoa; molecular phylogeny; Urmetazoa

Introduction
Sponges are sessile filter-feeding organisms with an extremely effective and complex network of water-conducting channels and choanocyte chambers lined with flagellated choanocyte cells. Until not too long ago the ground space, the mesohyl, between the external pinacoderm and the internal choanoderm [endopinacoderm], the two cell layers that surround sponges, was thought to consist of mostly functionally independent cells (Pechenik 2000). Such a set up would result in the formation of amorphous, unorganized creatures (Pechenik 2000). However, during the last few years the existence of cell surface-bound receptors and their extracellular as well as intracellular segments could be proved and it was concluded that the sponges possess molecules that allow the establishment of a distinct body plan. The discovery of the metazoan novelties first developed during evolution in sponges which comprise the cell-cell/-matrix, signal transduction-, immune-, neuronal- and morphogenetic

molecules helped to overcome the long-standing debate whether sponges are specialized protists or true Metazoa (Hyman 1940). The phylum Porifera is subdivided into three classes, the Hexactinellida, the Demospongiae and the Calcarea. Until very recently, the phylogenetic positions of these classes remained unresolved. Like any other metazoan also sponges have a defined Bauplan; this has most artistically been illustrated by Haeckel (1872). Unlike other Metazoa adult sponges are considered to have no pronounced anterior/posterior polarity; surely they do not have a dorsal ventral axis. In higher metazoans the patterning along the anterior-posterior axis is regulated among others by the famous family of homeobox genes. Homeobox-related genes that have been identified in sponges display, however, a more general function as transcription factors active in all sponge cells (Seimiya et al. 1998). The body plan of metazoans is defined and its orientation is fixed by the inorganic skeleton. In most sponges this

90

solid support, the spicules, is composed of hydrated, amorphous, noncrystalline silica [SiO2/H2O] as in the classes of Demospongiae and Hexactinellida, or of calcium carbonate [CaCO3] as in the class of Calcarea. The secretion of spicules occurs in specialized cells, the sclerocytes. While in Demospongiae silica is deposited around an organic filament, no organic axial structure is found in the spicules from Calcarea. Exciting new data on spicule formation and the enzymatic silicatein-mediated biosynthesis of silica has been summarized recently (Mller et al. 2005, 2006). In the following, the different steps which led to an understanding of the genetic repertoire of sponges and to the tuned expression of structural and morphogenetic molecules that form the basis for sponge individuals and their body plans (Bauplan) are discussed.

Position of the Porifera among the metazoans


The presently used phylum name Porifera dates back to 1836 (Grant 1836) and was originally Poriphera/Poriphora. The impact of sponges in biology was always remarkable, because they are the (major) taxon which can shed light on the evolution and the origin of the Metazoa. There was, however, one obstacle; before 1850 hardly any scientist had seen sponges in their natural environment, especially not in the ocean. After the first extensive expeditions at the end of the 19th century, e.g. the Challenger expedition, this situation changed. In 1876 Campbell (Campbell 1876), wrote in his log-letters, those beautiful glass-rope sponges, Hyalonema etc., have been found by our researchers to be the most characteristic inhabitants of the great depths all over the world, and with them ordinary siliceous sponges, some of which rival Hyalospongiae in beauty. Then Sollas (1888), who worked on the demosponge collection from the Challenger expedition wrote Now that von Lendenfeld has pointed it out nothing can be clearer, and no one, as he remarks, will raise any objection to the statement that the sponges are evidently Metazoa. But nevertheless many spongiologists were still under the impression of earlier works, which claimed that sponges are separate independent entities in systematics and should be looked upon as e.g. plant-animals (Esper 1794), animal-plants (Pallas, 1787) or Mesozoa (DeLage and Hrouard 1899). Today it should be accepted that this phylum must be grouped to the Metazoa, being qualitatively identical to the higher Metazoa [or, if pressed, to the Eumetazoa] and having only quantitatively different characters as other metazoan phyla (Mller 2001). In the Proceeding of the Second Sponge Symposium from 1978, Rasmont (1979) wrote At least five main differences can be recognized between the development of sponges and that of other Metazoa; among them morphogenetic interactions between sponge cells depend largely on long-ranged chemical messengers and the genetic individuality appears to be very different of that of other Metazoa. Around the year 1990 it became obvious that a further clarification of the phylogenetic position and the (potential) richness of the sponge morphological and functional characters would not be possible unless the basic genomic regulatory systems were known. As a consequence, sequencing of ribosomal genes was introduced to obtain more phylogenetic information. The 5S rRNA proved to be little reliable (Doolittle

and Brown 1994), while data from 18S rRNA as well as 28S rRNA molecules allowed phylogenetic inference (Halanych 1991). However, this approach revealed conflicting results. Based on sequence data of 18S rRNA alone both a polyphyletic (Field et al. 1988) and a monophyletic origin of Metazoa could be concluded (Christen et al. 1991). As reasons for this controversy, the following possibilities were claimed; (i) the molecular phylogenetic methods, basing on sequence data of rRNA, have reached their limits, (ii) there is a hidden paralogy, and/or (iii) lateral gene transfer is responsible. The introduction of molecular cloning of genes coding for informational proteins, increased in a rapid, self-accelerating manner our knowledge on the evolution to the Porifera; it furthermore supported the subdivision of this phylum into the three classes, Hexactinellida, Demospongiae and Calcarea. It is now clear that the Porifera must be grouped together with the other metazoan phyla into one monophyletic unit (Mller et al. 1994a, Mller 1995). Consequently the hypothetical ancestor of all metazoan phyla was named Urmetazoa (Mller 2001, Mller et al. 2001). Even though the present-day data on the strikingly high similarity/homology of sponge proteins with related molecules from evolutionary younger metazoan phyla are overwhelming, the earlier view of the sponge origin can still be found in the literature (e.g. Pechenik 2000). In the following, the different steps in the elucidation of the beauty of the sponge body plan, based on a complex genetic network, with the main focus on demosponges (mainly by using Suberites domuncula (Olivi, 1792) and Geodia cydonium (Jameson, 1811)) are outlined. The discussion is restricted primarily to results which are based on molecular and cell biological studies, because data obtained with cytological/ morphological techniques proved to be insufficient. The basic turning point to the modern integrating view of sponge biology/ body plan started with the paper of Mller et al. (1994a [Fourth Sponge Symposium in 1993]), describing the molecule [lectin] that for the first time indicated a monophyly of all metazoan animals, including the Porifera. Another publication of Mller et al. (1994b [3rd Brazilian Symposium on Extracellular Matrix, Angra dos Reis (Brazil), 1994]) formulated this hypothesis.

Cell-cell and cell-matrix adhesion in sponges (1973)


Sponges have long been used as classic model for basic studies to understand metazoan cell-cell adhesion. Wilson (1907) introduced this system to experimental biology where it then became a traditional model to study both cell-cell and cell-matrix adhesion (reviewed in: Burger and Jumblatt 1977, Mller 1982, Mller et al. 1988). The marine demosponges Microciona prolifera Bowerbank, 1862, G. cydonium and S. domuncula have been most thoroughly studied. In 1973 the first extracellular particle, termed aggregation factor [AF], which promotes the species-specific aggregation of sponge cells, had been described from G. cydonium (Mller and Zahn 1973) (Fig. 1). Shortly after the second AF was identified in M. prolifera (Henkart et al. 1973). The AF was characterized as high-molecular weight complexes with a sedimentation coefficient of 90S which are assembled from a series of proteins that are covalently and noncovalently bound to the core structure (summarized in: Mller 1982) (Fig. 1A and B). In its native form the AF appears as a sphere with

91

localized on the cell periphery. These systems are prerequisites for the establishment and stabilization of the functional arrangement of cells in the organism (Mller 1982).

Monophyletic origin of all metazoan phyla (1993)


Phylogenetic relationships especially with regard to the phylum Porifera had been formulated, but often uncertainties remained because of difficulties to distinguish between convergent or divergent features. During the 4th International Porifera Congress in 1993, we described the first protein-coding sequence, a galectin from the sponge G. cydonium; galectins exist only in Metazoa (Mller at al. 1994a). In parallel, it was stated/reported that sequencing of the 18S rRNA gene is not suitable to resolve deep branches in the phylogenetic tree and the data (rRNA-sequencing data) fail to support the monophyly of sponges and other Metazoa stronger than the monophyly of sponges and plants (Rodrigo et al. 1994). Our approach relied on amino acid [aa] sequence data obtained from genes identified mainly from the marine sponges G. cydonium and S. domuncula. We have selected mainly genes that code for proteins which are features of multicellularity, such as (i) extracellular adhesion molecules (galectins) with their typical carbohydrate binding site [LH(F) NPR~(G)~V~NT~(G)~W~(T)E~FPF], found in all vertebrate S-type lectins, and also found in the sponge lectin (Pfeifer et al. 1993), (ii) cell surface receptors, like integrin (Pancer et al. 1997) and speract receptors Scavenger Receptor, CysteineRich domain (SRCR) containing receptors (Blumbach et al. 1998), (iii) signal transduction molecules, like the receptor tyrosine kinase [RTK] (Schcke et al. 1994a), the serine/ threonine [Ser/Thr] kinases (Kruse et al. 1997, 1998), or (iv) transcription factors, the serum response factor (Scheffer et al. 1997). These molecular biological and cell biological findings have been taken as one major clue for the, now established, view that Metazoa, including Porifera, are of monophyletic origin, with the Urmetazoa as the hypothetical ancestor (Fig. 3). Based on these sequence data it was reasonable to place the Porifera into the kingdom Animalia together with the (Eu)Metazoa (Gamulin et al. 1994, Mller et al. 1994b, Mller 1995, Mller [ed.] 1998a, 1998b, Mller 1998c). This monophyletic origin of all metazoan phyla was subsequently often confirmed. Especially by taking sponge genes that code for RTK it is now established that modular proteins that were composed by exon-shuffling, are common to all metazoan phyla.

Fig. 1: Cell adhesion system in sponges. The aggregation factor [AF] from G. cydonium. A. Electron micrograph of the native AF; preparation shadowed with platinum. B. Electron micrograph of the core structure AF; shadowed with platinum. C. Working model in 1976 for the AF-aggregation receptor (AR)-mediated cell recognition in G. cydonium. The sunburst structure comprises the core of the AF (after Mller 1976).

a diameter of 1000 and a concave cup structure (Fig. 1A). Treatment of the AF with detergents yields a core structure that appears as sunburst with a circular center (diameter 1,000 ) and 25 radiating arms (Fig. 1B). In the presence of Ca2+ the AF interacts with a membrane component, termed aggregation receptor [AR]. A 140 kDa polypeptide was found to participate in the AF-mediated reaggregation process. This polypeptide interacts with a galectin that links individual AF molecules to the AR at the plasma membrane and consequently bridges two cells together (Fig. 1C). The major obstacle to the identification of the molecules participating in the very complex and dynamic cell-cell and cell-matrix recognition in sponges was the fact that the underlying molecules involved had not been obtained by molecular cloning. Even until 1994 it remained uncertain if the sponge adhesion molecules display high sequence relationship to functionally related molecules in higher Metazoa (Gamulin et al. 1994). With cloning of a galectin sequence (Pfeifer et al. 1993, Mller et al. 1997) as the first cell-cell adhesion molecule, and the integrin sequence as the first cell-matrix adhesion receptor in G. cydonium (Pancer et al. 1997, Wimmer et al. 1999a), it became overt that sponges contain molecules highly related to those known to also promote adhesion in Protostomia and Deuterostomia (Fig. 2). These molecular biological data confirmed earlier cell biological observations that sponges have developed a number of recognition systems

Evolutionary age of the Porifera (1994)


Also in 1994 (Schcke et al. 1994b) it was possible, based on the number of non-synonymous substitutions in sponge sequences, to estimate the time when the sponges diverged from the common ancestor of all metazoans; approximately 650-665 million years ago (MYA). Although the Porifera are the oldest Metazoa by fossil documentation, their earliest record is only from the Vendian about 600 MYA (reviewed in: Mehl et al. 1998). Hence, it still leaves us with a large stratigraphical gap down to the assumed age of the monophylum Metazoa,

92

Fig. 2: Different stages of morphogenesis in sponges (schematic model based on studies with the demosponges G. cydonium and S. domuncula). First, cell-cell and second, cell-matrix adhesion molecules allow the mechanical interaction between adjacent cells or cells and extracellular matrix molecules. AF in the extracellular space interacts with the AR and mediate the first mechanical contact. The AF-mediated cell-cell recognition is species-specific and very likely controlled by the complex SRCR/SCR-AR, that is embedded in the plasma membrane. Third, after this primary AF-AR-AF contact intracellular signal transduction pathways are activated, resulting in a selective gene expression. New insertion of adhesion receptors into the plasma membrane follows; e.g. integrin and other receptors, involved in tissue and skeleton formation. Together with these receptors their ligands are synthesized, e.g. collagen, molecules containing the fibronectin FN3 modules, or mucin-like molecules, which establish the cell-matrix adhesion system. During this phase growth factors are synthesized, e.g. the precursor and the mature epidermal growth factor (EGF), which interact with the newly synthesized receptors. Fourth, solute molecules are released which initiate axis formation. Fifth, after completion of these phases pattern formation can start, a process which is controlled by morphogenetic cell-surface receptors, like Frizzled, and by transcription factors, e.g. Forkhead. Also LIM-class homeodomain transcription factors are activated.

800 MYA, from which the Porifera are supposed to be the first group that split off. In molecular evolution an unsolved question is the validity of the molecular clock hypothesis which implies that the rate of molecular evolution is nearly constant per year among different evolutionary lineages. Consequently, this rate should be linked with the mutation rate and hence is closer correlated with the number of generations per unit time than to time itself. However, the standard value for the average rate of nonsynonymous substitutions in DNA can vary between 0.6 and

0.9 x 10-9 per site and year. Therefore, the calculated time of divergence of the different phyla might vary as well. Applying these rates of non-synonymous substitutions to the conserved amino acid (aa) stretches in the tyrosine kinase domains of the RTK and the sequences of two sponge S-type lectins, it could be estimated that the sponge molecules diverged from the common ancestral gene approximately 800 MYA. This figure is in good agreement with the generally accepted time of divergence found by traditional methods (Reitner and Wrheide 2002).

93

Fig. 3: Phylogenetic position of the Porifera between the Urmetazoa and the Urbilateria. The major evolutionary novelties which have to be attributed to the Urmetazoa are those molecules which mediate apoptosis and control morphogenesis, the immune molecules and primarily the cell adhesion molecules. The siliceous sponges with the two classes Hexactinellida and Demospongiae emerged first and finally the Calcarea, which possess a calcareous skeleton, appeared. These three classes of Porifera are living fossils that provide a reservoir for molecular biological studies. The Archaeocyatha, sponge related animals with a calcareous skeleton, became extinct. The Calcarea are very likely a sister group of the Cnidaria. From the latter phylum the Ctenophora evolved which comprise not only an oral/aboral polarity but also a biradial symmetry. Finally the Urbilateria emerged from which the Protostomia and the Deuterostomia originated. Very likely the Urmetazoa emerged between the two major snowball earth events, the Sturtian glaciation (710 to 680 MYA) and the Varanger-Marinoan ice ages (605 to 585 MYA). In the two poriferan classes Hexactinellida and Demospongiae the skeleton is composed of amorphous and hydrated silica, while the spicules of Calcarea are composed of Ca-carbonate. The latter biomineral is also prevalent in Protostomia and also in Deuterostomia. In vertebrates the bones are composed of Ca-phosphate [apatite]. The autapomorphic character for the Demospongiae is the spicule-synthesizing enzyme silicatein.

Intracellular signal transductions (2003)


With the increasing awareness of monophyly of all metazoans several pressing questions arose. The major enigma in development is pattern formation. Detailed investigations led to an understanding of the genetic networks constructing and controlling body plan formation in metazoan crown species and recently also answers for the body plan of sponges could be given (see: Mller 2005); most of these studies were performed with G. cydonium and S. domuncula. The structural and functional molecules were cloned and expressed. The cellcell and cell-matrix adhesion molecules found in sponges share amazingly high sequence and functional similarity to those of higher Metazoa (Fig. 2). The extracellular binding sites to

the ligands as well as the intracellular domains of these cell membrane receptors remained conserved throughout the animal kingdom. Functional studies proved that the receptors are provided with the properties of outside-in signaling (Wimmer et al. 1999b). One major step forward was the development of the primmorph system, a technique to grow sponge cells in culture (Mller et al. 1999a). With application of this system, solute morphogenic factors (e.g. myotrophin; Schrder et al. 2000), or secreted molecules (e.g. epidermal growth factor; Perovi-Ottstadt et al. 2004), as well as their receptors, which are involved in axis formation (Frizzled receptor; Adell et al. 2003b), and transcription factors that are required for polarity formation (e.g. the organizer-specific factor LIM homeobox

94

protein; Wiens et al. 2003b, or Forkhead; Adell et al. 2003a) were discovered.

The phylogenetic relationship of the sponge classes (1997)


It is generally agreed that multicellularity in Plantae, Fungi and Metazoa arose in the Proterozoic approximately 1,000 MYA. As the earliest Metazoa the Porifera evolved with the major taxa Hexactinellida, Demospongiae and Calcarea. The skeletal elements of the sponges, the spicules are composed in Demospongiae and Hexactinellida of hydrated, amorphous, noncrystalline silica while those of the class Calcarea are formed from calcium carbonate. The Demospongiae and Hexactinellida are the only two taxa within the kingdom of Metazoa which utilize silica instead of calcium in their mineral skeleton; calcium is otherwise the dominant inorganic skeletal component. According to fossil records, the Hexactinellida appeared first while Demospongiae and Calcarea developed later (Reitner and Mehl 1995). It can be argued that Porifera might not have been the first metazoan phylum which evolved; however, they are the only still extant witnesses of an evolutionary step that occurred during the maturation of the Metazoa, near the Proterozoic-Phanerozoic boundary close to 800 MYA. In this respect they have been considered as living fossils (Mller 1998c). Our group has analyzed genes of sponges, the Demospongiae S. domuncula and G. cydonium, the Calcarea, Sycon raphanus (Schmidt, 1862), as well as from the Hexactinellida, Aphrocallistes vastus (Schulze, 1887) and Rhabdocalyptus dawsoni (Lambe, 1892) in order to obtain an insight into the genome organization as well as the function of genes coding for functional proteins (Fig. 3). Hexactinellids are syncytial rather than cellular; approximately 75% of the tissue forms a multinucleate syncytium while the remaining tissue consists of uninucleate cells which are connected to the syncytium by perforated (aqueous) plugged junctions (Leys 1995). In two early approaches to resolve the phylogenic relationships of the three sponge classes (Schulze 1887, von Lendenfeld 1889) the Calcarea were considered to form the basis of the sponge taxon. While Schulze (1887) suggested that the Hexactinellida represent together with the Calcarea the earliest classes, von Lendenfeld (1889) proposed that the Demospongiae group together with the Calcarea and the Hexactinellida appeared later in evolution. More recent cladistic analyses including morphological data suggested that Porifera are monophyletic. However, for respect to the phylogenetic relationships among the sponge classes two contrary views have been presented. One considers cellular sponges, the Calcarea and the Demospongiae, as the sister group of the syncytial sponges, the Hexactinellida (Mehl and Reiswig 1991), while in the opposite view the siliceous sponges, Hexactinellida and Demospongiae, form the sister group to the calcareous sponges, the Calcarea (Bger 1988). Again, the sequencing data from rDNA were not conclusive enough to solve this question (Medina et al. 2001). On the other hand, studies on several sequences of informative molecules from the three poriferan classes, as well as detailed analyses of housekeeping proteins, e.g. heat shock

protein (Koziol et al. 1997) or -tubulin (Schtze et al. 1999), and of proteins involved in signal transduction, e.g. Ser/Thr kinases (Kruse et al. 1997 and 1998) or calmodulin (Schtze et al. 1999), revealed that among the three classes of Porifera the Hexactinellida are the phylogenetically oldest taxon, while the Calcarea is the class closest related to higher metazoan phyla (Fig. 4). Furthermore, data especially from studies with a series of Ser/ Thr kinases supported the position of Calcarea as sister group to higher metazoan phyla (Mller et al. 1998). The branching order originating from ancestral unicellular eukaryotes via Viridiplantae-Fungi to Porifera, the simplest metazoans, follows both the published fossil data and the sequence data obtained (Fig. 4). Since the Hexactinellida are syncytial animals and since our analyses indicate that they branched off from a common ancestor earlier than other sponges, this could imply that multicellularity came about by the division of a multinucleate syncytium rather than by aggregation of formerly single cells. However, it is also possible that the syncytial nature of the Hexactinellida reflects a reduction of a previously multicellular stage by fusion to form a syncytium. Additional support for an early origin of the Hexactinellida comes from paleontological evidence, which shows that the Hexactinellida were present in the Early Cambrian while the Calcarea and the Demospongiae arose later, in the Middle Cambrian (Reitner and Mehl 1995). The oldest sponge spicules found to date (approximately 600 MYA) come from China, and are mainly monaxonal spicules, but some also have definite crosses, which are typical of triaxones from hexactinellids. These data show that within the phylum Porifera, the class Hexactinellida diverged first from a common ancestor; while Calcarea and Demospongiae appeared later; the Calcarea are hence the sister group to the Cnidaria [paraphyletic relationship].

Primmorphs the sponge in vitro cell culture system (1998)


Sponge cells can be cultivated in vitro as primmorphs (Custdio et al. 1998, Mller et al. 1999a); they can be defined as a special form of in vitro cell culture, which allows the formation of a three-dimensional organization of aggregates that contain proliferating and differentiating cells. We have focused on the formation of primmorphs from the demosponges S. domuncula, Dysidea avara (Schmidt, 1862) (Mller et al. 2000) and occasionally G. cydonium. For this technique, sponge tissue samples are separated into single cells under shaking in Ca2+ and Mg2+-free artificial seawater containing EDTA. The cells are transferred after washing into medium, supplemented with Ca2+, Mg2+ and antibiotics. Immediately after transfer, the single cells form small ~20 cells containing aggregates which grow in size during the subsequent three days to 1,000 m large cell clumps. After usually five days primmorphs are formed. As the basal medium natural seawater is enriched to 0.2% with RPMI 1640medium. This innovative step has been patented (granted US patent 6,664,106). Primmorphs are characterized by the presence of proliferating cells and a distinct histology. The amount of DNA-synthesizing/ proliferating cells present in primmorphs reaches 20% to 30% depending on the age of the primmorphs. The diameter of the

95

Also the iron concentration, as Fe(+++), should be increased to 30 M for optimal growth conditions of primmorphs. One further growth promoting protein has been isolated from S. domuncula which was shown to stimulate proliferation of sponge cells; the myotrophin-like polypeptide (Schrder et al. 2000). Recombinant sponge myotrophin stimulated the overall protein synthesis by 5-fold (Schrder et al. 2000). Besides these chemical factors physical factors such as water current are critically important for primmorph survival and cell growth (reviewed in: Schrder et al. 2003). Incubation of the primmorphs under such conditions resulted in the formation of canals in the primmorphs and the expression of the homeobox gene Iroquois (Perovi et al. 2003).

Immune system (1998)


Fig. 4: Branching order of the three poriferan taxa at the basis of the Metazoa. During the transition from the common ancestor with the Fungi, the Urmetazoa evolved from which the Hexactinellida branched off first, followed by the Demospongiae. The third sponge taxon, the Calcarea, appeared which forms the sister group to the Urbilateria. This branching order, now well established, was first elucidated on molecular terms using representative protein sequences encoding the protein kinase C (PKC; the catalytic domain has been used for the analysis; Kruse et al. 1997). Sequences from the following organisms have been used: Metazoa cPKC from the deuterostomes Xenopus laevis [frog - cPKC_XL] and Lytechinus pictus [sea urchin - cPKC_ LP], from the protostomes cPKC from Drosophila melanogaster [fruit fly - cPKC_DM] and Aplysia californica [mollusc, cPKC_AC], also from Caenorhabditis elegans [cPKC_CE] and those from the sponges of the classes (i) Demospongiae, G. cydonium [cPKC_GC] and S. domuncula [cPKC_SD], (ii) Calcarea, S. raphanus [cPKC_SR], and (iii) the Hexactinellida, R. dawsoni [cPKC_RD] as well as from the yeast Saccharomyces cerevisiae [PKC_SC]. The phylogenetic tree was constructed on the basis of aa sequence alignment. Computing of the sequences by using the procedure of neighbour-joining applying the Neighbor program from the PHYLIP package PROTPARS [Protein-Parsinomy] (Felsenstein 1993) as described (Kruse et al. 1997).

cell aggregates increases steadily after approximately three days incubation; after a total treatment/incubation for five days primmorphs are formed from cell aggregates. During the phase of primmorph formation the aggregates contract to round 1 to 5 mm large bodies, leaving behind detritus and dead cells. In the initial phase the primmorphs remain round-shaped but after incubation of longer than three to four weeks many of them attach to the bottom of the culture dish. Microscopic analysis of the sections through primmorphs revealed that the cells present in the interior are surrounded by an almost complete single-cell layer of epithelial-like cells. The cells that compose the squamous epithelium of the primmorphs are pinacocytes as judged from their flattened, fusiform extensions and their prominent nucleus. The cells inside the primmorphs are primarily spherulous cells while the others may be termed amoebocytes and archaeocytes. Growth conditions could be optimized by supplementing the natural seawater/0.2% of RPMI1640medium with silicate. Natural seawater contains < 5 M silicate; however, the optimal concentration of silicate for cell proliferation and spicule formation is 60 M (Krasko et al. 2000, 2002).

The important contribution of Metchnikoff (1892) was the description of the phagocytotic activity of sponge cells, archaeocytes, as a mechanism to eliminate non-self particles and, even more advanced, to encapsulate the foreign material within cell aggregates of the sponge prior to the elimination by ablation. These abilities of sponges were discussed by Metchnikoff in the context of inflammation processes, proceeding in Metazoa during infection. The major step in the elucidation of the cellular mechanisms by which the sponges eliminate non-self and accept self came from elegant experimental transplantation studies. Smith and Hildemann in their extensive review (Smith and Hildemann 1986) have grouped sponge alloimmune responses into two major rejection processes. Some species may form barriers to separate from non-self tissue; e.g. the marine sponge Axinella verrucosa (Esper, 1794) (Buscema and van de Vyver 1983) or the freshwater sponge Ephydatia muelleri Lieberkhn, 1855, while others may react by cytotoxic factors which destroy the transplant; e.g. the marine sponges Callyspongia diffusa Sollas, 1885 (Hildemann et al. 1979) or G. cydonium (Pfeifer et al. 1992). The breakthrough in the discovery that immune mechanisms in sponges are highly similar to those, found in other metazoan phyla, came again after application of molecular biological techniques (see: Mller et al. 1999b). Defense against microbes/parasites: Almost all marine demosponges contain bacteria. Until now no conclusive data are available to say which bacterial strains are symbiotic and which are parasitic. One report at least suggests that the number of bacterial strains that are symbiotic or commensalic is limited (Althoff et al. 1998). All specimens of the marine demosponge Halichondria panicea (Pallas, 1766) collected from the Baltic Sea, the North Sea, as well as in the Mediterranean Sea were found to harbor one defined bacterium which belongs to the taxon of Roseobacter/Rhodobacter. Based on this finding it was suggested that this bacterial strain accepts at least a commensal relationship with the host. First data on the molecular mechanism by which the host (sponge) might discriminate between symbiotic or commensalic and parasitic bacteria have been obtained. We could demonstrate that defined bacterial strains can be engulfed by specific sponge cells, the bacteriocytes (Bhm et al. 2001). Furthermore, protein synthesis in tissue from S. domuncula is inhibited after incubation with the bacterial endotoxin lipopolysaccharide (LPS; Bhm et al. 2001). Since

96

Ser/Thr directed mitogen-activated protein (MAP) kinases are essential components of the LPS-mediated pathway, evidence of activation of these kinases in response to LPS was sought. Molecular biological and immunological studies confirmed that these pathways also exist for the Porifera, indicating that such defense pathways are highly conserved between sponges and humans (Bhm et al. 2001). One powerful tool to eliminate microbes is intracellular digestion. This cellular defense mechanism against foreign invaders is well developed from Porifera to insects and humans. Sponges possess specialized amoeboid cells, the archaeocytes (Metchnikoff 1892), which have in the past been considered as macrophages of sponges. Mammalian macrophages are the first cells to encounter non-self material. They express several receptors, scavenger receptors, that bind to bacteria or their constituents, and hence act as key molecules in innate immunity. Among them is the type I macrophage scavenger receptor which comprises highly conserved SRCR domains. With regard to sponges, molecules comprising SRCR domains have been discovered first in G. cydonium (Blumbach et al. 1998). Data strongly suggest that sponges comprise SRCRdomain containing cell-surface molecules which might be involved in the recognition of bacteria. In addition, it is likely that the ingested non-self bacteria are killed by both an oxidative and a nonoxidative (enzymatic) mechanism. Several cDNAs coding for lysosomal enzymes, have been isolated from sponges as well. Very recently, a further (putative) defense system against invading bacteria and/or viruses has been detected in Demospongiae: the (2-5)oligoadenylate synthetase [(2-5)A synthetase] system. In mammalian organisms, the (2-5)A synthetase(s) catalyzes the synthesis of a series of 2-5-linked oligoadenylates, termed (2-5)A [= pppA(2p5A)n [pnAn]] from ATP (Hovanessian 1991). In turn, (2-5)A acts as an allosteric activator of a latent endoribonuclease, the RNase L, which degrades single-stranded viral or cellular RNA. In mammalian organisms the (2-5)A system is activated by interferons. The first sponge species studied that was found to display higher levels of (2-5)A oligoadenylate synthetase and its products than vertebrate cells (Kuusksalu et al. 1995) was G. cydonium. The sponge (2-5)A synthetase was cloned (Wiens et al. 1999). This enzyme as well as its products are present in sponges and in the deuterostome lineage, but absent in protostomes. Recently, functional assays elucidated the role of the (2-5)A synthetase in sponges, especially with respect to a potential infection with foreign, pathogenic microorganisms. The sponge cellular system, which proved to be suitable for this approach, are the sponge primmorphs. The experiments showed that primmorphs synthesized (2-5)A in larger amounts if they were incubated with LPS, suggesting an activation of the synthetase through a LPS-initiated pathway (Grebenjuk et al. 2002). Histo(in)compatibility responses in sponges on tissue level: Studies of histo(in)compatibility response in sponges have been performed for 30 years. Initially it was reported that sponges have only a low capacity for allorecognition (Moscona 1963). However, after defining the system, it became apparent that sponges have a very high degree of precision when discriminating between self/self and self/nonself. We applied two transplantation techniques for our studies:

the insertion technique for G. cydonium and the parabiosis method. From G. cydonium tissue, pieces were removed with a cork borer from one specimen and were inserted into holes in the recipients (insertion technique) (Pancer et al. 1996; reviewed in: Mller and Mller 2003). All autografts fused and eventually no boundary line was seen; in contrast allografts initially fused together, but after approximately 3 to 5 days the rejected graft tissue formed a pronounced demarcation boundary and underwent apoptotic/necrotic degeneration and finally resorption. Histo(in)compatibility responses in sponges on cellular level: A cellular assay was developed to allow analysis of the histo(in)compatibility reactions on a cellular level (Mller et al. 2002). The basis of the assay was developed following the establishment of the primmorph system. In the mixed sponge cell reaction (MSCR) assay dissociated cells either from the same individual (autogeneic MSCR) or from different individuals (allogeneic MSCR) were mixed at equal cell concentrations. If cells from the same individual were mixed, autogeneic MSCR, 2 mm large aggregates were formed during the initial two days of incubation, which finally became 5 to 10 mm large primmorphs. In assays using cells from different specimens, they did not form single primmorphs but separated after two days, indicating that during the allogeneic MSCR the cells recognize non-self and form individual-specific aggregates. Molecules involved in histocompatibility response of sponges: Using transplantation models from both G. cydonium and S. domuncula (Mller et al. 1999b) it was established that macrophage-derived cytokine-like molecules are activated during allograft rejection. Among those sponge cytokines activated is the allograft inflammatory factor 1, a factor which has been described in rats and was identified as a cytokineresponsive macrophage molecule. The cDNA encoding the putative allograft inflammatory factor 1 (AIF-1) like molecule from S. domuncula has been cloned (Kruse et al. 1999). A strong upregulation has been determined in the rejection zone from allografts (Kruse et al. 1999). In parallel with this change in expression, a second characteristic molecule was identified which resulted in increased expression of the Tcf-like transcription factor (TCF) after transplantation in S. domuncula (Mller et al. 2006). Also the sponge TCF polypeptide shares highest similarity to those protostome and deuterostome transcription factors that are involved in diverse developmental processes. Further molecules/factors very likely involved in histo(in)compatibility reactions are the glutathione peroxidase and the endothelial-monocyte-activating polypeptide (EMAP). In vertebrates EMAP (type II) causes cell activation and expression of adhesion molecules in endothelial cells as well as in monocytes and granulocytes from human and mouse resulting in angiogenesis. The putative EMAP-related polypeptide was cloned from the marine sponge G. cydonium; it has a deduced molecular mass of 16 kDa and shows high sequence similarity (again) to the human and murine EMAP (Pahler et al. 1998). The glutathione peroxidase (GPX) is activated in humans/ vertebrates during the early phases of inflammation that occur during graft recognition or during wound healing in mammals when reactive oxygen species (ROS) are formed. It is the

97

major enzyme involved in the detoxification of ROS during these processes. The cDNA encoding the putative sponge GPX is known from S. domuncula (Kruse et al. 1999). As in the previous experiments using the AIF-1 like molecule from S. domuncula, the expression of the gene encoding the GPXrelated protein was low in the controls. However in the zones between grafts (the attachment zones), the expression of the S. domuncula GPX increased gradually with time, and reached a maximal level of 6.5-fold. This finding suggested again that during graft fusion and rejection in sponges, ROS are generated which amplify the immune response, as they do in cytokineactivated macrophages in vertebrates. Finally a pre-B-cell colony-enhancing factor was found in S. domuncula (Mller et al. 1999b). In the primmorph system of S. domuncula, the expression of the gene encoding this cytokine-like molecule increased after exposure of the cells to membranes from another species, such as those from G. cydonium. This indicated further that sponges have a molecular mechanism for the recognition of non-self. Molecules in sponges comprising polymorphic Ig-like domains: The most striking similarity between molecules involved in the human adaptive immunity and sequences isolated from G. cydonium are among those which contain immunoglobulin (Ig)-like domains, the receptor tyrosine kinase (RTK) and the sponge adhesion molecules (SAM). The G. cydonium RTK molecule possesses in the deduced polypeptide structure two complete Ig-like domains (Mller and Schcke 1996). Two other SAM species have been found which do not encode a tyrosine kinase but also contain in the extracellular part two Ig-like domains (GC-SAM) (Blumbach et al. 1999). The Ig-like domains found in GC-SAM long form (L) and GC-SAM short form (S) as well as in the RTK display high sequence similarity to the V domain of mammalian immunoglobulin domains (Blumbach et al. 1999). Studies with the two G. cydonium genes GC-SAML and GC-SAMS were performed during auto- and allografting; the results revealed that those genes undergo a differential expression (Blumbach et al. 1999).

have been identified in G. cydonium and/or S. domuncula (Wiens et al. 2002, 2003b).

Stem cells (1966/2003)


By definition, stem cells (i) have the capacity of selfreplication and (ii) give rise to more than one type of mature daughter cells. In general, three levels of stem cells can be distinguished, (i) totipotent stem cells that can rise to an intact organism, including the germinal tissues, (ii) pluripotent stem cells are capable to give rise to cells, derived from the germ layers, and (iii) multipotent stem cells that give rise to a single organ/tissue system. In sponges, the plasticity of the differentiation states of cells dominates over a pointof-no-return differentiation. While Borojevic (1966) provided experimental data indicating that the origin of the differentiation paths to the terminally differentiated cells starts with archaeocytes, Diaz (1977) proposed that the choanocytes have the capacity to differentiate to the archaeocytes. The presently accepted hypothesis is that the archaeocytes, present in early embryos or gemmules, are the totipotent stem cells. In the freshwater sponge Ephydatia fluviatilis Linn 1758 the skeletal cells, sclerocytes, very likely originate from archaeocytes (Weissenfels 1989). The fate of the sclerocytes is unknown; degeneration as well as a movement, away from the spicule, has been discussed (Bergquist 1978, Simpson 1984). For a series of sponge species it could be shown that oocytes originate from archaeocytes (see: Witte and Barthel 1994). These findings imply that both types of cells, the archaeocytes and the germ cells have the same stem cell propensity; they are totipotent stem cells. For this reason we group here the sponge cells only to totipotent stem cells, including the pluripotent stem cells, and the multipotent progenitor cells. Through the multipotent stem cell stages the sponge cells proceed to the terminally differentiated, somatic cells (see: Fig. 5). The terminally differentiated cells, the collencytes/sclerocytes and the myocytes, very likely undergo cell death, after formation of the structural elements they produce the spicules and the fibrils (Mller et al. 2005). At present the study of embryonic stem cells in sponges is limited, since no technique to induce mass production of embryos under controlled conditions has yet been successful. As a substitution, the three-dimensional cell culture has been established for S. domuncula. Under suitable conditions dissociated, single cells form special types of cell aggregates, the primmorphs. They contain cells of high proliferation and differentiation capacity. Gene expression pattern of archaeocytes (stem cells); reproductive cells: Until recently it was not possible to define cell types in sponges in a strict manner. Now molecular markers have been worked out, allowing also the distinction of the different levels of stem cells. Stem cells are self-renewing populations of cells that undergo symmetric and/or asymmetric divisions either to self-renew or to differentiate. This minimal definition does not allow a clear distinction of stem cells from other dividing and differentiating cells. Recently genetic expression markers have been identified, which can be applied for the identification of embryonic cells and tissue in sponges.

Apoptosis (2000)
Apoptosis represents the morphological manifestation of programmed cell death and paradoxically it is a prerequisite for metazoan life. Thus, apoptosis is responsible for the demise of cells during many physiological processes. Obviously apoptosis must be regulated by a complex network of various molecular signaling pathways. Research during the past 20 years has led to the identification of major functional groups of molecules involved in apoptotic pathways. These include members of the Bcl-2 superfamily, members of the TNF family, caspases and their activators. Yet, the evolutionary conservation of those elements of the apoptotic machinery was only established from nematode to man. Recently the present day knowledge on apoptosis in sponge was compiled (Wiens and Mller 2006). The key molecules are: The poriferan Bcl-2 homologues (Wiens et al. 2000a, 2001a, 2004), the death domain proteins, which are inducers of apoptotic cell death, belonging to the family of Fas, TRADD (Wiens et al. 2000b) as well as the the poriferan caspases that

98

Fig. 5: Sponge stem cell system. Schematic outline of the postulated development of the toti-/pluri-/multipotent sponge embryonic stem cells, the archaeocytes, to the germ cells on one side and to the three major differentiated cell types, the epithelial-, the contractile- and the skeletal cells. It is indicated that during these transitions progenitor cells characteristic for these lineages have to be passed. The (potential) factors, e.g. noggin and the mesenchymal stem cell-like protein (MSCP) on the path to the skeletal cells, which trigger the differentiation are shown. In addition it is outlined that committed progenitor cells are formed which respond to the silicate/Fe(+++) stimulus through differentiation to skeletal cells, the sclerocytes (=skeletal cells). Embryonal stem cells are present in the blastocyst embryo, the blastomeres, and also in the gemmules, there as the thesocytes.

Like in any other metazoan, also in sponges fertilized eggs develop during a series of cell divisions to morulae, blastulae and perhaps even to gastrulae. Subsequently the embryos develop to mature larvae, which can be classified into several types (Leys 2003). The first study, using molecular markers to determine the restriction of gene expression during embryogenesis in a sponge appeared recently (Perovi-Ottstadt et al. 2004). We found that in oocytes, morulae and blastulae/ larvae from S. domuncula, distinct genes are expressed, among them a sponge-specific receptor tyrosine kinase (RTKvs). In addition, the sex-determining protein FEM1 and the sperm associated antigen-related protein are highly expressed; in adult animals the levels of expression of these genes are very low (Perovi-Ottstadt et al. 2004) (Fig. 6). Marker genes for totipotent cells: Using the biological systems of larvae and gemmules, the question for molecular markers of stem cells according to the stringent definition could be approached by screening for genes which are expressed in dormant germ cells and in dormant cells of gemmules. Two candidate genes have been identified, which are highly expressed either in oocytes or in cells of gemmules, the receptor tyrosine kinase RTKvs (oocytes and early larvae; Perovi-Ottstadt et al. 2004) and the embryonic development

protein EED (gemmules; Mller 2006). In tissue of adult S. domuncula both genes are expressed only in scattered cells of the pinacoderm. In situ hybridization demonstrated that RTKvs_SUBDO is highly expressed in eggs and early stages of embryos in S. domuncula. In the adults RTKvs_SUBDO-expressing cells are detectable only rarely around the aquiferous canals. EED2_ SUBDO is highly expressed in gemmules, while only a few isolated cells are found in the other tissue. In view of these data, we suggest that the cells expressing these two genes represent archaeocytes, which are in the functional state in either fertilized eggs or cells constituting early embryos (as in RTKvs_SUBDO), or form the gemmules (EED2_SUBDO). Future studies must show if precursor archaeocyte-cells exist which express RTKvs_SUBDO and EED2_SUBDO. To date we propose that these two genes are expressed in primordial, perhaps totipotent, cells of S. domuncula. Gene expression pattern of archaeocytes (stem cells) sclerocyte lineage [skeletal cells]: Sclerocytes are the cells which produce the siliceous spicules, the skeletal elements of sponges. During the progression from the totipotent archaeocytes, via the multipotent cells to the terminally differentiated somatic sclerocytes, marker genes are expressed. The first cDNAs

99

identified in S. domuncula whose deduced proteins share sequence similarity to mammalian stem cell markers were the mesenchymal stem cell-like protein (MSCP-l) and noggin (Mller et al. 2003a). They can be considered as marker genes for multipotent stem cells. The mesenchymal stem cell-like protein (MSCP) is expressed in vertebrates in mesenchymal stem cells (van den Bos et al. unpublished [see: accession number MN_016647]). These authors provided evidence that MSCP is expressed in osteogenic mesenchymal stem cells. The S. domuncula gene MSCP encoding the mesenchymal stem cell-like protein was isolated by PCR (Mller et al. 2003a). Functional studies revealed that the expression of this gene is under positive control of the morphogenetic inorganic elements silicon and ferric iron (Krasko et al. 2002). The other potential gene involved in the differentiation of stem cells in sponges is noggin. Noggin is a glycoprotein that binds bone morphogenetic proteins selectively and opposes their effects. The noggin-like protein from S. domuncula was deduced from the SDNOGG-l cDNA (Mller et al. 2003b). Expression studies revealed a higher level of the SDNOGG-l transcripts in primmorphs in the presence than in the absence of silicate/Fe(+++). Finally, a deduced protein with similarity to the vertebrate glia maturation factor should be mentioned. This protein is restricted in vertebrates to the nervous system. The sponge related protein is again closer related to the human molecule than to the corresponding molecules from D. melanogaster or S. cerevisiae (Mller et al. 2003b). Northern blot studies, supported by in situ hybridization analyses, revealed that the expression of these genes follows a sequential order, after exposure of the animals/primmorphs to silicate/Fe(+++). At first noggin is expressed, followed by silicatein and finally the glia maturation factor-like molecule. At present no molecules have been identified, which might act as cis- or trans regulators for this sequential expression, and control the temporal and spatial expression of these genes. Gene expression pattern of archaeocytes (stem cells) - pinacocyte lineage [epithelial layer]: The surface layer constituted of pinacocytes can be looked upon as an epithelium. One marker gene for the differentiation of the archaeocytes/stem cells to the pinacocytes has been isolated; the Iroquois (marker gene for the pinacocyte lineage) that codes for a putative homeobox gene has been isolated from S. domuncula (Perovic et al. 2003). Expression of the putative Iroquois transcription factor was found in these cells adjacent to the canal system; it is upregulated in primmorphs which are cultivated in strong water current (Perovic et al. 2003). The restriction of the Iroquois expression to a specific tissue region, here the epithelial layer of the aquiferous system, adds a further piece to the understanding of the complexity of tissue organization in sponges. Gene expression pattern of archaeocytes (stem cells) - myocyte lineage: Myocytes in sponges are functionally characterized as cells which synthesize the organic skeletal elements, e.g. collagen. During the progress of archaeocytes to myocytes, myotrophin is expressed in S. domuncula (Schrder et al. 2000). Myotrophin was first found in mammalian systems; in cardiac myocytes myotrophin stimulates protein biosynthesis, suggesting a crucial role in the formation of cardiac hypertrophy. The closest similarity of the sponge

Fig. 6: Sequential expression of (putative) stem cell marker genes in S. domuncula. In primmorphs as well as in germ cells a high expression two genes can be identified, the sponge-specific receptor tyrosine kinase (RTKvs) and the embryonic ectoderm development protein (EED). They might be considered as markers for totipotent stem cells. At exposure of the primmorphs to a water current, the transcription factor Iroquois is expressed; this process is seen primarily in epithelial cells. Noggin as well as silicatein gene expression is provoked after addition of silicate/Fe(+++) to the culture medium; the expression is prominent in the skeletal (spicule)-forming cell lineage. In contractile cells (myocytes), myotrophin is expressed. As underlay of the bars, early drawings of a larva of Aplysilla sulfurea Schulze, 1878. (above; DeLage 1892) and a cross section through an entire sponge (Craniella schmidtii Sollas, 1886), showing embryos within the parent (Sollas 1888), are given.

molecule is with the human sequence (identity 50% / similarity 72%; Schrder et al. 2000). Recombinant sponge myotrophin stimulates protein synthesis by 5-fold (Schrder et al. 2000). Since myotrophin is neither expressed during embryogenesis nor in gemmules it might be characterized as a marker gene for the myocyte lineage. After incubation of single cells with myotrophin the primmorphs show an unusual elongated, oval shape. Furthermore, in the presence of myotrophin sponge cells up-regulate collagen gene expression. We assume that the sponge myotrophin causes in homologous cells the same/ similar effect as the cardiac myotrophin in mammalian cells, where it is also involved in initiation of cardial ventricular hypertrophy. Focusing on the stem cell system in sponges the main lessons are; (i) sponge cells progress from a primordial stage to terminally differentiated stages, (ii) they contain totipotent stem cells, (iii) during the progression from stem cells to differentiated cells genes are expressed, among which some

100

share high sequence similarity to those identified in vertebrates (Fig. 6). At present it is the notion that the plasticity of stem cells is high, because of the high regeneration/repair capacity of somatic sponge cells.

Axis formation in sponges (2003)


Pattern formation requires the definition of the main axes (reviewed in: Mller 2005). One basic requirement for a metazoan body plan is the close interactions between adjacent cells via junctions. Our screening for a gene encoding a tight junction scaffold protein from a sponge, here S. domuncula, was successful; the scaffold protein membrane-associated guanylate kinase with inverted arrangement (MAGI) had been identified (Adell et al. 2004). The sponge MAGI scaffold protein comprises the characteristic six PDZ domains that are involved in protein:protein interaction, two WW domains that bind to proline-rich peptide motifs and the conserved guanylate kinase motif. In addition, the existence of one tetraspan receptor, tetraspanin, in S. domuncula has been reported (Mller et al. 1999d). The tetraspanins belong to a group of hydrophobic proteins, comprising four transmembrane domains with a series of conserved aa residues in the extracellular loops (Fig. 7B). By in situ hybridization it is shown that the MAGI gene is highly expressed in the epithelial cell layer and in the cells surrounding the canals. Focusing of the axis formation, diploblastic animals, Porifera and Cnidaria, are characterized by one main body axis [apical/oscular-basal], while the triploblasts have two axes [in addition to the antero-posterior axis, the dorsal-ventral]. Many signaling pathways are involved in those polarity forming processes. Two large groups can be distinguished; pathways which originate from secreted signaling molecules, and those which are controlled by transcription factors. Signaling molecules: Wnt pathway: The Wnt signaling pathway is a cell communication system which regulates cellfate decisions, tissue polarity and morphogenesis (Fig. 7A). The Frizzled protein is the membrane receptor for the Wnt secreted glycoproteins. Through the canonical Wnt signaling pathway, the activated Frizzled binds to Dishevelled (Dsh), which leads to the stabilization and accumulation of -catenin in the nucleus, where it activates the TCF/lymphoid enhancer factor (LEF) transcription factor. Besides this canonical Wnt signaling pathway two further related pathways have been identified. A signaling downstream of Dsh which includes the Rho and JNK cascade, the non canonical Wnt signaling pathway. And, the Wnt/calcium pathway that stimulates intracellular calcium release in a G-protein-dependent manner. In vertebrates the canonical Wnt signaling pathway is involved in axis specification, non canonical Wnt signaling in the formation of cell polarity and convergent extension, and the Wnt/calcium pathway in tissue separation. Recent studies demonstrated that genes encoding the Frizzled receptor (Adell et al. 2003a), as well as genes expressing proteins downstream of this receptor are present already in sponges. In situ hybridization analysis in adult S. domuncula specimens showed expression in the cortex region and in the epithelial layer of the aquiferous canals. Furthermore, Northern blot analysis revealed an upregulation of its levels of expression during the formation of sponge primmorphs.

Transcription factors: T-box Forkhead: During development sets of genes, most of them transcription factors, that are responsible for cell fate and pattern determination, are expressed. Among them are T-box (Adell et al. 2002, 2003b), Forkhead and Homeobox gene families; they have been found to be extremely conserved on sequence and functional level in all metazoans. Members of the T-box family, e.g. Brachyury, are involved in the formation and differentiation of the third germ layer, the mesoderm, in triploblastic animals. Recently, two T-box genes have been isolated from the sponge S. domuncula; a Brachyury gene and a homologue of the Tbx2-34-5 genes from chordates, which in the latter taxa are involved in the formation of the limbs. Since the larvae of S. domuncula cannot be routinely cultivated it is at present not possible to conduct experiments with them. Therefore, studies are restricted to adult animals and cultured sponge primmorphs. Interestingly it could be demonstrated that the expression of the Brachyury gene is upregulated in differentiating sponge cells during formation of canal-like structures, suggesting that already in Porifera the primordial axis is genetically fixed. This assumption was subsequently confirmed by the isolation and phylogenetic characterization of five members of the winged-helix/Forkhead gene family from the sponge S. domuncula. Forkhead proteins form a subfamily within the large group of helix-turn-helix proteins. They are responsible for a wide range of functions and key roles in early developmental processes, during organogenesis and also for the function of the major organs and tissues in the adult animals. HNF3, the founding member of this gene family, is responsible for the formation of terminal structures that develop into the gut. The expression patterns of Forkhead and T-box/Brachyury genes during late blastulae and early gastrulae, support the assumption that these two sets of genes are required for the morphogenetic movements occurring during processes identical or phylogenetically preceding gastrulation. Moreover, the overexpression of Brachyury is seen from Porifera and Coelenterata onwards to the triploblasts in distinct regions of the body, usually adjacent to the organizers. In sponges the water enters the animals through many porocytes on their surface, the inhalant openings, into the canals formed by the endopinacoderm and then passes to the choanocyte chambers from where the water is driven to the central atrium and finally pressed through the oscule back into the environment. Generally, the number of oscules is restricted and many species comprise only one major oscule. Several lines of morphological/cytological and molecular biological evidence indicate that the aquiferous canal system in sponges represents the organizational center of the sponges. During embryogenesis amphiblastula or parenchymella larvae are formed (see: Leys 2003) that have in their interior cavity choanocyte chambers; these chambers are the central organlike structure which directs and controls the water current. Subsequently canals are formed which finally fuse with the outer pinacoderm layer. 3D-cell culture experiments likewise revealed that an increase of the water current in the culture fluid results in canal formation, controlled by the homeodomain protein Iroquois (Perovi et al. 2003). In order to determine if the oscule, the morphologically most prominent region in the sponge acts also as an organizer-like region ablation/

101

Fig. 7: A. Wnt signaling pathway. The extracellular Wingless (Wg)/Wnt ligand binds to the Frizzled receptor (Fz) which regulates cell-fate decisions through the Dishevelled (Dsh) molecule that is composed of three functional domains (DIX, PDZ, DEP). From there two pathways either lead to the activation of the TCF/LEF transcription factor or to the JNK kinase cascade. The resulting selective gene expression causes a planar cell polarity. Those molecules which have been already identified in sponges are highlighted in bold. B. Tight junction proteins in S. domuncula; schematic representation of the molecules involved. Tight junctions are sealing the epithelial layer of metazoan organisms to control the lateral-extracellular transport in the aqueous milieu. By the formation of tight junctions cells undergo a polarization. The tight junctional cell membrane-spanning receptors, here tetraspanin, associate with the scaffold protein, the PDZ protein MAGI. The scaffold protein MAGI plays a crucial role in the organization of the membrane receptor molecules and the effector molecules; the latter compose the cytoskeleton and the signal transduction molecules.

transplantation studies were performed with S. domuncula; the specimens comprise only one oscule and histological sections show a large atrium in which the exhaling canals end. The regeneration capacity of the oscule region is different from that of other regions of the body. After removal of the oscule this area regenerates and is sealed only by an intact epithelium with a double pinacoderm layer but no new oscule is formed even at other parts of the surface. If however, the oscule is excised and transplanted to another site an intact oscule with an atrium is formed (reviewed in: Mller 2005). It could also be demonstrated that the oscule region comprises the highest level of expressed genes indicative for organizer regions. Among the overexpressed genes in the tissue surrounding the oscule are the LIM-homeodomain protein, Brachyury, Frizzled receptor and the secreted Frizzled-related proteins, noggin or Iroquois. These data are strong arguments for the assumption that regionalized organizer centers are present from Porifera to the crown triploblastic species and are localized close to the openings to the body cavity (see: Mller 2005). After accepting the monophyletic origin of all metazoans, including Porifera, and that all animals have common basic elements of the immune system and the body plan, it was not

too surprising that also ancestral homeobox genes are present in sponges. The developmental processes resulting in the formation of a body axis require a head center; e.g. in bilaterians, the Spemanns organizer (Spemann 1936). The genes which are involved in the establishment of the head organizer during embryogenesis have been grouped into three classes of homeobox genes, the Paired-class, the Antennapedia-class and the LIM-class genes. In this area a rapid progress was made in sponges in the last few years. A paired-class (Pax-2/5/8)-gene had been isolated from the freshwater sponge E. fluviatilis, which encodes a complete although substantially degenerated homeodomain (Seimiya et al. 1998). In S. domuncula a cDNA encoding a LIM/homeobox protein has been isolated which comprises high sequence similarity to the related LIM homeodomain proteins in other animals (Wiens et al. 2003a): its potential function was elucidated in the primmorph model. If they are cultured on a homologous galectin matrix on which they form canal-like structures, morphogenetic processes are triggered that also involve the LIM/homeobox protein/ transcription factor. In addition, retinoic acid plays an important role in local signaling of homeodomain factor-mediated vertebrate development. As the first metazoan nuclear hormone receptor

102

the retinoid X receptor (RXR) was identified in Porifera and its role was studied in the primmorph system. Like in vertebrates or in Drosophila also in S. domuncula retinoic acid is formed from -carotene via cleavage by the ,-carotene-15,15dioxygenase to retinal and further oxidization to retinoic acid. Retinoic acid (9-cis-retinoic acid) binds to RXR resulting in a regulation of transcriptional activity of morphogenetic genes, including also a homeobox gene (Wiens et al. 2003c). In Porifera the zygote increases in size and develops flagellae after fertilization. Depending on the taxon morphologically slightly different types of larvae are formed and released from the maternal body. During this phase the embryo polarizes, a process which is morphologically primarily obvious in the localization of the cilia and the formation of the body cavity. Then gastrulation takes place driven by an asymmetric and tangentially arranged cleavage. The epithelia within the body cavity invaginate under formation of the choanocyte chambers. The embryos become sessile and the young sponge forms an oscule, through which the body cavity opens to the external milieu; with this process the young sponge forms a oscular/ apical-basal axis. The oscule region as mentioned above can be considered as an organizer, since there the characteristic vertebrate organizer genes are expressed. In addition, this region is provided with a regeneration capacity which is distinguished from the rest of the animal. There are especially the organizer-specific genes, Lim-homeodomain, Brachyury, Frizzled receptor, noggin and Iroquois, which are expressed along the aquiferous system and the oscule. This water canal system is the combined route for feeding, secretion and gas exchange. Strong evidence has been collected in the last two years which indicate that these organizer genes are not only expressed around the canals and the oscule but also display morphogenetic activity. X-ray analyses of the skeleton of the Lake Baikal sponge Lubomirskia baikalensis (Pallas, 1776) reveal that the architecture of the specimens is supported by a highly ordered arrangement of the spicules within the body (Fig. 8); Kaluzhnaya et al. (2005a, 2005b). Lubomirskia baikalensis follows an organized growing pattern, reflecting a radiate accretive growth process. This pattern is maintained throughout the body of the animals and can be seen at the tips, the growing parts of the specimens, and at the basis. During growth new layers/rings of new longitudinal spicule bundles are added at the tip of the branches. A similar highly regular arrangement of the spicules can also be seen in the hexactinellid Euplectella aspergillum (Owen, 1841). There is no doubt that such highly ordered growth processes in the Porifera are genetically controlled. The discovery that organizer-specific genes are present at the tips of the branches, around the oscules, and the fact that growth along the oscular/ apical-basal axis proceeds in a radiate accretive pattern, suggest that at the growth lines, which are readily seen between the preceding and the succeeding growth layers, genes are expressed which form serial modules along this oscular/apical axis (Fig. 8). Such serial modules should, however, not be termed segments, because this would imply that they were built by a more complex genetic network, as seen in insects.

The power of genomics


There was never a faster progress in the understanding of the differentiation capacity of sponge cells than during the last 10 years when the power of molecular biology was developed and applied. In the past biochemical data were not sufficient to provide solid evidence for the phylogenetic position of the Porifera within the multicellular animals. With the application of molecular biological techniques it became clear that during the transition from the fungal state to the colonial state of multicellular animals two sets of innovative molecules had to be developed, the cell-cell and the cell-matrix adhesion systems. Surprisingly the receptors and ligands involved in these processes and so far identified from the Porifera to the crown taxa of Metazoa share no similarity to the Fungi or the Plantae. These evolutionary novelties allowed the cells to form complex aggregates which made interaction in a tuned manner possible. The basis for a specialized integration of the cells within these aggregates was an efficient signal transduction that allowed extracellular ligands to cause a modulation of the cell metabolism after complex interaction with membraneassociated receptors. These processes affected either the intermediate metabolism or the gene replication/expression machinery. Only operationally separated from these adhesion/signal transduction systems are the two further qualities of the multicellular animals systems, the immune system and the programmed cell death/apoptosis. These achievements allowed the aggregates to form individuals which have the capability to form functional units composed of cells with different properties. Having reached this evolutionary step the multicellular animals could distinguish between self-self and self/non-self and were able to adjust their cellular homeostasis. In addition, data suggest that the transcription factors already existed in the Urmetazoa which allowed to establish a simple form of body axis. These evolutionary novelties had to be created within a period of approximately only 200 million years, from the common ancestor of Fungi and Metazoa to the appearance of the Urmetazoa as the common ancestor of all metazoan phyla. This time period is comparatively short with respect to the subsequent 600 million years of evolution of Metazoa. The finding that the basic molecular strategies to form the integrated Urmetazoa have not changed qualitatively during this time, but were only prone to a processes of specialization is surprising. Until 10 years ago the opposite view was dominating, stating that the Porifera are only Parazoa [alongside animals] or Mesozoa [middle animals]. The latter terms imply qualitative assessments that in view of the molecular data compiled proved not to be helpful for a further understanding of the evolution of Metazoa. Taken together, the hypothetical ancestor of Metazoa, the Urmetazoa (Mller 2001, Mller et al. 2001), was already provided with the basic regulatory gene repertoire, like cell adhesion molecules [aggregation factor or galectin] and cell differentiation factors [involved in collagen synthesis] as well as with a highly elaborated immune system (Mller et al. 1999b) which allowed a pattern formation due to an expression of morphogens, a Pax-A like homeodomain protein, Hox-like molecules or a Lim-class homeodomain protein. In addition, recent studies in our laboratory demonstrated that

103

Fig. 8: Schematic view of pattern formation in Porifera. During embryogenesis the fertilized egg develops to a larva with a central cavity (ca). During and after settlement of the larva first choanocyte chambers (cc) are formed and the body cavity opens up to the external milieu through an oscule (os). The growth of the adult proceeds along the apical-basal axis in a compartmental manner; next to the photo of a branch, a schematic outline is given. The data suggest that the growth of those sponges which display an arborescent morphology, like the freshwater sponge L. baikalensis, proceeds by addition of serial modules along the body axis. The expression levels of those genes that code for structural proteins (the four silicateins and the MBL) as well as the mago nashi follow an apical-basal gradient; while the expressions of the catabolic enzyme cathepsin L and the house-keeping gene tubulin follows an opposite direction or remains constant. Interestingly enough, the inhibitor of the Wnt-pathway, the soluble frizzled molecule, comprises a higher level of expression at the basis, compared to the top of the branches. Finally, it is proposed that the body axis is controlled by paired-class and LIM-class homeoproteins (HP).

S. domuncula expresses Forkhead/T-box genes (Adell et al. 2003a) and also a retinoic acid receptor (Wiens et al. 2003c). Furthermore, apoptotic genes are expressed (e.g. Wiens et al. 2001) which are considered to be involved in the formation of the sponge body cavities. Based on these data it can be deduced that sponges are provided with amazingly rich and diversified regulatory molecules that allow pattern formation.

References
Adell T, Wiens M, Mller WEG (2002) Sd-Bra and SD-Tbx: first T-box transcription factors from Porifera. Boll Mus Ist Biol Univ Genova 66-67: 5 Adell T, Nefkens I, Mller WEG (2003a) Polarity factor Frizzled in the demosponge Suberites domuncula: identification, expression and localization of the receptor in the epithelium/pinacoderm. FEBS Letters 554: 363-368 Adell T, Grebenjuk VA, Wiens M, Mller WEG (2003b) Isolation and characterization of two T-box genes from sponges, the phylogenetically oldest metazoan taxon. Dev Genes Evol 213: 421-434 Adell T, Gamulin V, Perovi-Ottstadt S, Wiens M, Korzhev M, Mller IM, Mller WEG (2004) Evolution of metazoan cell junction proteins: The scaffold protein MAGI and the transmembrane receptor tetraspanin in the demosponge Suberites domuncula. J Mol Evol 59: 41-50

Althoff K, Schtt C, Krasko A, Steffen R, Batel R, Mller WEG (1998) Evidence for a symbiosis between bacteria of the genus Rhodobacter and the marine sponge Halichondria panicea: harbor also for putatively-toxic bacteria? Mar Biol 130: 529-536 Bergquist PR (1978) Sponges. Hutchinson, London Blumbach B, Pancer Z, Diehl-Seifert B, Steffen R, Mnkner J, Mller I, Mller WEG (1998) The putative sponge aggregation receptor: isolation and characterization of a molecule composed of scavenger receptor cysteine-rich domains and short consensus repeats. J Cell Sci 111: 2635-2644 Blumbach B, Diehl-Seifert B, Seack J, Steffen R, Mller IM, Mller WEG (1999) Cloning and expression of new receptors belonging to the immunoglobulin superfamily from the marine sponge Geodia cydonium. Immunogenetics 49: 751-763 Bger H (1988) Versuch ber das phylogenetische System der Porifera. Meyniana 40: 67-90 Bhm M, Hentschel U, Friedrich A, Fieseler L, Steffen R, Gamulin V, Mller IM, Mller WEG (2001) Molecular response of the sponge Suberites domuncula to bacterial infection. Mar Biol 139: 1037-1045 Borojevic R (1966) tude exprimentale de la diffrentiation des cellules de l ponge au cours de son dveloppement. Dev Biol 14: 130-153 Burger MM, Jumblatt J (1977) Membrane involvement in cellcell interactions: a two component model system for cellular recognition that does not require live cells. In: Lash JW, Burger

104

MM (eds). Cell and tissue interactions. Raven Press, New York. pp. 155-172 Buscema M, van de Vyver G (1983) Variability of allograft rejection processes in Axinella verrucosa. Dev Comp Immunol 7: 613-616 Campbell LG (1876) Log Letters from The Challenger. MacMillan, London Christen R, Ratto A, Baroin A, Perasso R, Grell KG, Adoutte A (1991) An analysis of the origin of metazoans, using comparisons of partial sequences of the 28S RNA, reveals an early emergence of triploblasts. EMBO J 10: 499-503 Custdio MR, Prokic I, Steffen R, Koziol C, Borojevic R, Brmmer F, Nickel M, Mller, WEG (1998) Primmorphs generated from dissociated cells of the sponge Suberites domuncula: A model system for studies of cell proliferation and cell death. Mech Ageing Develop 105: 45-59 Delage Y (1892) Embryognie des ponges. Arch Zool Exp 2: 345498 DeLage Y, Hrouard E (1899) Trait de zoologie concrte. Le Soudier, Paris Diaz JP (1977) Transformation histologiques et cytologiques posttraumatiques chez la demosponge Suberites massa Nardo. Bull Mus Natl Hist Nat (Paris) 445: 375-396 Doolittle WF, Brown JR (1994) Tempo, mode, the progenote, and the universal root. Proc Natl Acad Sci USA 91: 6735-6742 Esper EJC (1794) Die Pflanzentiere. Raspesche Buchhandlung. Nrnberg Felsenstein J (1993) PHYLIP, ver. 3.5. University of Washington, Seattle Field KG, Olsen GJ, Lane DJ, Giovannoni SJ, Ghiselin MT, Raff EC, Pace NR, Raff RA(1988) Molecular phylogeny of the animal kingdom. Science 239: 748-753 Gamulin V, Rinkevich B, Schcke H, Kruse M, Mller IM, Mller WEG (1994) Cell adhesion receptors and nuclear receptors are higly conserved from the lowest Metazoa (marine sponges) to vertebrates. Biol Chem Hoppe-Seyler 375: 583-588 Grant RE (1836) Animal kingdom. In: Todd RB (ed). The cyclopaedia of anatomy and physiology, vol 1. Sherwood-GilbertPiper, London Grebenjuk VA, Kuusksalu A, Kelve M, Schtze J, Schrder HC, Mller WEG (2002) Induction of (2-5)oligoadenylate synthetase in the marine sponges Suberites domuncula and Geodia cydonium by the bacterial endotoxin lipopolysaccharide. Europ J Biochem 269: 1382-1392 Haeckel E (1872). Atlas der Kalkschwmme. Verlag von Georg Reimer, Berlin Halanych KM (1991) 5S ribosomal RNA sequences inappropriate for phylogenetic reconstruction. Mol Biol Evol 8: 249-253 Henkart P, Humphreys S, Humphreys T (1973) Characterization of sponge aggregation factor. A unique proteoglycan complex. Biochemistry 12: 3045-3050 Hildemann WH, Johnston IS, Jokiel PL (1979) Immunocompetence in the lowest metazoan phylum: transplantation immunity in sponges. Science 204: 420-422 Hovanessian AG (1991) Interferon-induced and double-stranded RNA-activated enzymes: a specific protein kinase and 2,5oligoadenylate synthetases. J Interferon Res 11: 199-205 Hyman LH (1940) The Invertebrates; vol. 1. McGraw Hill, New York Koziol C, Leys SP, Mller IM, Mller WEG (1997) Cloning of Hsp70 genes from the marine sponges Sycon raphanus (Calcarea)

and Rhabdocalyptus dawsoni (Hexactinellida). An approach to solve the phylogeny of sponges. Biol J Linn Soc 62: 581-592 Kaluzhnaya OV, Belikov SI, Schrder HC, Rothenberger M, Zapf S, Kaandorp JA, Boreiko A, Mller IM, Mller WEG (2005a) Dynamics of skeleton formation in the Lake Baikal sponge Lubomirskia baicalensis. Part I. Biological and biochemical studies. Naturwissenschaften 92: 128-133 Kaluzhnaya OV, Belikov SI, Schrder HC, Wiens M, Giovine M, Krasko A, Mller IM, Mller WEG (2005b) Dynamics of skeleton formation in the lake baikal sponge Lubomirskia baicalensis. Part II. Molecular biological studies. Naturwissenschaften 92: 134138 Krasko A, Batel R, Schrder HC, Mller IM, Mller WEG (2000) Expression of silicatein and collagen genes in the marine sponge Suberites domuncula is controlled by silicate and myotrophin. Europ J Biochem 267: 4878-4887 Krasko A, Schrder HC, Batel R, Grebenjuk VA, Steffen R, Mller IM, Mller WEG (2002) Iron induces proliferation and morphogenesis in primmorphs from the marine sponge Suberites domuncula. DNA Cell Biol 21: 67-80 Kruse M, Mller IM, Mller WEG (1997) Early evolution of Metazoan serine/threonine- and tyrosine kinases: Identification of selected kinases in marine sponges. Mol Biol Evol 14: 1326-1334 Kruse M, Leys SP, Mller IM, Mller WEG (1998) Phylogenetic position of the Hexactinellida within the phylum Porifera based on amino acid sequence of the protein kinase C from Rhabdocalyptus dawsoni. J Mol Evol 46: 721-728 Kruse M, Steffen R, Batel R, Mller IM, Mller WEG (1999) Differential expression of allograft inflammatory factor 1 and of glutathione peroxidase during auto- and allograft response in marine sponges. J Cell Sci 112: 4305-4313 Kuusksalu A, Pihlak A, Mller WEG, Kelve M (1995) The (2-5) oligoadenylate synthetase is present in the lowest multicellular organisms, the marine sponges: demonstration of the existence and identification of its reaction products. Eur J Biochem 232: 351357 Leys SP (1995) Cytoskeletal architecture and organelle transport in giant syncytia formed by fusion of hexactinellid sponge tissues. Biol Bull 188: 241-254 Leys SP (2003) Comparative study of spiculogenesis in demosponge and hexactinellid larvae. Micr Res Tech 62: 300-311 Medina M, Collins AG, Silberman JD, Sogin ML (2001) Evaluating hypothesis of basal animal phylogeny using complete sequences of large and small subunit rRNA. Proc Natl Acad Sci USA 98: 97079712 Mehl D, Reiswig HM (1991) The presence of flagellar vanes in choanomeres of Porifera and their possible phylogenetic implications. Z zool Syst Evolut-Forsch 29: 312-319 Mehl D, Mller I, Mller WEG (1998) Molecular biological and palaeontological evidence that Eumetazoa, including Porifera (sponges), are of monophyletic origin. In: Watanabe Y, Fusetani N (eds). Sponge science - multidisciplinary perspectives. SpringerVerlag, Tokyo. pp 133-156 Metchnikoff (1892) Leons sur la pathologie compare de inflammation. G. Masson, Paris Moscona AA (1963) Studies on cell aggregation: demonstration of materials with selective cell-binding activity. Proc Natl Acad Sci USA 49: 742-747 Mller WEG (1982) Cell membranes in sponges. Intern Rev Cytol 77: 129-181

105

Mller WEG (1995) Molecular phylogeny of Metazoa (animals): monophyletic origin. Naturwissenschaften 82: 321-329 Mller WEG (editor) (1998a) Molecular evolution: evidence for monophyly of Metazoa. Progr Molec Subcell Biol 19. Springer Verlag, Berlin-Heidelberg Mller WEG (editor) (1998b) Molecular evolution: towards the origin of Metazoa. Progr Molec Subcell Biol 21. Springer Verlag, Berlin-Heidelberg Mller WEG (1998c) Origin of Metazoa: sponges as living fossils. Naturwissenschaften 85: 11-25 Mller WEG (2001) How was metazoan threshold crossed: the hypothetical Urmetazoa. Comp Biochem Physiol 129: 433-460 Mller WEG (2005) Spatial and temporal expression patterns in animals. In: Meyers RA (ed). Encyclopedia of molecular cell biology and molecular medicine. vol. 13. Wiley-VCH, Weinheim. pp. 269-309 Mller WEG (2006) Review: the stem cell concept in sponges (Porifera): metazoan traits. Semin Cell Dev Biol. 17(4): 481-491 Mller WEG, Zahn RK (1973) Purification and characterization of a species-specific aggregation factor in sponges. Exp Cell Res 80: 95-104 Mller WEG, Schcke H (1996) Characterization of the receptor protein-tyrosine kinase gene from the marine sponge Geodia cydonium. Prog Molec Subcell Biol 17: 183-208 Mller WEG, Mller IM (2003) Origin of the metazoan immune system: identification of the molecules and their functions in sponges. Integr Comp Biol 43: 281-292 Mller WEG, Mller I, Zahn RK, Kurelec B (1976) Species-specific aggregation factor in sponges. VI. Aggregation receptor from the cell surface. J Cell Sci 21: 227-241 Mller WEG, Diehl-Seifert B, Gramzow M, Friese U, Renneisen K, Schrder HC (1988) Interrelation between extracellular adhesion proteins and extracellular matrix in reaggregation of dissociated sponge cells. Int Rev Cytol 111: 211-229 Mller WEG, Spreitzer I, Milanovic S, Schrder HC, Rinkevich B, Gamulin V, Bretting H (1994a) Identification and characterization of S-type lectins from the marine sponge Geodia cydonium. In: van Soest RWM, van Kempen TMG, Braeckman JC (eds). Sponges in time and space: biology, chemistry, paleontology. Balkema, Rotterdam. pp. 389-393 Mller WEG, Mller IM, Gamulin V (1994b) On the monophyletic evolution of the Metazoa. Brazil J Med Biol Res 27: 2083-2096 Mller WEG, Blumbach B, Wagner-Hlsmann C, Lessel U (1997) Galectins in the phylogenetically oldest metazoa, the sponges [Porifera]. Trends Glycosci Glycotechnol 9: 123-130 Mller WEG, Kruse M, Koziol C, Leys SP (1998) Evolution of early Metazoa: phylogenetic status of the Hexactinellida within the phylum of Porifera [sponges]. Progr Molec Subcell Biol 21: 141-156 Mller WEG, Wiens M, Batel R, Steffen R, Borojevic R, Custdio MR (1999a) Establishment of a primary cell culture from a sponge: primmorphs from Suberites domuncula. Mar Ecol Progr Ser 178: 205-219 Mller WEG, Blumbach B, Mller IM (1999b) Evolution of the innate and adaptive immune systems: relationships between potential immune molecules in the lowest metazoan phylum [Porifera] and those in vertebrates. Transplantation 68: 1215-1227 Mller WEG, Bhm M, Batel R, de Rosa S, Tommonaro G, Mller IM, Schrder HC (2000) Application of cell culture for the production of bioactive compounds from sponges: Synthesis of

avarol by primmorphs from Dysidea avara. J Nat Prod 63: 10771081 Mller WEG, Schrder HC, Skorokhod A, Bnz C, Mller IM, Grebenjuk VA (2001) Contribution of sponge genes to unravel the genome of the hypothetical ancestor of Metazoa (Urmetazoa). Gene 276: 161-173 Mller WEG, Krasko A, Skorokhod A, Bnz C, Grebenjuk VA, Steffen R, Batel R, Schrder HC (2002) Histocompatibility reaction in tissue and cells of the marine sponge Suberites domuncula in vitro and in vivo: Central role of the allograft inflammatory factor 1. Immunogenetics 54: 48-58 Mller WEG, Wiens M, Mller IM, Schrder HC (2003a) The chemokine networks in sponges: potential roles in morphogenesis, immunity and stem cell formation. Progr Molec Subcell Biol 34: 103-143 Mller WEG, Korzhev M, Le Pennec G, Mller IM, Schrder HC (2003b) Origin of metazoan stem cell system in sponges: first approach to establish the model (Suberites domuncula). Biomol Engineer 20: 369-379 Mller WEG, Rothenberger M, Boreiko A, Tremel W, Reiber A, Schrder HC (2005) Formation of siliceous spicules in the marine demosponge Suberites domuncula. Cell Tissue Res 321: 285-297 Mller WEG, Belikov SI, Tremel W, Perry CC, Gieskes WWC, Boreiko A, Schrder HC (2006) Siliceous spicules in marine demosponges (example Suberites domuncula). Micron 37: 107120 Pahler S, Krasko A, Schtze J, Mller IM, Mller WEG (1998) Isolation and characterization of a cDNA encoding a potential morphogen from the marine sponge Geodia cydonium, that is conserved in higher metazoans. Proc R Soc Lond B 265:421-425 Pallas PS (1787) Charakteristik der Thierpflanzen. Raspesche Buchhandlung, Nrnberg Pancer Z, Kruse M, Schcke H, Scheffer U, Steffen R, Kovcs P, Mller WEG (1996) Polymorphism in the immunoglobulin-like domains of the receptor tyrosine kinase from the sponge Geodia cydonium. Cell Adhes Commun 4: 327-339 Pancer Z, Kruse M, Mller I, Mller WEG (1997) On the origin of adhesion receptors of metazoa: cloning of the integrin subunit cDNA from the sponge Geodia cydonium. Molec Biol Evol 14: 391-398 Pechenik JA (2000) Biology of the invertebrates. McGraw Hill, Boston Perovi S, Schrder HC, Sudek S, Grebenjuk VA, Batel R, tifanic M, Mller IM, Mller WEG (2003) Expression of one sponge Iroquois homeobox gene in primmorphs from Suberites domuncula during canal formation. Evol Develop 5: 240250 Perovi-Ottstadt S, etkovi H, Gamulin V, Schrder HC, Kropf K, Moss C, Korzhev M, Diehl-Seifert B, Mller IM, Mller WEG (2004) Molecular markers for germ cell differentiation in the demosponge Suberites domuncula. Int J Dev Biol 48: 293-305 Pfeifer K, Schrder HC, Rinkevich B, Uhlenbruck G, Hanisch FG, Kurelec B, Scholz P, Mller WEG (1992) Immunological and biological identification of tumor necrosis factor in sponges: role of this factor in the formation of necrosis in xenografts. Cytokine 4: 161-169 Pfeifer K, Haasemann M, Gamulin V, Bretting H, Fahrenholz F, Mller WEG (1993) S-type lectins occur also in invertebrates: high conservation of the carbohydrate recognition domain in the lectin genes from the marine sponge Geodia cydonium. Glycobiology 3: 179-184

106

Rasmont R (1979) Les ponge: des metazoaires et des societes de cellules. In: Lvi C, Boury-Esnault N (eds). Biologie des spongiaires. Centre National de la Recherche Scientifique, Paris. pp. 21-29 Reitner J, Mehl D (1995) Early Paleozoic diversification of sponges: new data and evidences. Geol Palont Mitt Innsbruck 20: 335347 Reitner J, Wrheide G (2002) Non-lithistid Demospongiae origins of their palaeobiodiversity and highlights in history of preservation. In: Hooper JNA, van Soest RWM (eds). Systema Porifera: a guide to the classification of sponges. Kluwer Academic/Plenum Publishers, New York. pp 52-70 Rodrigo AG, Bergquist PR, Bergquist PL, Reeves RA (1994) Are sponges animals? An investigation into the vagaries of phylogenetic inference. In: van Soest RWM, van Kempen TMG, Braeckman JC (eds). Sponges in time and space: biology, chemistry, paleontology. Balkema, Rotterdam. pp. 389-393 Schcke H, Schrder HC, Gamulin V, Rinkevich B, Mller IM, Mller WEG (1994a) Molecular cloning of a receptor tyrosine kinase from the marine sponge Geodia cydonium: a new member of the receptor tyrosine kinase class II family in invertebrates. Mol Membr Biol 11: 101-107 Schcke H, Mller IM, Mller WEG (1994b) Tyrosine kinase from the marine sponge Geodia cydonium. In: Mller WEG (ed). Use of aquatic invertebrates as tools for monitoring of environmental hazards. Gustav Fischer verlag, Stuttgart. pp 201-211 Scheffer U, Krasko A, Pancer Z, Mller WEG (1997) High conservation of the serum response factor within metazoa: cDNA from the sponge Geodia cydonium. Biol J Linn Soc 61: 127-137 Schrder HC, Krasko A, Batel R, Skorokhod A, Pahler S, Kruse M, Mller IM, Mller WEG (2000) Stimulation of protein (collagen) synthesis in sponge cells by a cardiac myotrophin-related molecule from Suberites domuncula. FASEB J 14: 2022-2031 Schrder HC, Brmmer F, Fattorusso E, Aiello A, de Rosa S, Ammar MSA, Mller WEG (2003) Sustainable production of bioactive compounds from sponges: primmorphs as bioreactors. In: Mller WEG (ed). Marine molecular biotechnology, vol. 1: sponges (Porifera). Springer, Berlin. pp. 163-197 Schulze FE (1887) Zur Stammesgeschichte der Hexactinelliden. Akademie der Wissenschaften, Berlin Schtze J, Custdio MR, Efremova SM, Mller IM, Mller WEG (1999) Evolutionary relationship of Metazoa within the eukaryotes based on molecular data from Porifera. Proc R Society Lond 266: 63-73 Seimiya M, Naito M, Watanabe Y, Kurosawa Y (1998) Homeobox genes in the freshwater sponge Ephydatia fluviatilis. Progr Molec Subcell Biol 19: 132-155 Simpson TL (1984) The cell biology of sponges. Springer-Verlag, New York Smith LC, Hildemann W H (1986) Allogeneic cell interactions during graft rejection in Callyspongia diffusa (Porifera; Demospongia); a study with monoclonal antibodies. Proc R Soc Lond 226: 465-377 Sollas WJ (1888) Report on the Tetractinellida. Rep Sci Res Voy H.M.S. Challenger, Zool 25(63): 1-458 Spemann H (1936) Experimentelle Beitrge zu einer Theorie der Entwicklung. Springer Verlag, Berlin von Lendenfeld R (1889) A monograph of the horny sponges. Royal Society, London

Weissenfels N (1989) Biologie und Mikroskopische Anatomie der Swasserschwmme (Spongillidae). Gustav Fischer Verlag, Stuttgart Wiens M, Mller WEG (2006) Cell death in Porifera: molecular players in the game of apoptotic cell death in living fossils. Can J Zool 84: 307-321 Wiens M, Kuusksalu A, Kelve M, Mller WEG (1999) Origin of the interferon-inducible (2-5)oligoadenylate synthetases: cloning of the (2-5)oligoadenylate synthetase from the marine sponge Geodia cydonium. FEBS Letters 462: 12-18 Wiens M, Krasko A, Mller CI, Mller WEG (2000a) Molecular evolution of apoptotic pathways: cloning of key domains from sponges (Bcl-2 homology domains and death domains) and their phylogenetic relationships. J Mol Evol 50: 520-531 Wiens M, Krasko A, Mller IM, Mller WEG (2000b) Increased expression of the potential proapoptotic molecule DD2 and increased synthesis of leukotriene B4 during allograft rejection in a marine sponge. Cell Death Diff 7: 461-469 Wiens M, Diehl-Seifert B, Mller WEG (2001) Sponge Bcl-2 homologous protein (BHP2-GC) confers selected stress resistance to human HEK-293 cells. Cell Death Diff 8: 887-898 Wiens M, Luckas B, Brmmer F, Ammar MSA, Steffen R, Batel R, Diehl-Seifert B, Schrder HC, Mller WEG (2002) Okadaic acid: a potential defense molecule for the sponge Suberites domuncula. Mar Biol 142: 213-223 Wiens M, Mangoni A, DEsposito M, Fattorusso E, Korchagina N, Schrder HC, Grebenjuk VA, Krasko A, Batel R, Mller IM, Mller WEG (2003a) The molecular basis for the evolution of the metazoan bodyplan: extracellular matrix-mediated morphogenesis in marine demosponges. J Mol Evol 57: 1-16 Wiens M, Krasko A, Perovic S, Mller WEG (2003b) Caspasemediated apoptosis in sponges: cloning and function of the phylogenetic oldest apoptotic proteases from metazoa. Biochim Biophys Acta 1593: 179-189 Wiens M, Batel R, Korzhev M, Mller WEG (2003c) Retinoid X receptor and retinoic acid response in the marine sponge Suberites domuncula. J Exp Biol 206: 3261-3271 Wiens M, Perovic-Ottstadt S, Mller IM, Mller WEG (2004) Allograft rejection in the mixed cell reaction system of the demosponge Suberites domuncula is controlled by differential expression of apoptotic genes. Immunogenetics 56: 597-610 Wilson HV (1907) On some phenomena of coalescence and regeneration in sponges. J Exp Zool 5: 245-258 Wimmer W, Blumbach B, Diehl-Seifert B, Koziol C, Batel R, Steffen R, Mller IM, Mller WEG (1999a) Increased expression of integrin and receptor tyrosine kinase genes during autograft fusion in the sponge Geodia cydonium. Cell Adhes Commun 7: 111-124 Wimmer W, Perovic S, Kruse M, Krasko A, Batel R, Mller WEG (1999b) Origin of the integrin-mediated signal transduction: functional studies with cell cultures from the sponge Suberites domuncula. Europ J Biochem 178: 156-165 Witte U, Barthel D (1994) Reproductive cycle and oogenesis of Halichondria panicea (Pallas) in Kiel bight. In: van Soest RWM, van Kempen TMG, Braeckman JC (eds). Sponges in time and space: biology, chemistry, paleontology. Balkema, Rotterdam. pp. 297-305

Porifera research: Biodiversity, innovation and sustainaBility - 2007

107

Diversity and evolution of deep-sea carnivorous sponges


Jean Vacelet
Aix-Marseille Universit, CNRS UMR-6540 DIMAR, Centre dOcanologie de Marseille. Station Marine dEndoume. Rue Batterie des Lions, 13007. Marseille, France. jean.vacelet@univmed.fr Abstract: The carnivorous habit of feeding that has been discovered in a cavernicolous species of Cladorhizidae is probably general for all the representatives of this deep-sea family, which numbered approximately 90 species at the end of the 20th century. Recent reports have shown that the number of species is actually considerably higher and that carnivory probably also occurs in several representatives of other Poecilosclerida families. A few specimens collected by trawling in the Pacific and Atlantic oceans have been described as new species. A larger sample collected from manned submersibles on rocky substrates near active hydrothermal sites in the south Pacific has provided a remarkably high proportion of new species. However, it is at present difficult to determine whether the abundance and diversity of carnivorous sponges in this collection is linked to the vicinity of hydrothermal sites, which provides solid substrata and general organic enrichment, and also stimulates a special sampling effort by direct methods. Carnivorous sponges cannot be considered as true members of the hydrothermal fauna, as they are apparently absent from the rich animal communities that thrive in the immediate environment of active smokers. The new species from the South Pacific include several representatives of Abyssocladia, previously synonymized with Phelloderma (Myxillina), increasing the microsclere heterogeneity of carnivorous sponges. Moreover, some other deepsea poecilosclerids, Euchelipluma spp. (Guitarridae) and some Esperiopsis spp. (Esperiopsidae) also appear to be carnivorous. This may suggest that carnivory appeared independently in several evolutionary lines of poecilosclerids. Conversely, however, the polyphyly of carnivorous sponges is not supported by a number of shared characters. The two hypotheses are discussed, but it is suggested, given the important morphological adaptations of these sponges, their ambiguous relationships with extant families of poecilosclerids and our rapidly increasing knowledge regarding their diversity, that it would be premature to drastically change the classification before having more information, especially of reproduction and molecular characters. Keywords: Cladorhizidae, Carnivorous sponges, Evolution, Deep Sea, Poecilosclerida

Introduction
The discovery that a representative of the Cladorhizidae, Asbestopluma hypogea Vacelet and Boury-Esnault, 1996 was carnivorous (Vacelet and Boury-Esnault 1995) has led to renewed interest in these strange deep-sea sponges that were previously only studied from a taxonomic point of view. Several lines of evidence suggest that this unexpected feeding habit is general in the Cladorhizidae as defined in Systema Porifera (Hajdu and Vacelet 2002) with three valid genera, Cladorhiza Sars, 1872, Asbestopluma Topsent, 1901 and Chondrocladia Thompson, 1873. It has been shown that in addition to A. hypogea several cladorhizids contain crustacean debris in the course of being digested (Kbler and Barthel 1999, Vacelet and Boury-Esnault 2002, Reiswig and Lee 2007). Furthermore, all the cladorhizids display morphological characters that are seemingly related to carnivory. They display a peculiar symmetrical shape, generally stipitate with lateral processes lined by hook-shaped microscleres. Most of them seem to be devoid of the sponge diagnostic attributes, i.e. an aquiferous system with canals, ostia, osculum and choanocyte chambers. An aquiferous system is present only in the genus Chondrocladia, in which, however, it is modified

and apparently not used for water filtration, but for the inflation of turgescent spheres at the surface of which prey capture is performed. Furthermore, recent observations suggest that this feeding regime also occurs in some other poecilosclerids that may have other family level affinities. These carnivorous sponges, which do not concur with the conventional definition of the phylum as given by Bergquist (1978) a sedentary, filter-feeding metazoan which utilizes a single layer of flagellated cells (choanocytes) to pump a unidirectional water current through its body, have developed an organization that is unique in the Metazoa, feeding on macro-prey by cells acting individually, without any digestive cavity (Vacelet and Duport 2004). The evolution, most likely from normal sponges, biology, ecology and diversity of such a remarkable derivation from a taxon that is considered as the most basal in the evolution of Metazoa, are fascinating new topics of research. The aim of this paper is to examine what is known to date of the diversity, classification and ecology of the carnivorous poecilosclerid sponges. How many taxa there are, whether they are monophyletic, or polyphyletic as carnivorous plants, and whether they are significant components of the deep-sea ecosystems, will be the main questions addressed.

108

Biodiversity of carnivorous sponges


At the end of the 20st century the Cladorhizidae numbered approximately 90 species. Most of them were described without any reference to their histological organization. This lack of information could be due to the poor preservation usual for deep-sea animals collected by dredging or trawling. It may be stressed too that the describers of cladorhizids were sponge taxonomists expecting a system of apertures, canals and choanocyte chambers, which in this case was absent or significantly modified. Several of them, however, such as Lundbeck (1905, p. 47), expressed their surprise that neither pores nor oscula have ever been mentioned. Careful observers such as Ridley and Dendy (1887) wrote The Crinorhiza forms appear to be without oscula and pores, nor have we succeeded in finding flagellated chambers, although some of the specimens were in very fair condition. It seems just possible, therefore, that, as originally suggested by Sars in the case of the first known species of the genus, Cladorhiza abyssicola, these sponges have some method of obtaining their supplies of nutriment which is quite different from that found in other sponges; this is, however, extremely unlikely. The same authors interpreted the lining of hook-like microscleres, which are now understood to be trapping devices for prey, as an efficient protection against parasites and other enemies. Recent observations on species that are definitely carnivorous, Asbestopluma hypogea and Chondrocladia gigantea (Hansen, 1885) have provided new information on their histology and organization. Moreover, a few recent taxonomic studies and studies in progress indicate that the diversity of cladorhizid sponges, and more generally of carnivorous sponges, in the deep sea is much higher than previously assumed. Since the beginning of the 21st century, 15 species have been described as new (Vacelet and Boury-Esnault 2002, Cristobo et al. 2005, Lehnert et al. 2005, Vacelet 2006, Reiswig and Lee 2007), increasing significantly the number of known species and resurrecting the genus Abyssocladia Lvi, 1964 which has been tentatively transferred from Phellodermidae to Cladorhizidae. The new species were collected in the deep south Pacific and south Atlantic, with a very high ratio of new species in the various collections, indicating that these large areas certainly still contain a large number of undescribed species. The study that I recently published on specimens from the deep Pacific (Vacelet 2006) is particularly indicative of the poor knowledge that we have of this fauna. This collection includes 9 species, of which 9 are new, although this area admittedly very large has been explored for sponges by the Challenger, Vitiaz and Galathea expeditions (Ridley and Dendy 1887, Koltun 1958, 1959, 1970, Lvi 1964). Deep-sea sponges have often been described from very few specimens, so that their intraspecific variability is poorly known, possibly wrongly resulting in an exaggerated splitting of species. This does not appear to be the case, as the new species are significantly different from any known species, and variability is low when several specimens are present. An example is Abyssocladia agglutinans Vacelet, 2006, which is known by two specimens that are exactly similar although distant by 557 km. In this study, such a high proportion of new species could be ascribed to the collection of the specimens by manned

submersibles on submarine ridges, in deep-sea environments where active hydrothermal vents favour general fauna enrichment and where hard substrates are relatively common. This could provide a higher diversity than the methods that were used in the deep Pacific by the Challenger, Vitiaz and Galathea expeditions during which the specimens were collected by blind trawling generally on mud bottoms. However, a preliminary study of cladorhizids collected by trawling off New Zealand (Kelly and Vacelet, in progress), including the Kermadec Trench which has been previously thoroughly explored by the Galathea expedition (Lvi 1964), also reveals a high ratio of undescribed species. Similar results were obtained by Cristobo et al. (2005) for the genus Chondrocladia in the south Atlantic. A collection presently under study (Fourt, Vacelet and Boury-Esnault, unpublished report to IFREMER) from the deep North Atlantic, an area which has been more thoroughly explored than the deep Pacific, also contains an unexpectedly high proportion of new species of Cladorhizidae, although not as high as in the Pacific. It is thus obvious that the diversity of cladorhizid sponges in the deep sea is far higher than is known to date and that many species, possibly genera, have yet to be discovered. It must be stressed, however, that caution must be exercised when describing such fragile sponges that are most often incomplete, in which some spicule categories are often precisely located, and which often collect pieces of other sponges due to the adhesive properties of their prey-trapping surfaces. None of the new species described in the genera Asbestopluma, Cladorhiza and Abyssocladia display any trace of aquiferous system or choanocyte chamber. The best preserved specimens display a regular arrangement of the microscleres that line the lateral filaments, with the alae of chelae or the teeth of sigmancistras outwardly directed, an arrangement which allows the capture of the thin setae of crustacean prey. Moreover, a few specimens contain crustacean debris, especially clear in Abyssocladia huitzilopochtli Vacelet, 2006, also found in two new Chondrocladia spp. These facts confirm that all the sponges presently classified in Cladorhizidae are very likely carnivorous. Indisputable general evidence, however, is difficult to provide in these fragile deep-sea animals, which easily lose the lateral filaments or appendages on which the prey are trapped and on which experimentation is not easy. Even in the best preserved specimens, prey are rarely visible, which is not surprising, considering that carnivorous sponges are sit-andwait predators that very likely do not eat frequently in the oligotrophic deep sea. Furthermore, it appears that Asbestopluma, Chondrocladia and Cladorhiza spp. are not the only carnivorous sponges. Several deep-sea poecilosclerids that are, or were, classified in diverse families due to their microsclere spicules, but that display morphology similar to that of Cladorhizidae, may also be carnivorous. As already pointed out, Abyssocladia, previously synonymized with Phelloderma Ridley and Dendy, 1886 (Phellodermidae, Myxillina), is now reconsidered as a valid genus of Cladorhizidae with seven species (Vacelet 2006) and several new species in the course of description. A carnivorous feeding habit is highly likely in Euchelipluma Topsent, 1909, which has been classified in the Guitarridae

109

due to the presence of placochelae similar to those highly diagnostic of filter-feeding sponges belonging to Guitarra Carter, 1874. The genus contains three species, E. pristina Topsent, 1909, E. (Desmatiderma) arbuscula Topsent, 1928 and E. elongata Lehnert et al., 2006. All display a pinnate shape, with regularly arranged lateral filaments lined by microscleres with the alae and teeth outwardly directed. This skeleton organization, the shape of the sponge and the seemingly absence of aquiferous system suggest a carnivorous feeding habit, which has been confirmed by the observation of crustaceans debris in some specimens of E. pristina (Vacelet 1999, Vacelet and Segonzac 2006). Another example may be found in the Esperiopsidae, where deepsea Esperiopsis spp. of the group of E. villosa Carter, 1874, including E. symmetrica Ridley and Dendy, 1886 and E. desmophora Hooper and Lvi, 1989, have a morphology that is highly suggestive of carnivorous feeding. The similarities in shape and skeleton arrangement, including a reinforcement by desmas which is rather unsual in poecilosclerids, between Esperiopsis desmophora and the Ordovician sponge Saccospongia baccata Bassler suggest that carnivory may be very ancient in poecilosclerid sponges. It thus appears that there is in fact a very high biodiversity of carnivorous sponges in the deep sea. There are now more than one hundred described representatives of Cladorhizidae which very likely have this feeding habit, but this number is certainly much higher, and it appears too that carnivory has been developed in some representatives of other poecilosclerid families as construed in the present consensual classification. So far, however, this feeding habit is restricted to the order Poecilosclerida. A role of exotyles present in diverse orders of demosponges in trapping large particles has been suggested by Hajdu (1994), but this does not indicate a carnivorous feeding habit in sponges possessing exotyles, in which an aquiferous system has generally been reported. The high specific diversity and the biogeographical distribution of the carnivorous sponges are difficult to correlate with peculiarities in the reproduction mechanisms and to the dispersal ability, as the reproduction of these species is poorly known. In the Mediterranean, Asbestopluma hypogea has been able to colonize several littoral caves most probably from deep-sea canyons (Bakran-Petricioli et al. 2007), suggesting relatively high dispersal ability.

rhizoids as an anchoring base, or on rocky bottom where their diversity has certainly been more seriously underestimated. However, it remains unknown which is the more favourable type of bottom. In a few cases, it seems that the same species, or very close species, may live on both types of substratum, developing either an enlarged fixation base on rocks or rhizoids in mud. The high diversity found in collections taken by manned submersibles from rocky bottom near active hydrothermal vents might suggest that this previously poorly sampled environment is their preferred habitat. It would appear that carnivorous sponges, as generally filter-feeding sponges, do not take part in the rich oases of life thriving in the immediate proximity of active hydrothermal vents. One exception is Cladorhiza methanophila (Vacelet et al. 1995, 1996) which constitutes unusually large populations near methane sources of a mud volcano in the Barbados because of symbiosis with methanotroph bacteria. The number and diversity of carnivorous sponges, however, could be enhanced at a certain distance from such sites both by the unusual prevalence of rocky substrates due to volcanic activity, and by a general enrichment of the deep-sea ecosystem. No quantitative data are available to date in relation to this question. The abundance of Chondrocladia lampadiglobus Vacelet, 2006 on the East Pacific Ridge between 2586 and 2684 m depth has been estimated at 1-2.6 individuals per km of path by the manned submersible Nautile, but this estimation was made some hundreds of meters from active vents and we do not know if this is general on the East Pacific Ridge. So far hydrothermal sites have been the favorite targets of exploration from manned submersibles and ROVs, introducing an evident bias. In a Pacific abyssal plain rich in polymetallic nodules, the density of Cladorhizidae has been estimated respectively at 16, 4 and 5 individuals per hectare for Chondrocladia, Cladorhiza and Asbestopluma (Tilot 1992). Given their relatively small number and small size, it would not appear at present that carnivorous sponges play an important role in the deep-sea food chains.

Evolution and classification


The diversity of microscleres and of organization in carnivorous poecilosclerids raises a puzzling problem of evolution and classification. It has been pointed out by Hajdu and Vacelet (2002) that the family Cladorhizidae as construed in Systema Porifera in the suborder Mycalina lacks a clear synapomorphy and that it could be polyphyletic. However, this has to be reexamined, as several shared characters of Cladorhizidae were also found in other sponges, such as Euchelipluma, Abyssocladia and Esperiopsis spp. that now appear to belong to a set of carnivorous sponges. The three cladorhizid genera Asbestopluma, Cladorhiza and Chondrocladia display a special shape, generaly stipitate, pinnate or branching (Table 1), and their megascleres, although differing in size and shape according to their localization in the sponge, are referable to a single category (mycalostyles) with the same skeleton arrangement. Asbestopluma and Cladorhiza share the general organization, with a stipitate, often pinnate shape and absence of aquiferous system, but not Chondrocladia, which has kept the sponge aquiferous system although its function and organization are

Ecology
All carnivorous sponges are deep-sea species that were previously considered as well adapted to the most foodpoor mid-basin areas (Gage and Tyler 1991). They may be considered as sit-and-wait predators, spending a minimal amount of energy during long periods between rare feeding opportunities. Three species of Cladorhizidae have been found at more than 8000 m depth and Asbestopluma occidentalis Lambe, 1883 is the deepest known sponge, living in hadal depth at 8840 m (Koltun 1970). A few cladorhizids, however, are able to live at only 100 m depth in high latitudes, and the Mediterranean cavernicolous species, Asbestopluma hypogea, lives at a few meters depth in a cold-water littoral cave, but most likely colonizes this habitat from a deep-sea population. They live either on muddy bottom, where they often develop

110

Table 1: Characters of the genera of carnivorous sponges (including some unpublished data). +: present in all species. : present in some species only. * present in a single species. Abyssocladia Stipitate, pinnate or branching Stipitate, with inflated spheres Rhizoids Aquiferous system Mycalostyles Basal substrongyles Basal desmas Spinose tylostyles or oxeas Microtylostyles Surface lining by microscleres Palmate anisochelae Anchorate anisochelae Anchorate isochelae Palmate isochelae Arcuate isochelae Abyssochelae Placochelae Sigmas Sigmancistras Forceps + Asbestopluma Chondrocladia + + + + + + + Cladorhiza + + + * * + + + + + * + + ? + * + Esperiopsis (pars) + Euchelipluma +

+ + * + +

+ + + *

significantly modified. The chelae microscleres of the three genera, however, are different, being palmate anisochelae in Asbestopluma, anchorate/unguiferate anisochelae in Cladorhiza and anchorate isochelae in Chondrocladia, indicating possible polyphyly according to the present interpretation of chelae (Hajdu et al. 1994). The value of microscleres, and especially of cheloids in sponge classification has often been the subject of debate. However, the most common opinion today for Poecilosclerida is that summarized by van Soest (2002, p. 518): Because of complex morphology, chelae are considered to reflect phylogenetic relationships both at the family and genus level. This seemingly phyletic heterogeneity of sponges conventionally classified in Cladorhizidae is even more evident for the whole set of carnivorous sponges with the recent additions. Carnivorous sponges now very likely include: (i) three species of Euchelipluma, presently classified in Guitarridae due to the presence of the diagnostic placochelae; (ii) several species of Esperiopsis of the villosa group, currently classified in the Esperiopsidae, with palmate isochelae (Vacelet 2006); (iii) seven described species of Abyssocladia and several new species under study from New Zealand and the North Atlantic, currently classified in the Cladorhizidae although they have palmate or arcuate isochelae. The case of Abyssocladia is particularly puzzling because the genus was previously synonymized, on the basis of possession of special isochelae (abyssochelae), with Phelloderma Ridley and Dendy, 1886 in family Phellodermidae van Soest and Hajdu, 2002 in the suborder Myxillina in which the chelae are not palmate, but arcuate. However, it now appears that the type of isochelae, arcuate or palmate, is rather uncertain in Abyssocladia as the genus stands now with the inclusion of the newly described

species and of species in the process of description, as will be explained below in greater detail. The heterogeneity of the Cladorhizidae, and more generally of carnivorous poecilosclerids, could be interpreted in two different ways: (i) carnivory has developed relatively recently in several different lines of evolution of Poecilosclerida, some or all of the characters that they share being homoplasies due to their special mode of life, with the consequence that the various genera of carnivorous sponges are to be classified in these different lines, matching up several families of Mycalina, possibly of Myxillina; (ii) carnivory developed very early in Poecilosclerida, possibly before the divergence of Mycalina and Myxillina, the shared characters being symplesiomorphic, with the consequence that they are to be classified in a single high level taxon, possibly a distinct suborder. The present set of data, summarized in Table 1, with the shared and distinctive characters of carnivorous sponges, will be examined and discussed below.
Fig. 1: Representatives of four genera of carnivorous sponges illustrating the diversity of microscleres in sponges with a similar morphology and organization. All have similar megascleres (mycalostyles) with or without addition of strongyles. A. Abyssocladia naudur Vacelet, 2006, paratype, abyssochelae, sigma and sigmancistra. B. Asbestopluma agglutinans Vacelet, 2006, holotype and paratype, anisochelae 1, anisochelae 2, and sigmancistra. C. Cladorhiza segonzaci Vacelet, 2006, holotype and paratypes, anchorate anisochelae, sigma and sigmancistra. D. Euchelipluma pristina Topsent, 1919, specimen from Barbados, isochela, placochela and sigmancistra. A, B and C from Vacelet (2006). D from Vacelet and Segonzac (2006).

111

112

All the presumed carnivorous sponges share a certain number of morphological characters. They display a special outer morphology, absence or significant modification of the aquiferous system, an unusual microsclere arrangement and the same type of megasclere skeleton. A stipitate shape with symmetrical lateral expansions is found in all genera. For instance, the feather-like shape with a more or less laterally compressed axis bearing symmetrical laterals filaments lined by prey-trapping microscleres is found in all genera except Chondrocladia, and species classified in different genera or families may be remarkably similar in shape (Fig.1). The absence of canal system and choanocyte chambers also appears to be general, again with the exception of Chondrocladia. The main skeleton is always composed of more or less modified mycalostyles, frequently strongly fusiform, building longitudinal axes, showing only size differentiation according to their position in the main or secondary axes, without an ectosomal differentiation. In several species of the various genera, the base of the main axis is reinforced by mycalostyles modified in short strongyles. In three species, Euchelipluma arbuscula, Asbestopluma (Helophloeina) stylivarians (Topsent, 1928), Esperiopsis desmophora, again belonging to various genera and families, these strongyles are themselves modified in desmas. As far as may be inferred from the description of often poorly preserved specimens, most, possibly all species have on their lateral extensions a lining of sigmoid or cheloid microscleres arranged with teeth and alae outwardly directed. Carnivorous sponges are the only poecilosclerids possessing true sigmancistras, which are sometimes difficult to differentiate from sigmas but most often clearly distinct. Sigmancistras are recorded in all genera except Esperiopsis, although their occurrence is not general in all the species. They have been reported for only two Asbestopluma spp. and four Chondrocladia spp., but they are more frequent in Cladorhiza and occur in all species of Abyssocladia and Euchelipluma. Sigmancistras have been supposed by Hajdu (1994) to be the primitive condition of development of diverse poecilosclerid microscleres (cyrtancistra, diancistra, clavidisc), an hypothesis which may underline the possible antiquity of carnivory in Poecilosclerida. An interesting issue for other shared characters of carnivorous sponges could be the reproductive phenomena. Reproduction is very poorly known in these deep-sea sponges, although large embryos have been reported fairly often (for instance by Lundbeck 1905), but without precise description. These reports and preliminary observations suggest that carnivorous sponges could share some peculiarities. In several genera the embryos have been described as large, including fascicles of megascleres and a variety of microscleres, and with a special envelope which suggested to Topsent (1909) that they were gemmules rather than embryos in Cladorhiza spp. According to personal unpublished observations in Asbestopluma hypogea, young embryos have multiflagellated cells (Fig. 2), a character which is very unusual in sponges (multiflagellated cells are known only in the trichimella larva of Hexactinellida). This is confirmed by preliminary observations in another species of Asbestopluma (Leys, pers. comm.). The spermatogenesis of A. hypogea also appears very unusual (Vacelet 1996) (Fig. 2), possibly in relation with the absence of choanocyte chambers from which the sperm

cells of sponges generally derive. Spermatocysts develop in the body, and then migrate towards the end of the lateral processes, where the mature cysts become free. In the mature cyst, sperm cells are surrounded by two envelopes, the inner one unicellular, the outer one made by closely intertwined cells. Two tufts of forceps are diametrically protruding on the mature cyst, and may serve either as flotation devices for dispersal of the whole cyst or for capture by the Velcro-like lining of the filaments of another individual. Preliminary light microscope observations suggest that similar phenomena may occur in Cladorhiza methanophila (Vacelet and BouryEsnault 2002), in Chondrocladia gigantea (Hansen, 1885) (Kbler and Barthel 1999), and in Euchelipluma pristina (unpublished). These shared characters of carnivorous sponges, however, are in contrast with the characters of the chelae microscleres, which is an important character for the suborder classification of Poecilosclerida. The chelae are arcuate in the suborder Myxillina, and palmate in the suborder Mycalina (Hajdu et al. 1994, Hooper and van Soest 2002). The presence of palmate anisochelae, anchorate anisochelae and anchorate isochelae in the three genera of Cladorhizidae as defined in Systema Porifera, was already rather puzzling. The addition of Euchelipluma, Abyssocladia and some Esperiopsis spp., in which are present placochelae and isochelae grading from palmate to arcuate, greatly increases the microsclere diversity of carnivorous poecilosclerids. Moreover, most of the abyssochelae and isochelae of Abyssocladia, as well as the isochelae of Euchelipluma are difficult to assign precisely to the arcuate or palmate type. In several species, isochelae and abyssochelae may be palmate to arcuate, or clearly palmate, or clearly arcuate (confirmed by Hajdu, pers. comm.). In a few other Abyssocladia and Asbestopluma, some microsclere characters do not agree with the present distinction between microscleres of Mycalina and Myxillina. Abyssocladia dominalba Vacelet, 2006 has small anisochelae with one end palmate and the other arcuate, in addition to arcuate isochelae and palmate to arcuate abyssochelae. An Asbestopluma sp. from New Zealand, in the course of description by Kelly and Vacelet, has arcuate, possibly anchorate, anisochelae in addition to the normal palmate anisochelae. An Abyssocladia sp., which will also be described from New Zealand, has isochelae intermediate between the arcuate and the anchorate types. This supports the hypothesis of polyphyly of carnivorous poecilosclerids, which would mean that several shared characters were obtained by homoplasic evolution in several lines of Mycalina, in relationship with carnivorous feeding. Is that likely? This feeding mode does not in fact need any aquiferous system and could induce a certain number of common features. A plumose or pinnate shape, which is most propitious for the passive capture of swimming prey, needs strong axes of fusiform spicules, ideally reinforced at the base of the stalk by intermingled spicules such as a cover of short vermiform strongyles or of desmas. Prey capture also requires a lining of the lateral expansions by hook-like cheloid microscleres, able to trap the setae or appendages of invertebrate prey. Diverse shapes of chelae and sigmas are propitious for such a role, and chelae, sigmancistras and placochelae appear particularly well adapted. Possible similarities in reproduction processes could

113

Fig. 2: Reproductive stages in Asbestopluma hypogea. A. Semi-thin section through an embryo, showing internal cells with yolk inclusions, an outer layer of multiflagellated cells (arrow) and a maternal envelope. B. TEM view of a multiflagellated cell of the same embryo. C. Mature sperm cyst at the tip of a filament, showing sperm cells (Sp), an inner envelope (En) and an outer envelope (Oe) made of closely intertwined cells, and two tufts of forceps (desilicified) within their sclerocytes (Fo).

also be induced by the absence of aquiferous system and life in the deep sea. Such a hypothesis of convergent evolution of different characters linked to a carnivorous feeding habit from different families of Poecilosclerida cannot be ruled out. However, it is rather unlikely that the microscleres appeared

independently in several lines of evolution, for instance the placochelae in Euchelipluma and in Guitarridae. Moreover, this interpretation would mean the allocation of each genus to a precise family, which in fact appears difficult. The affinities of Asbestopluma with Mycalidae, of Esperiopsis

114

with Esperiopsidae and of Euchelipluma with Guitarridae rely only on a single category of microscleres and on similar megascleres which, however, are very differently arranged in filter-feeding representatives of these families. Furthermore, Cladorhiza, Chondrocladia and Abyssocladia would remain with uncertain affinities in the present classification system. The second hypothesis is more in agreement with the characters shared by carnivorous poecilosclerids, such as special morphology, megasclere nature and arrangement, microsclere arrangement, and the absence or modification of aquiferous system. Only the genus Chondrocladia would appear to be distinctive by its morphology and the preservation of an aquiferous system. However, a taxon including all the carnivorous sponges, even excluding Chondrocladia, would not be in agreement with the present classification of Poecilosclerida. The highly diverse microsclere complement found in the various genera would match more or less adequately several families of Mycalina, possibly also of Myxillina. This could mean that the present basis of the classification of Poecilosclerida in suborder is not appropriate. It could also mean that carnivory is very ancient in sponges with a possible support from the similarities between the Ordovician Saccospongia baccata and Esperiopsis desmophora and appeared in an early ancestor of Poecilosclerida with mycalostyle megascleres and very diverse cheloid microscleres, to be considered as symplesiomorphies. These microscleres could subsequently have evolved differently in the diverse lines of Poecilosclerida, developing a clearer distinction between palmate, arcuate and anchorate types of chelae. It is interesting to note that in carnivorous poecilosclerids the cheloid microscleres have an obvious function, for which both their shape and arrangement are well designed, while in filter-feeding poecilosclerids they have no apparent function. Was this function lost in filterfeeding sponges, or was it relatively recently designed by carnivorous sponges from preadapted microscleres of presently unknown usefulness? I consider that there is presently not enough strong evidence for one or another of these hypotheses, which rest on morphological and spicule characters. As suggested by the surprising specific diversity found in the recent collections, we may in the near future expect the discovery in the deep sea of a large number of undescribed species, which will bring new information. The data on reproduction are still very incomplete and poorly understood, but the uniqueness of preliminary observations in a few species suggests that they will provide significant information. Furthermore, indications from gene sequences will certainly become available soon and will bring new data on the molecular phylogeny of Poecilosclerida, which is at present insufficiently achieved and includes very few exploitable data for carnivorous sponges. My feeling is that it would be preferable to wait for this new information before offering an interpretation of the phylogeny or a formal proposal for the classification of carnivorous Poecilosclerida. However, in predictive mode, the present set of data appears to me more in agreement with the hypothesis of monophyly or paraphyly of carnivorous poecilosclerids, possibly to be considered as a distinct suborder of Poecilosclerida, rather than with a polyphyletic interpretation.

Acknowledgements
I am grateful to Michel Segonzac (Ifremer) and Michelle Kelly (NIWA) for entrusting me with the study of deep-sea specimens. I also acknowledge the technical help of Chantal Bzac, Centre dOcanologie de Marseille. This paper benefited from fruitful discussions and comments from Nicole Boury-Esnault and Eduardo Hajdu.

References
Bakran-Petricioli T, Vacelet J, Zibrowius H, Petricioli D, Chevaldonn P, Rada T (2007) New data on the distribution of the deep-sea sponges Asbestopluma hypogea and Oopsacas minuta in the Mediterranean Sea. Mar Ecol Evol Persp 28(suppl. 1): 1023 Bergquist PR (1978) Sponges. Hutchinson & Co, London Cristobo FJ, Urgorri V, Rios P (2005) Three new species of carnivorous deep-sea sponges from the DIVA-1 expedition in the Angola Basin (South Atlantic). Organisms Diversity & Evolution 5: 203-213 Gage JD, Tyler PA (1991) Deep-sea biology. Cambridge University Press, Cambridge. Hajdu E (1994) A phylogenetic interpretation of hamacanthids (Demospongiae, Porifera), with the redescription of Hamacantha popana. J Zool 232: 61-77 Hajdu E, Vacelet J (2002) Family Cladorhizidae. In: Hooper JNA, van Soest RWM (eds). Systema Porifera: a guide to the classification of sponges, vol. 1. Kluwer Academic/Plenum Publishers, New York. pp. 636-641 Hajdu E, van Soest RWM, Hooper JNA (1994) Proposal for a phylogenetic subordinal classification of poecilosclerid sponges. In: van Soest RWM, van Kempen TMG, Braekman JC (eds). Sponges in time and space: biology, chemistry, paleontology. Balkema, Rotterdam. pp. 123-140 Hooper JNA, Lvi C (1989) Esperiopsis desmophora n. sp. (Porifera: Demospongiae): a desma-bearing Poecilosclerida. Memoir Queensl Mus 27: 437-441 Hooper JNA, van Soest RWM (2002) Order Poecilosclerida Topsent, 1928. In: Hooper JNA, van Soest RWM (eds). Systema Porifera: a guide to the classification of sponges, vol. 1. Kluwer Academic/ Plenum Publishers, New York. pp. 403-408 Koltun VM (1958) Cornacuspongida of sea waters washing the South Sakhalin and the South Kurile Islands region. Issled dalvenost Mor SSSR 5: 42-77 Koltun VM (1959) Corneosiliceous sponges of the northern and far eastern seas of the USSR. Zoological Institute of the Academy of Sciences of the USSR, Moscow-Leningrad Koltun VM (1970) Sponge fauna of the north-western Pacific from the shallows to the ultra-abyssal depths. Inst Oceanol Acad Sci USSR 86: 165-221 Kbler B, Barthel D (1999) A carnivorous sponge, Chondrocladia gigantea (Porifera: Demospongiae: Cladorhizidae), the giant deepsea clubsponge from the Norwegian trench. Memoir Queensl Mus 44: 289-298 Lehnert H, Watling L, Stone R (2005) Cladorhiza corona sp. nov. (Porifera: Demospongiae: Cladorhizidae) from the Aleutian Islands (Alaska). J Mar Biol Assoc UK 85: 1359-1366 Lvi C (1964) Spongiaires des zones bathyale, abyssale et hadale. Galathea Rep 7: 63-112

115

Lundbeck W (1905) Porifera. Pars II: Desmacidonidae (pars). Danish Ingolf-Exp 6(2): 1-219 Reiswig HM, Lee WL (2007) A new species of Cladorhiza (Porifera: Cladorhizidae) from S. California (USA). In: Custdio MR, LboHajdu G, Hajdu E, Muricy G (eds). Porifera research: biodiversity, innovation, sustainability. Srie Livros 28. Museu Nacional, Rio de Janeiro. pp. 517-523 Ridley OS, Dendy A (1887) Report on the Monaxonida collected by H.M.S. Challenger during the years 1873-1876. Rep Sci Res Voy H.M.S. Challenger, Zool 20(59): 1-275 Tilot V (1992) La structure des assemblages mgabenthiques dune province nodules polymtalliques de lOcan Pacifique tropical Est. Thesis, Universit de Bretagne occidentale, Brest Topsent E (1909) tude sur quelques Cladorhiza et sur Euchelipluma pristina n. g. et n. sp. Bull Inst ocanogr Monaco 151: 1-23 Topsent E (1928) Une mycaline productrice de desmes Desmatiderma arbuscula n. g., n.sp. Bull Inst ocanogr Monaco 519: 1-8 Vacelet J (1996) Deep-sea sponges in a Mediterranean cave. In: Uiblein F, Ott J, Stachowitsch M (eds). Deep-sea and extreme shallow-water habitats: affinities and adaptations, 11. Austrian Academy of Sciences, Vienna. pp. 299-312 Vacelet J (1999) Outlook to the future of sponges. Memoir Queensl Mus 44: 27-32 Vacelet J (2006) New carnivorous sponges (Porifera, Poecilosclerida) collected from manned submersibles in the deep Pacific. Zool J Linn Soc 148: 553-584

Vacelet J, Boury-Esnault N (1995) Carnivorous sponges. Nature 373: 333-335 Vacelet J, Boury-Esnault N (2002) A new species of carnivorous deep-sea sponge (Demospongiae: Cladorhizidae) associated with methanotrophic bacteria. Cahiers Biol Mar 43: 141-148 Vacelet J, Boury-Esnault N, Fiala-Mdioni A, Fisher CR (1995) A methanotrophic carnivorous sponge. Nature 377: 296 Vacelet J, Duport (2004) Prey capture and digestion in the carnivorous sponge Asbestopluma hypogea (Porifera: Demospongiae). Zoomorphology 123: 179-190 Vacelet J, Fiala-Mdioni A, Fisher CR, Boury-Esnault N (1996) Symbiosis between methane-oxidizing bacteria and a deep-sea carnivorous cladorhizid sponge. Mar Ecol Prog Ser 145: 77-85 Vacelet J, Segonzac M (2006) Porifera. In: Desbruyres D, Segonzac M, Bright M (eds). Handbook of deep-sea hydrothermal vent fauna, 18. Denisia. pp. 35-46 van Soest RWM, Hajdu E (2002) Family Phellodermidae fam. nov. In: Hooper JNA, van Soest RWM (eds). Systema Porifera: a guide to the classification of sponges, vol. 1. Kluwer Academic/Plenum Publishers, New York. pp. 621-624 van Soest RWM (2002) Suborder Myxillina Hajdu, van Soest & Hooper, 1994. In: Hooper JNA, van Soest RWM (eds). Systema Porifera: a guide to the classification of sponges, vol. 1. Kluwer Academic/Plenum Publishers, New York. pp. 515-520

Porifera research: Biodiversity, innovation and sustainaBility - 2007

117

South American continental sponges: state of the art of the research


Ceclia Volkmer-Ribeiro
Museu de Cincias Naturais, Fundao Zoobotnica do Rio Grande do Sul. Rua Dr. Salvador Frana, 1427. 90690-000, Porto Alegre, RS, Brazil. Research fellow of CNPq. cvolkmer@fzb.rs.gov.br Abstract: An intensive survey and study of the South American freshwater sponges started around four decades ago. A large number of results came out which now allows a first overview. An outstanding number of new species of freshwater sponges and even new genera were described. The new materials presented remarkable differences when compared with species from the other continents. This new sponge continental fauna has ever since been subjected to constant checking due to continued and extensive surveys, redescriptions and habitat descriptions. The diversity of the South American sponge fauna is remarkable being possibly the richest in the world. All the review efforts dedicated to this branch of Demosponges are enhancing confidence in the perception of how thrustworthy some characters are and how much their degree of variation is linked to specific habitats, thus enabling the application of this taxonomic knowledge. Keywords: Continental sponges, research, South America, state of the art

Build and rebuild your software


Evolution has been the main word leading the research of all of us as biologists and naturalists in our efforts to understand how living beings have organized themselves and how living processes of all sorts play around us. Lately, with the growing success of replicating life in our labs, evolution includes the idea of how we are setting up life around the world and even around the planetary system. One but not the least of the resulting consequences is that taxonomists are being urged to come up with applied propositions stemming from their taxonomic research. This taxonomic perfectioning takes place and weight as our scientific life goes by and particularly if it stays long enough as that of a large number of us has been staying. Looking back and forth becomes a daily routine with special emphasis on ideas, hypotheses and conclusions, challenged by every new evidence, findings and reading. One starts with grain-size evidences, cements them together, builds up his/her initial operating system and from then on builds and rebuilds oneselfs rationale. Plain common sense, but a painful process And here it is when Science turns into Art and Philosophy. Into art because a scientists always present inquiring impulses are driven by a desire to grasp and reproduce the perfection offered by Nature. Into philosophy because only this endeavour will heal the human mind in its failure to reach that goal.

The gains of the continental approach


A whole new continental fauna of Demosponges, that of the South American continent, was disclosed mainly over the last half of the 20th century. This continental fauna may be comparable in importance to the findings of fossil marine

sponge faunas preserved in rocky outcrops which lately emerged as continental areas. The main difference between these lies on the fact that those marine faunas are a snapshot of the past and not an ongoing movie as the present continental sponge faunas are. And, as such, research on continental sponge faunas benefits from playbacks whenever collecting sites are re-visited to check hypotheses, improve descriptions and study niches (niche sensu Hutchinson 1967). But more than offering easy access to previous collecting sites, continental sponge faunas offer the chance to study biological/ ecological (adaptation),versus geologic (phylogeny) evolution, when continental drift is considered and continental plates are understood as analogous to huge islands. A large isolation degree in respect to time and space causing easier determination and follow up of geographic barriers and vicariance effects (Nelson and Platnick 1981) appears then as important aspects to drive the search for evolving characters in these continental Demosponge faunas aiming to the perfection of species identification, the detection of endemisms and the estimation of the time consumed along all these processes. All such aspects are obviously harder to detect when marine demosponges are considered. Three main realms should be in fact considered in what respects sponges: the marine, the epicontinental and the continental one. The first and third ones need no further explanation. The second one has to do with those seas in the process of continental enclosure, so turning into brackish and then fresh water. This epicontinental marine fraction of the present world waters has seldom been surveyed for sponges, in spite of the fact that other phyla were studied in detail and seen to pass by a drastic reduction in biodiversity, like, for instance the Echinoderms in the Baltic Sea (Hutchinson 1967). The presently known rich marine versus poor freshwater

118

poriferan biodiversity (Hooper and van Soest 2002) certainly allows for the expectation that the present epicontinental sponge faunas may also offer very interesting and intriguing selection processes and local extinctions prior to adapting to freshwater.

Continental surveying of an unknown sponge fauna


The first descriptions of South American continental sponges were produced in the 19th century and were based on a few specimens gathered in the Orinoco, Amazon and Uruguay Rivers by foreign explorers and deposited mainly in English and German Museums. The next main taxonomic efforts date to the middle of the last century, when Bonetto and Ezcurra de Drago started to survey the Argentinean continental sponges (Ezcurra de Drago 1971) and VolkmerRibeiro the Brazilian ones (Volkmer-Ribeiro 1981) resulting at present in a number of twenty nine new species, six new genera and one new extant family described, plus one new fossil species and family defined. At this time significant freshwater sponge collections were initiated by these authors at respectively the Instituto Nacional de Limnologia - INALI, Santa F, Argentina and the Museu de Cincias Naturais, Fundao Zoobotnica do Rio Grande do Sul, Rio Grande do Sul, Brazil. Bonetto and Ezcurra de Drago as well as Volkmer-Ribeiro initially faced a confusing situation in what respected the taxonomy of the worlds freshwater sponges long ago split by Carters taxonomic proposals (Jewell 1952). The problem was however overcome due to the remarkable differences that the new materials presented when compared with the descriptions available for the species already known. In that way an outstanding number of new species was described which is resisting the continued studies and surveys that are being carried out until the present. The appearance of Penney and Raceks (1968) comprehensive revision of the worlds freshwater sponges offered next a sound basis for revisional studies of species and genera and proposition of new genera. The master lines established by Penney and Racek were followed by both of the authors, Volkmer-Ribeiro and Ezcurra de Drago, resulting in the validation and enlightening of prior proposals for diverse continental sponge genera by Gray (1867), according to detailed redescriptions (VolkmerRibeiro and De Rosa-Barbosa 1972, Volkmer-Ribeiro 1984, Volkmer-Ribeiro and Costa 1992, Volkmer-Ribeiro and Tavares 1995, Tavares and Volkmer-Ribeiro 1997) of new materials with South American species, which had been only briefly described before.

South American continental plates. In this way genera with exclusive Nearctic-Neotropical distribution came into light such as Corvomeyenia Weltner, 1913 (Volkmer-Ribeiro et al. 2005) and Anheteromeyenia Schrder, 1927 (VolkmerRibeiro 1986a), or with predominant occurrence in these two continents like Racekiela Bass and Volkmer-Ribeiro, 1998 (Bass and Volkmer-Ribeiro 1998).

One genus and five continental plates drifted apart


The continued concern with the search for trustworthy diagnostic specific characters present in dried preserved materials (so that comparative studies could keep on encompassing old preserved materials) was deepened next with the revision of Metania Gray 1867, which has a tropical distribution. Such an effort sprung from the large number of specimens of this genus collected particularly in the Brazilian and Venezuelan Amazonia. At this time the search for what would become trustworthy diagnostic characters for species identification was centered on the large isolated sponge faunas at the plates of South America, Africa, Australia, India and Indonesia split from Gondwana and drifted apart. The resulting study brought to attention the existence of a gondwanian fauna of freshwater sponges (Volkmer-Ribeiro 1986b, Volkmer-Ribeiro and Costa 1992, 1993, Silva and Volkmer-Ribeiro 2001), which Volkmer-Ribeiro and De Rosa-Barbosa (1979) had already indicated by extending the occurrence of the family Potamolepidae from the Ethiopian to other gondwanian plates. An array of characteristics came to light again which confirmed Grays (1867) insight of the value of gemmoscleres, microscleres and the gemmule structure for the definition of genera and species.

Timing the birth of a continental sponge fauna


Also, from the studies on Metania it was possible for the first time to envision the time elapsed in attaining speciation and generic diversification for a group of continental sponges i.e. the Cretaceous drifting apart of the Gondwanian plates. Given the established paradigm that geologic time scales are those required to produce measurable change, particularly of gemmoscleres and microscleres shape and size, as well as on number of spicule categories present, a continued revisional effort was extended to all genera of South American continental sponges, allowing a more confident redefinition of monospecific genera (Acalle, Volkmer-Ribeiro and De Rosa-Barbosa 1972, Uruguaya and Sterrastrolepis, VolkmerRibeiro and De Rosa-Barbosa 1979, and the recognition of new genera (Oncosclera Volkmer-Ribeiro, 1970, Saturnospongilla Volkmer-Ribeiro, 1976, Corvoheteromeyenia, Ezcurra de Drago, 1979, Racekiela Bass and Volkmer-Ribeiro, 1998) as well as the description of new species, among which those where monospecific genera descriptions had been based on: Saturnospongilla carvalhoi, Sterrastrolepis brasiliensis. At present the largest number of records in the South American freshwater sponges are from Brazil and Argentina but reports have also been produced for Suriname (Ezcurra de Drago 1975), Venezuela (Bonetto and Ezcurra de Drago 1973, Volkmer-Ribeiro and Pauls 2000), Chile (Ezcurra de Drago 1974, Kilian and Wintermann-Kilian 1976) and Uruguay

Two neighboring continental plates


A milestone mark at this point was the study of the Edward Potts collection of type materials of the species he described for the United States and Canada (Potts 1887) and deposited at the Academy of Natural Sciences of Philadelphia (VolkmerRibeiro and Traveset 1987). That collection had not been taken in consideration by Penney and Racek (op. cit.). The idea was that comparative descriptions of species restricted to the Nearctic/Neotropical regions would favor any future vicariance studies due to the vicinity of the North and the

119

(Berroa Beln 1968). The South American continental plate has so been crossed from north to south and east to west leading to the idea that a large number of habitats has yet to be surveyed for continental sponges in this remarkably diverse continent. Now, quite a different picture has emerged of this South American fauna showing that it is one of the richest, if not the richest in the world.

Applied sponge taxonomy


Continuous checking of habitat characteristics versus species occurrence is a tool never discarded by specialists in their search for the confirmation of a species status and the description of ecomorphic variations of characters (VolkmerRibeiro 1973, Poirrier 1974). The performance of the abovementioned procedures, besides submitting species and genera to constant revisional efforts generates confidence in species definitions and provides a series of applications for this taxonomic knowledge. The first concerns the monitoring of freshwater habitats with respect to the integrity of their biodiversity or their restoration with environmental recovering practices. The knowledge now available encompasses the detection of several sponge assemblages in some typical South American habitats, such as Coastal ponds, lakes and lagoons (VolkmerRibeiro and Machado 2007), Cerrado (Savannah) ponds where an assemblage of five species thrive or where they formed spongillite deposits in the past (Volkmer-Ribeiro et al. 1998). Also, particular sponge assemblages have been detected in large South American rivers, as for instance the rocky bottoms of the middle Uruguay river (Ezcurra de Drago and Bonetto 1969), the rocky tributaries of the middle Paran river, as well as in the macrophyte stands of its middle floodplain (Ezcurra de Drago 1993, 2003), in Amazonian river rocky bottoms and in their marginal seasonally flooded forests (Batista et al. 2003). The application of this taxonomic tool is also proving to be rewarding onto environmental and climatic paleointerpretations, following the identification of sponge species based on the spicules detected in columns of recovered lake sediments of quaternary age (Siffeddine et al. 1994, Volkmer-Ribeiro and Turcq 1996, Turcq et al. 1998, Cndido et al. 2000, Volkmer-Ribeiro et al. 2007, Parolin et al. 2007). While the surveys are being extended across the continent some restricted local endemisms remain unchallenged. As a result the first official State and National recognitions and red listings of sponge species under threat were attained, upon the following of IUCN standard procedures. Oncosclera jewelli, Anheteromeyenia ornata and Drulia browni integrate the red list of endangered fauna of Rio Grande do Sul State, Brazil (Volkmer-Ribeiro 2003). Oncosclera jewelli, A. ornata, Uruguaya corallioides, Sterrastrolepis brasiliensis, Corvoheteromeyenia australis, Corvoheteromeyenia heterosclera, Corvospongilla volkmeri, Heteromeyenia insignis, Houssayella iguazuensis, Racekiela sheilae and Metania kiliani are listed with the Brazilian endangered freshwater invertebrates and fishes (Brasil 2004). This fact offers support to national and regional policies aiming at the

preservation of particular freshwater habitats and the species they contain. Lately, surveys for the South American continental sponges have also focused on river, lake, lagoonal and pond waters contained in preserved areas such as State and National Parks and Ecological Stations with the aim of establishing parameters for biomonitoring and bioindication, at the same time improving the knowledge of the species they contain and the use of such preserved areas as biodiversity banks (Volkmer-Ribeiro et al. 1988, 1999, 2005, Tavares et al. 2005, Volkmer-Ribeiro and Almeida 2005). A further application of this taxonomic tool is within the context of archeological studies. Spicules (cauxi), present in archeological Amazonian pottery are revealing unsuspected native technologies and histories of the sustainable management of natural resources (in this case biosilica produced by the sponges), besides allowing the tracing of cultural trends and past native population migrations within the continent (Volkmer-Ribeiro and Gomes 2006, VolkmerRibeiro and Viana 2006).

What next?
Homo sapiens may be producing a more extensive modification of the Earths surface than any other animal species did before. One such profound environmental change is the damming of large rivers in order to produce hydroelectric power, particularly throughout the Tropical and Sub-tropical realms. South America is a continent where the damming of large rivers has boomed over the last thirty years. Huge lakes have been formed in areas where this permanent freshwater habitat was previously absent, such as in the Amazonian Region, famous for its seasonal vrzea lakes. In regard to the rich Amazonian sponge fauna, surveys have extended to this new habitat in order to detect the invasion by sponges and the exclusion/adaptation forces in action. Results have shown that colonization is being carried by some species previously detected in the riverine rocky bottoms when the prior Impact Assessment surveys were done. All harder substrates located in the lake waters (excluding the anoxic ones), including the trunks of the forest flooded by the lake, are being used by those sponges that had occupied more extensively the original river bottoms (Volkmer-Ribeiro and Hatanaka 1991). The monitoring of the occupation of these dammed waters by sponges is being continued bearing in mind to offer taxonomic substrate for further research purposes encompassing from basic sponge biology and ecology to the production of biosilica or biocompounds by sponges. The mapping of substrates occupied by sponges in these dammed waters, allied to their continued recruitment as a consequence of the permanent ingression of upstream gemmules, renders these living stocks ideal for continued observation/monitoring and experimentation. These natural systems are better than laboratory aquaria, where freshwater sponge species other than those belonging to Ephydatia are barely kept alive for a few days. Another area of research being pursued based on the taxonomic knowledge currently available is the area of medicine, as there are several historical and some current records of dermal diseases caused by contact with sponge

120

spicules, particularly in the Amazonian Region. The discovery of freshwater sponge spicules acting as agents of ocular pathology in the Araguaia River area (Brazilian Amazonia) has only recently been reported (Volkmer-Ribeiro et al. 2006, Volkmer-Ribeiro and Batista 2007). Other pathologies related to freshwater sponge spicules inferred from archeological work have also been compiled and discussed in the aforementioned publications. The spreading of the geographic surveys of the South American continental sponges is obviously an ongoing process. Hopefully at the same speed as global economic enterprises are reaching them and their habitats. Renewed efforts should, from now on, focus on the aquatic habitats contained in preserved areas which, as a rule, benefit of previous selections aiming the protection of continental biomes. The invertebrate faunas of such biomes are yet poorly known and their study will certainly come up with the detection of new species.

Acknowledgments
The author is indebted to the organizers of the 7th International Sponge Symposium (Armao dos Bzios, RJ) for the invitation to present this opening speech as well as to two anonymous referees for the suggestions presented. She heartily thanks Dr. Eduardo Hajdu for a minutious reading of the MS and valuable improvements indicated. She acknowledges the continued support CNPq. has provided to the research projects proposed along the last three decades.

References
Bass D, Volkmer-Ribeiro C (1998) Radiospongilla crateriformis (Porifera, Spongillidae) in the West Indies and taxonomic notes. Iheringia Sr Zool 85: 123-128 Batista TCA, Volkmer-Ribeiro C, Darwich A, Alves LF (2003) Freshwater sponges as indicators of floodplain lake environments and of river rocky bottoms in Central Amazonia. Amazoniana 17(3/4): 525-549 Berroa Beln C (1968) Nomina de las esponjas dulceacucolas de la fauna del rio Uruguay, Amrica Del Sur. Physis 27(75): 285-289 Bonetto AA, Ezcurra de Drago I (1973) Aportes al conocimiento de las esponjas del Orinoco. Physis 32(84): 19-27 Brasil (2004) Instruo Normativa n 5, de 21 de maio de 2004. Dirio Oficial da Republica Federativa do Brasil, Braslia 28 de maio de 2004, Seo 1: 136-142 Candido JL, Volkmer-Ribeiro C, Simes Filho FL, Turcq BJ, Chauvel A (2000) Microsclere variations of Dosilia pydanieli (Porifera, Spongillidae) in Caracaran lake (Roraima - Brazil). Palaeoenvironmental implication. Biocincias 8(2): 77-92 Ezcurra de Drago I (1971) Porifera. In: Hulbert SH (ed). Biota acutica de Sudamrica Austral. San Diego State University, San Diego. pp. 57-61 Ezcurra de Drago I (1974). La presencia de Spongilla (Eunapius) fragilis Leidy en Chile (Porifera, Spongillidae). Physis B 33(87): 249-252 Ezcurra de Drago I (1975) Freshwater sponges of Suriname. Stud Fauna Suriname 15: 175-183 Ezcurra de Drago I (1979) Um nuevo gnero sudamericano de esponjas: Corvoheteromeyenia gen. nov. (Porifera Spongillidae). Neotropica 25(74): 109-118

Ezcurra de Drago I (1993) Distribucin geogrfica de las esponjas argentinas (Porifera: Spongillidae, Potamolepidae y Metaniidae). Relaciones zoogeogrficas, vias de poblamiento. In: Boltovskoy A, Lpez HL (eds). Conferencias de Limnologia, Instituto de Limnologia Dr. R. A. Ringuelet. La Plata, Buenos Aires. pp. 115-125 Ezcurra de Drago I (2003) Biodiversidad de Porifera en el litoral argentino. Grado de competncia com el bivalvo invasor Limnoperna fortunei (Dunker, 1857) (Bivalvia, Mytilidae). In: Aceolaza FG (ed). Temas de la Biodiversidad del Litoral argentino, INSUGEO, Miscelneas 12, Tucumn. pp. 5-12 Ezcurra de Drago I, Bonetto AA (1969) Algunas caracteristicas del bentos en los saltos del rio Uruguay, con especial referencia a la Ecologia de los Porferos. Physis 28(77): 359-369 Gray JE (1867) Notes on the arrangement of sponges, with the description of some new genera. Proc Zool Soc London 1867: 492558 Hooper JNA, van Soest RWM (2002) Systema Porifera: a guide to the classification of sponges. Kluwer Academic/Plenum Publishers, New York Hutchinson GE (1967) A treatise on limnology. Volume II. Introduction to lake biology and the limnoplancton. John Wiley & Sons, New York Jewell ME (1952) The genera of North American freshwater sponges; Parameyenia, new genus. Trans Kansas Acad Sci 55: 445-457 Kilian E, Wintermann-Kilian G (1976) Die Spongilliden Sdamericas derzeitiger Stand der Kenntniss iher Verbreitung. In: Descimon H (ed). Biogeographie et evolution en Amerique tropicale. Publications du Laboratoire de Zoologie de lcole Normale Superieure 9: 75-97 Nelson G, Platnik NI (1981) Systematics and biogeography: cladistics and vicariance. Columbia University Press, New York Parolin M, Volkmer-Ribeiro C, Stevaux JC (2007) Sponge spicules in peaty sediments as paleoenvironmental indicators of the holocene in the Upper Paran River, Brazil. Rev Bras Paleontol 10(1): 17-26 Penney JT, Racek AA (1968) Comprehensive revision of a worldwide collection of freshwater sponges (Porifera: Spongillidae). Bull U S natn Mus 272: 1-184 Poirrier M (1974) Ecomorphic variation in gemmoscleres of Ephydatia fluviatilis Linnaeus (Porifera: Spongillidae) with comments upon its systematics and ecology. Hydrobiologia 44(4): 337-347 Potts E (1887) Contributions towards a synopsis of the American forms of freshwater sponges with descriptions of those named by other authors and from all parts of the world. Proc Acad Nat Sci Philadelphia 39: 158-270 Sifeddine A, Frhlich F, Fournier M, Martin L, Servant M, Soubis F, Turcq B, Suguio K, Volkmer-Ribeiro C (1994) La sedimentation lacustre indicateur de changements des paloenvironments au cours des 30.000 dernires annes (Carajs, Amazonie, Brsil). C R Acad Sci Paris 318(II): 1645-1652 Silva CMM, Volkmer-Ribeiro C (2001) Key to the Ethiopian species of the genus Metania Gray, 1867 (Porifera: Metaniidae) with redescription of Metania rhodesiana and M. godeauxi, comb. n. Bull Inst r Sci nat Belg Biologie 71: 127-138 Tavares MCM, Volkmer-Ribeiro C (1997) Redescrio das esponjas de gua doce Oncosclera navicella (Carter, 1881) (Potamolepidae) e Spongilla spoliata Volkmer-Ribeiro & Maciel, 1983 (Spongillidae). Biocincias 5(1): 97-111

121

Tavares MCM, Volkmer-Ribeiro C, Hermany G (2005) Seasonal abundance in a sponge assembly at a Southern Neotropical inner delta. J Coast Res 42: 335-342 Turcq B, Sifeddine A, Martin L, Absy ML, Soubies F, Suguio K, Volkmer-Ribeiro C (1998) Amazon Forest fires: a lacustrine report of 7.000 years. Ambio 27(2): 139-142 Volkmer-Ribeiro C (1973) Redescription and ecomorphic variations of the freshwater sponge Trochospongilla minuta (Potts, 1887). Proc Acad Nat Sci Philadelphia 125(8): 137-144 Volkmer-Ribeiro C (1970) Oncosclera - a new genus of freshwater sponges (Porifera-Spongillidae) with redescription of two species. Amazoniana 2(4): 435-442 Volkmer-Ribeiro C (1976) A new monotipic genus of neotropical freshwater sponges (Porifera-Spongillidae) and evidence of a speciation via hybridism. Hydrobiologia 50(3): 271-281 Volkmer-Ribeiro C (1981) Porifera. In: Hurlbert SH, Rodrigues G, Santos ND (eds). Aquatic biota of tropical South America. Part 2: Anarthropoda. San Diego State University, San Diego. pp. 86-95 Volkmer-Ribeiro C (1984) Evolutionary study of the genus Metania Gray, 1867 (Porifera: Spongillidae): II. Redescription of two Neotropical species. Amazoniana 8(4): 541-553 Volkmer-Ribeiro C (1986a) Acanthodiscus new genus and genus Anheteromeyenia redefined (Porifera, Spongillidae). Iheringia, Sr Zool 81: 31-43 Volkmer-Ribeiro C (1986b) Evolutionary study of the freshwater sponge genus Metania Gray, 1867: III. Metaniidae, new family. Amazoniana 9(4): 493-509 Volkmer-Ribeiro C (2003) Porferos. In: Fontana CS, Bencke GA, Reis RE (orgs). Livro vermelho da fauna ameaada de extino no Rio Grande do Sul. Edipucrs, Porto Alegre. pp. 43-48 Volkmer-Ribeiro C, Almeida FB de (2005) As esponjas do Lago Tup. In: Santos-Silva E, Aprile FM, Scudeller VV, Melo S (orgs). BioTup: meio fsico, diversidade biolgica e sociocultural do Baixo Rio Negro, Amaznia Central. INPA, Manaus. pp.123-134 Volkmer-Ribeiro C, Batista TCA (2007) Levantamento de cauxi (Porifera, Demospongiae), provvel agente etiolgico de doena ocular em humanos, Araguatins, Rio Araguaia, Estado do Tocantins, Brasil. Rev Bras Zool 24(1): 133-143 Volkmer-Ribeiro C, Correia MMF, Brenha SLA, Mendona MA (1999) Freshwater sponges from a Neotropical sand dune area. Mem Qld Mus 44: 643-649 Volkmer-Ribeiro C, Costa PR (1992) On Metania spinata (Carter, 1881) and Metania kiliani n. sp.: Porifera, Metaniidae VolkmerRibeiro, 1986. Amazoniana 7(1): 7-16 Volkmer-Ribeiro C, Costa PR (1993) Redescription of the Oriental and Australian species of the genus Metania Gray, 1867 (Porifera: Metaniidae). Iheringia, Sr Zool 74: 81-101 Volkmer-Ribeiro C, De Rosa-Barbosa R (1972) On Acalle recurvata (Bowerbank, 1863) and an associated fauna of other freshwater sponges. Rev Bras Biol 32(3): 303-317 Volkmer-Ribeiro C, De Rosa-Barbosa R (1978) A new genus and species of Neotropical freshwater sponges. Iheringia, Sr Zool 52: 103-107 Volkmer-Ribeiro C, De Rosa-Barbosa R (1979) Neotropical freshwater sponges of the Family Potamolepidae Brien, 1967. In:

Lvi C, Boury-Esnault N (eds) Biologie des spongiaires. Centre National de la Rechereche Scientifique, Paris. pp: 503-511 Volkmer-Ribeiro C, De Rosa-Barbosa R, Machado VS (2005) Corvomeyenia epilithosa sp. nov. (Porifera, Metaniidae) no Parque Nacional da Serra Geral, Rio Grande do Sul, Brasil. Rev Bras Zool 22(4): 844-852 Volkmer-Ribeiro C, Ezcurra de Drago I, Parolin M (2007) Spicules of the freshwater sponge Ephydatia facunda indicate lagoonal paleoenvironments at the Pampas of Buenos Aires Province, Argentina. J Coast Res 50: 449-452 Volkmer-Ribeiro C, Gomes DMC (2006) Ferraz Egreja: implicaes zooarqueolgicas no estudo do antiplstico cermico. In: Vialou AV (org.) Pr-histria do Mato Grosso, Volume 2: Cidade de Pedra. EDUSP, So Paulo. pp. 203-206 Volkmer-Ribeiro C, Hatanaka T (1991) Nota Cientifica: composio especfica e substrato da espongofauna (Porfera) no Lago da Usina Hidroeltrica-Tucuru, Par, Brasil. Iheringia, Sr Zool 71: 177-178 Volkmer-Ribeiro C, Lenzi HL, Orfice F, Pelajo-Machado M, Alencar LM de, Fonseca CF, Batista TCA, Manso PPA, Coelho J, Machado M (2006) Freshwater sponge spicules: a new agent of ocular pathololgy. Mem Inst Oswaldo Cruz 101(8): 899-206 Volkmer-Ribeiro C, Machado VS (2007) Freshwater sponges (Porifera, Demospongiae) indicators of some coastal habitats in South America: redescriptions and key to identification. Iheringia, Sr Zool 97(2): 157-167 Volkmer-Ribeiro C, Motta JFM, Callegaro VLM (1998) Taxonomy and distribution of Brazilian spongillites. In: Watanabe Y, Fusetani N (eds). Sponge sciences: multidisciplinary perspectives. SpringerVerlag, Tokyo. pp. 271-278 Volkmer-Ribeiro C, Mansur MCD, Mera PAS, Ross SM (1988) Biological indicators in the aquatic habitats of the Ilha de Maraca. In: Milliken W, Ratter JA (eds). Marac The biodiversity and environment of an Amazonian rainforest. Chichester, John Willey & Sons Volkmer-Ribeiro C, Parolin M (2005) Segundo registro de Sterrastrolepis brasiliensis Volkmer-Ribeiro & De Rosa-Barbosa (Demospongiae, Potamolepidae) com descrio do habitat e de assemblia, Bacia do Rio Paran, Brasil. Rev Bras Zool 22(4): 1003-1013 Volkmer-Ribeiro C, Pauls SM (2000) Esponjas de agua dulce (Porifera, Demospongiae) de Venezuela. Acta Biol Venez 20(1): 1-28 Volkmer-Ribeiro C, Tavares MCM (1995) Redescrio de Drulia uruguayensis Bonetto & Ezcurra de Drago, 1968 com redefinio do gnero Drulia Gray, 1867 (Porifera: Metaniidae). Biocincias 3(1): 183-205 Volkmer-Ribeiro C, Traveset A (1987) Annotated catalog of the type specimens of Potts species of freshwater sponges. Proc Acad Nat Sci Philadelphia 139: 223-242 Volkmer-Ribeiro C, Turcq B (1996) SEM analysis of siliceous spicules of a freshwater sponge indicate paleoenvironmental changes. Acta Microscop 5(B): 186-187 Volkmer-Ribeiro C, Viana SA (2006) Cermica arqueolgica com cauixi. In: Viana SA (coord). Pr-Histria no Vale do Rio Manso/ MT. Universidade Catlica de Gois, Goinia. pp. 309-327

Porifera research: Biodiversity, innovation and sustainaBility - 2007

123

The Sponge Barcoding Project: aiding in the identification and description of poriferan taxa
Gert Wrheide(1*), Dirk Erpenbeck(1,2), Christian Menke(1)
Abteilung Geobiologie, Geowissenschaftliches Zentrum, Georg-August Universitt Gttingen, Goldschmidtstr. 3, D37077 Gttingen, Germany. gert.woerheide@geo.uni-goettingen.de, derpenb@gwdg.de, cmenke@davion.de (2) Biodiversity Program, Queensland Museum, South Brisbane, Queensland, Australia
(1)

Abstract: Sponges are among the most ancestral metazoans and are often notoriously difficult to identify due to their depauperate suite of complex morphological characters. However, as a group they are highly diverse, ecologically important and of significant commercial importance to the pharmaceutical and biomaterials industry. Therefore, means of unambiguous identification are urgently needed. Sponge barcodes promise a set of indispensable tools to aid taxonomists and ecologists in confirmation of identification of sponge species, and have the potential to enhance the discovery of drug-producing species. Here, we introduce the Sponge Barcoding Project (SBP) and present the structure of the Sponge Barcoding Database (SBD). Keywords: DNA barcoding, DNA taxonomy, sponges

Introduction
Sponges are among the most ancestral metazoans (e.g., Medina et al. 2001) and may hold many clues to our understanding of the evolution of early animal and developmental processes (Martindale 2005). They are highly diverse, abundant in nearly every aquatic habitat, some freshwater and most marine, and play numerous important ecological roles, e.g. in nutrient cycling (Lesser 2006) or as bioeroding organisms in coral reefs (Lopez-Victoria and Zea 2005). Their significant commercial importance to the pharmaceutical and biomaterials industry is increasingly being recognized, e.g. as producers of highly potent secondary metabolites (reviewed in e.g. Faulkner 2000) useful for drug development (Munro et al. 1994). Many sponge species are notoriously difficult to identify, often even by taxonomic experts, because morphological characters for comparative morphology are scarce and prone to homoplasies, highly variable or otherwise unsuitable for unambiguous identification. In addition, many sponges discovered in large scale biodiversity surveys remain undescribed (Hooper and Ekins 2005), partly also due to the lack of skilled taxonomists. As a result of uncertainties in morphological systematics, sponge species have frequently been regarded as widely distributed (cosmopolitan). However, genetic approaches, mostly using allozymes, have clearly shown that such cosmopolitan sponge species are rare and appear to result from over-conservative systematics, lumping morphologically similar but evolutionary distinct lineages into one widely distributed morpho-species (e.g. Klautau et al. 1999). The question of how to describe and distinguish such genetically distinct and reproductively isolated lineages remains, due to the difficulty of relating those genetic differences to morphological delineation of species.

Secondly, however, what is a species in sponges? While the use of fixed differences in diagnostic morphological characters (e.g. spicules and architecture) is practical and has served reasonably well to catalogue diversity, it is doubtful that such a typological system reflects the real biological diversity. Sponge alpha-taxonomy still is a quite artificial system solely based on morphological differences without considering evolutionary history and/or reproductive isolation. However, this issue will not be discussed in depth here. For further discussion see Sol-Cava and Wrheide (2007). Nonetheless, correctly identifying reproductively isolated and evolutionary distinct lineages of sponges remains pertinent for understanding a broad range of subjects such as marine ecology, biodiversity, dispersal, animal evolution and discovery of pharmaceutically / biotechnologically valuable taxa. Nonetheless, conventional morphological taxonomy alone clearly is at its limit with the task of distinguishing closely related but evolutionary distinct sponge lineages, especially in character poor taxa such as e.g. Halichondrida. The utilization of additional characters, such as informative signature DNA sequences (also known as DNA barcodes, Hebert et al. 2003a, 2003b), and the establishment of a DNA sequence-aided taxonomic system might provide an opportunity to overcome these shortcomings and aid our future endeavours to strive for more comprehensive species discoveries and descriptions as well as the deeper understanding of evolutionary factors that shape species distributions in space and time. In any case, a DNA sequence-based taxonomic system should by no means replace but rather complement conventional taxonomy based on comparative morphology the DNA sequences will be regarded as additional characters to described morphological (and biochemical) features.

124

The pitfalls and conceptual weaknesses of DNA barcoding have been discussed extensively in the literature (e.g. Moritz and Cicero 2004, Meyer and Paulay 2005, Hickerson et al. 2006) and shall not be repeated here as they are also summarized by Sol-Cava and Wrheide (2007). Despite all acknowledged shortcomings of DNA barcoding, a group of sponge biologists convened after a round table discussion at the 7th International Sponge Conference and agreed that time was ripe to act and initiated the Sponge Barcoding Project. This project was proposed by the first author (GW) during his talk, with an international steering committee consisting of Gert Wrheide (project coordinator, Germany), Andrea Blanquer (Spain), Paco Cardenas (Norway), Christina Maria Diaz (Venezuela), Sandra Duran (Spain), Dirk Erpenbeck (Australia, Germany), Dennis Lavrov (USA), Jose Lopez (USA), Grace McCormack (Ireland), Shirley Pomponi (USA) and Bob Thacker (USA) (in alphabetical order). The aim of the present contribution is to briefly outline the Sponge Barcoding Project (SBP), its recently launched website (www.spongebarcoding.org) and the Sponge Barcoding Database (SBD).

Scope
Phylum Porifera (sponges) consists of more than 8,000 described species, with an estimated species number of 15,000 (Hooper and van Soest 2002). The Sponge Barcoding Project (SBP) aims at establishing a DNA sequence-based reference system to aid future species discovery and description. It will work towards covering species from all sponge taxa, from classes Demospongiae, Hexactinellida, and Calcarea, ranging in habitat from the marine intertidal to the deep-sea, as well as freshwater, and from different biogeographic regions. In the long term the SBP intends to sequence DNA signature sequences of about 8,000 taxa, with an initial phase of 3 years focusing on 2,000 species covering all genera. This will provide a platform from which more extensive sampling can be directed. To establish a solid taxonomic framework, the SBP will start with recently described type specimens curated in associated museums and supplement these with unequivocally identifiable species. Fresh material of such taxa will be collected by individual groups involved in the SBP and will be taxonomically identified by an expert before making DNA signature sequences publicly available via the projects website and database. Ongoing pilot studies will evaluate the efficiacy of the methods using closely related taxa and investigate the relationship between intra- vs. interspecific diversity (i.e., the Barcoding gap, Meyer and Paulay 2006).

Background and approach


This is the first worldwide barcoding project on any diploblast taxon, and intends to cover the complete taxonomic range of Porifera. Several smaller pilot studies have recently been conducted independently, with various levels of resolution and success (Duran and Rtzler 2006, Wrheide 2006). The standard mtDNA cytochrome oxidase subunit 1 (COI) barcoding fragment, which is used for almost all current (eukaryotic) barcoding initiatives, spans over

a ca. 650 nucleotide region close to the 5 end (Erpenbeck et al. 2006). This mitochondrial protein displays sufficient variability in most bilaterian species. However, in diploblastic animals mitochondrial proteins display a lower evolutionary rate (Wrheide et al. 2000, Shearer et al. 2002) and it has been shown, that frequently co-occurring, congeneric sibling sponge species are difficult to separate with COI fragments (Wrheide 2006) due to very low variability. However, a more variable downstream fragment appears to bear adequate resolution (Erpenbeck et al. 2006). Therefore, a concerted effort is now needed to evaluate the usefulness of DNA signature sequences for poriferan species discovery and description, and warrants comprehensive, phylum-wide coverage. Due to the fact that the highly conserved COI-barcoding primers are prone to amplify sponge commensals and/or symbionts, the primary task in the first phase of the project is to optimize sponge-specific primer design. Additionally, to resolve closely related species, we will supplement the standard ca. 650 bp fragment with 440 bp of downstream sequence. The addition of an unlinked marker such as either rDNA ITS or the C2D2 region of the 28S rDNA gene might prove pivotal to accomplish the projects aims. The second task will then be, after sufficient initial data have been gathered, to evaluate the potential of those DNA signature sequences for species distinction, i.e. the error rate associated with certain thresholds of genetic distances commonly used for species designation (see also Meyer and Paulay 2005, Hickerson et al. 2006). It is imaginable that once a sufficiently and densely covered reference system has been established and evaluated, identification of any given specimen, using the standard barcoding marker, at least to genus level should be possible. From there, species designation would be contingent on more variable signature sequences such as rDNA ITS or a fragment of the 28S rDNA. However, all this will take place in combination with conventional comparative morphology. Therefore, we will focus the initial phase of the SBP on appropriately identified and curated type specimens to build a taxonomically sound and solid backbone for a DNA sequenceaided taxonomy. Samples will be obtained from associated partners e.g. at the Zoological Museum in Amsterdam/ Netherlands (Dr. Rob van Soest - >18,000 specimens) and from the Queensland Museum in Brisbane/Australia (Prof. John Hooper - >34,000 specimens), or from collections of SBP partners such as the Harbor Branch Oceanographic Institution (Dr. S. Pomponi - >34,000 specimens) in Florida, USA. Initial assessments of DNA quality from these collections indicate they are of an adequate suitability. DNA sequencing will be carried out either by individual SBP partners (all of whom have significant prior expertise in the field and appropriate capacities), or can be pooled at certain DNA sequencing facilities (e.g. at the coordinating institution, the Gttingen Centre for Biodiversity and Ecology, Germany). All efforts will be undertaken to support developing countries in their efforts to produce DNA barcodes from specimens of their sponge fauna. Sequences and associated data (voucher and taxonomic information) will be made publicly available at the projects database (see below) on its website (www.spongebarcoding.org) and submitted to the Barcode of Life Data Systems and Genbank/EMBL databases.

125

Such a system could also enable a reverse taxonomic system, in that in large-scale biodiversity surveys, all collected samples are genotyped for DNA signature sequences, followed by pooling all specimens with identical or highly similar sequences. This will enable focused morphological work on distinct genetic lineages.

point of entry for the Sponge Barcoding Database, which is outlined in more detail below.

Technical concept and implementation of the Sponge Barcoding Database


The Sponge Barcoding Database (SBD) is developed with the aim to function as the primary access point for DNA signature sequences together with providing information on conventional morphological taxonomic characters to aid species discovery, description and characterization. The unique combination of sponge-specific conventional taxonomic information and DNA signature sequences is the distinguishing feature, in which the SBD differs from other database systems, such as Genbank (www.ncbi.nlm.nih.gov/) or the Barcode of Life Data Systems (www.barcodinglife.com/). While records of the SBD will be

The Sponge Barcoding Website (www.spongebarcoding.org)


The Sponge Barcoding Website (Fig. 1) has been set up to serve the projects aims and provide a centralized platform for data exchange. Information about the approach, progress and the people involved can be found on separate pages, as well as a list of local (taxonomic or geographic) campaigns, as well as a guide on how to get involved in the project (Fig. 1). The central place for data access is the Data page, the

Fig. 1: The entry page of the Sponge Barcoding Projects website (www.spongebarcoding.org), accessed on 31 May 2007.

126

Fig. 2: The structure of the Sponge Barcoding Database. Rectangles represent tables in the database with ovals connected to them denoting their attributes (i.e. table columns), diamonds denote relationships between the tables and their multiplicities given as numbering on each line (1 or n). For example, for any given species, there can be arbitrarily many specimen records, but each specimen is of exactly one species. For each specimen, arbitrarily many sequences can be saved, but each sequence will be obtained from only one specimen.

linked with both databases, both do not provide the desired flexibility and have the desired options available for the SBD, e.g. they do not provide fields to store more detailed (morphological) taxonomic descriptions. An additional backbone for nomenclatorial and taxonomical entries is the cross-linking to the World Porifera Database (WPD, www.marinespecies.org/porifera/). This data base is edited by Rob van Soest, Nicole Boury-Esnault, Dorte Janussen and John Hooper and has gone online in 2005. The WPD will provide the ultimate taxonomic authority with regards to accepted species names. The structure of the SBD (Fig. 2) was developed with flexibility and avoidance of redundancy in mind. For example, if multiple DNA sequences will be provided for one specimen, then the specimen information is saved only once. To achieve this, the database consists of multiple tables (Fig. 2): - A species table containing species names and an ID for each species. The Species ID (WPD-ID) will directly be obtained and linked to the record in the World Porifera Database, which can also directly be accessed through the SBP website. - A specimen table containing all the information related to one collected specimen, e.g. collection date, location and a morphological description. Each specimen record has its own, unique record number in the SBD (SBD-ID). Specimen records are linked to the species table by their species IDs. This table also contains fields for filenames of associated pictures. The associated images themselves are saved outside of the database in the file system.

- A sequence table containing sequence strings and related information like sequence type and the Genbank Accession Number. Sequence records are linked to the specimen table by the SBD-ID. This table also stores filenames of sequencing chromatograms and quality values, which will have to be submitted along with the sequence string to allow quality assessment. Chromatograms can be viewed from the query results page. - A separate table linked to the specimen table that contains relevant literature references e.g. for molecular applications of the sequence. (References to original descriptions and/or revisions are found in the WPD). - A user table and an edit history table. For any change made to a specimen record, an entry containing the specimen record number, the ID of the user that committed the change and a comment is inserted into the edit history. This allows tracking of any changes made to the specimen data. The splitting of the database into multiple tables increases its flexibility. A specimen record can have arbitrarily many associated sequences and reference entries without any redundant saving of information. A field submitted as in the specimen table contains the genus and species name as valid at the time of entry into the database. Additionally, the WPD-ID will be stored, and records displayed to the user after queries will dynamically load the current valid genus and species names from the WPD and display the currently accepted name in addition to the name as submitted. This allows records to still be found by their original name even after a name has been synonymized e.g. after a taxonomic

127

Fig. 3: Record #167 from the Sponge Barcoding Database, accessible via the data button on www.spongebarcoding.org. This is an example of a Reference record that contains all neccessary information (see text for details). Several subcategories (e.g., morphological description, reference, and associated DNA sequences can be enlarged to display their full content by clicking on show/hide). Accessed on 31 May 2007.

128

revision. The database currently displays two categories of records to the end user (additional maintenance categories are available to the editors): REFERENCE: records from described species with a full taxonomic description, DNA signature sequence(s), and verification of voucher material by a recognized taxonomic expert (Fig. 3). SUBMITTED: records from described species that either lack full taxonomic description or verification by a taxonomic expert, or DNA signature sequences from as yet undescribed and unverified species. Categorical Submitted records should only be used for comparative purposes and NOT for species identification. To avoid database inconsistency, the deletion of records needs to cascade through the database. If a specimen record is deleted, all associated reference and sequences must in turn also be deleted. The database does this automatically. Likewise, any image or chromatogram files are automatically deleted from the file system upon removal of their corresponding database entry. The databases user interface is written in PHP as a website. Web enabled data viewing and editing allows for flexible access to the database from many different platforms. The PHP interface also takes care of user authentication and error checking.

Acknowledgments
We acknowledge funding by the German Research Foundation (DFG). D.E. acknowledges financial support of the European Union under a Marie-Curie outgoing fellowship (MOIF-CT-2004 Contract No 2882).

References
Duran S, Rtzler K (2006) Ecological speciation in a Caribbean marine sponge. Mol Phylogenet Evol 40: 292-297 Erpenbeck D, Hooper JNA, Wrheide G (2006) CO1 phylogenies in diploblasts and the Barcoding of Life - are we sequencing a suboptimal partition? Mol Ecol Notes 6: 550553. Faulkner DJ (2000) Highlights of marine natural products chemistry (1972-1999). Nat Prod Rep 17:1-6 Hebert PD, Cywinska A, Ball SL, deWaard JR (2003b) Biological identifications through DNA barcodes. Proc Roy Soc Lond B Biol Sci 270: 313-321 Hebert PD, Ratnasingham S, deWaard JR (2003a) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc Roy Soc Lond B 270(Suppl 1): S96-9

Hickerson M, Meyer C, Moritz C (2006) DNA barcoding will often fail to discover new animal species over broad parameter space. Syst Biol 55: 729-739 Hooper JNA, Ekins M (2005) Collation and validation of museum collection databases related to the distribution of marine sponges in northern Australia. Report to the National Oceans Office, Australia (Contract number C2004/020). pp. 235 Hooper JNA, van Soest RWM (2002) Systema Porifera: a guide to the classification of sponges. Kluwer Academic/Plenum Publishers, New York Klautau M, Russo CAM, Lazoski C, Boury-Esnault N, Thorpe JP, Sol-Cava AM (1999) Does cosmopolitanism result from overconservative systematics? A case study using the marine sponge Chondrilla nucula. Evolution 53: 1414-1422 Lesser MP (2006) Benthic-pelagic coupling on coral reefs: Feeding and growth of Caribbean sponges. J Exp Mar Biol Ecol 328: 277288 Lopez-Victoria M, Zea S (2005) Current trends of space occupation by encrusting excavating sponges on Columbian coral reefs. Mar Ecol 26: 33-41 Martindale MQ (2005) The evolution of metazoan axial properties. Nat Rev Genet 6: 917-927 Medina M, Collins AG, Silberman JD, Sogin ML (2001) Evaluating hypotheses of basal animal phylogeny using complete sequences of large and small subunit rRNA. Proc Natl Acad Sci USA 98: 9707-9712 Meyer CP, Paulay G (2005) DNA barcoding: error rates based on comprehensive sampling. PLoS Biology 3: e422 Moritz C, Cicero C (2004) DNA Barcoding: promise and pitfalls. PLoS Biology 2: e354 Munro MHG, Blunt JW, Lake RJ, Litaudon M, Battershill CN, Page MJ (1994) From seabed to sickbed: what are the prospects? In: van Soest RWM, van Kempen TMG, Braekman JC (eds). Sponges in time and space: biology, chemistry, paleontology. Balkema, Rotterdam. pp. 473-484. Shearer TL, Van Oppen MJH, Romano SL, Worheide G (2002) Slow mitochondrial DNA sequence evolution in the Anthozoa (Cnidaria). Mol Ecol 11: 2475-2487 Wrheide G (2006) Low variation in partial cytochrome oxidase subunit I (COI) mitochondrial sequences in the coralline demosponge Astrosclera willeyana across the Indo-Pacific. Mar Biol 148: 907-912 Wrheide G, Degnan BM, Hooper JNA (2000) Population phylogenetics of the common coral reef sponges Leucetta spp. and Pericharax spp. (Porfera: Calcarea) from the Great Barrier Reef and Vanuatu. In: Hopley D, Hopley P, Tamelander J, Done T (eds). Ninth International Coral Reef Symposium, Bali, vol. Abstracts, p. 21

RESEARCH ARTICLES

Porifera research: Biodiversity, innovation and sustainaBility - 2007

131

Spongivory by juvenile angelfish (Pomacanthidae) in Salvador, Bahia State, Brazil


Brbara R. Andra(1), Daniela Batista(1,2), Cludio L.S. Sampaio(3), Guilherme Muricy(1*)
Departamento de Invertebrados, Museu Nacional, Universidade Federal do Rio de Janeiro. Quinta da Boa Vista, s/no., So Cristvo. 20940-040 Rio de Janeiro, RJ, Brasil. muricy@acd.ufrj.br (2) Departamento de Biologia Marinha, Universidade Federal Fluminense, Niteri, Rio de Janeiro, Brasil (3) Departamento de Sistemtica e Ecologia, Universidade Federal da Paraba, Joo Pessoa, Brasil.
(1)

Abstract: Adult angelfish of the genera Pomacanthus and Holacanthus (Family Pomacanthidae) are considered the most important spongivorous fishes of the Caribbean, with sponges comprising more than 70% of their gut contents. However, despite their commercial importance as ornamental fish, little is known about the diet of juvenile angelfish, which are generally considered to be cleaners. The goal of this study was to identify through gut content analysis the sponge species eaten by juveniles of the angelfish Pomacanthus paru, Holacanthus ciliaris and Holacanthus tricolor in Salvador, Bahia state, Brazil. We also estimated the frequency of occurrence of each sponge species in the diet of juvenile angelfish, and tested the correlation between fish size and number of sponge species preyed. In Salvador, 34 species of sponges were found in the gut contents of 14 out of 16 specimens of juvenile angelfish. Twenty-two species of sponges were eaten by Holacanthus tricolor, 15 by H. ciliaris, and 14 by Pomacanthus paru. There was a significant positive correlation between fish size and number of preyed sponge species, but the coefficient of determination was low and even the smallest fishes had sponges in their gut contents. These findings indicate that juveniles of all three species of angelfish are generalists in the consumption of sponges in Brazil, a diet similar to that of the adults. The most frequent sponges in juvenile angelfish gut contents were Tedania ignis, Mycale sp., and Spirastrella sp., all with 37.5% of frequency in the 16 stomachs analyzed. Tedania ignis was not consumed by H. ciliaris in Salvador, but it was consumed by the other fish species, being the most frequent prey of H. tricolor and P. paru. Juvenile angelfish probably adapt to a cleaning behavior or to benthic feeding according to local environmental conditions. The development of sponge-based artificial foods may allow longer maintenance and reproduction of angelfish in aquaria, thus helping to protect their natural populations. Keywords: Holacanthus, Pomacanthus, Porifera, Predation, Southwestern Atlantic

Introduction
Sponges are important structural and functional components of coral reefs, where they participate in numerous ecological relationships such as competition, commensalism, symbiosis, bioerosion and predation (reviewed by Diaz and Rtzler 2001, Wulff 2001, 2006a). Although reef sponges are abundant and many species live exposed to predators, few animals are known to feed on them, probably due to their physical (spicules) and chemical (secondary metabolites) defenses (Pawlik et al. 1995, Hill et al. 2005, Wulff 2006a, 2006b). Among invertebrates, spongivory is shown by opisthobranchs (e.g., Glossodoris pallida, Archidoris montereyensis, Peltodoris atromaculata, Tylodina perversa), sea stars (e.g., Oreaster reticulatus, Echinaster echinophorus), sea urchins (e.g., Eucidaris tribuloides and Lytechinus variegatus) and hermit crabs (Reiswig 1973, Wulff 1995, 2006b, Becerro and Paul 1998, Wadell and Pawlik 2000, Santos et al. 2002, Gemballa and Schermutzki 2004). Some associated copepods, amphipods, isopods and alpheid shrimps also consume their host sponges (Pawlik 1983, Ros and Duffy 1999, Mariani and Uriz 2001). The main vertebrate sponge-feeders are the

sea turtle Erethmochelys imbricata (Meyland 1988) and reef fishes belonging to the families Pomacanthidae, Ostraciidae, Tetraodontidae, Ephippidae and Monacanthidae. Among these, the family Pomacanthidae contains the most important sponge-feeding fishes in the Caribbean, particularly in the genera Pomacanthus and Holacanthus (Randall and Hartman 1968, Wulff 1994). The family Pomacanthidae includes 88 species distributed in all tropical seas (Allen et al. 1998, Debelius et al. 2003). Within the family there is a diverse range of feeding specializations, including herbivory, planktivory, cleaning activity, and omnivory (Bhlke and Chaplin 1968, Hourigan et al. 1989, Allen et al. 1998, Deloach 1999, Bellwood et al. 2004). Ontogenetic variation in diet contents has been observed in many pomacanthids (Thresher 1980, Deloach 1999) as well as in other reef fish families such as Blenniidae, Kyphosidae and Scaridae (Bellwood 1988, Sturm and Horn 1998, Muoz and Ojeda 2000). In the Caribbean, sponges comprise over 70% of the diet of adults of the common pomacanthid species Pomacanthus paru (French angelfish), Pomacanthus arcuatus (Gray angelfish), Holacanthus ciliaris (Queen angelfish) and Holacanthus tricolor (Rock beauty)

132

(Randall and Hartman 1968, Dunlap and Pawlik 1996). In Brazil, adults of Pomacanthus paru also feed mostly on sponges and algae, in variable proportions according to the locality studied (Batista 2006). The feeding behavior of juveniles, however, appears to be different from that of adults in these species. Juveniles of H. ciliaris, P. paru and P. arcuatus may act as cleaners until they have approximately 10-15 cm in total length (Feder 1966, Thresher 1980, Deloach 1999, Sazima et al. 1999). The primary food of P. paru and P. arcuatus juveniles was reported to be filamentous algae, with copepods picked from client fishes and few free-living copepods making up to 25% of their diet (Deloach 1999). In Abrolhos Archipelago, Brazil, juveniles of the French angelfish P. paru removed ectoparasites of 31 species of reef fishes (Sazima et al. 1999). Food items in their stomachs included caligid (15-30%) and harpaticoid copepods (5-10%), together with both red and green algae (30-70%). Juveniles of H. ciliaris also feed on algae until they reach sexual maturity (Deloach 1999). The diet of H. tricolor juveniles is largely unknown, but it is assumed that it is a combination of drifting plankton, small benthic invertebrates, and possibly the mucus and parasites of larger fishes (Thresher 1979, 1980, Gasparini and Floeter 2001). Brazil is one of the five leading exporting countries of tropical aquarium fishes in the world, and the interest in marine ornamental organisms has increased substantially from mid- to late 1990s (Gasparini et al. 2005, Floeter et al. 2006). Juvenile angelfish are preferred over the adults for ornamental purposes due to their smaller size and beautiful color patterns. In Cear State, NE Brazil, angelfish juveniles have been captured and exported in large numbers: 43,730 specimens of H. ciliaris, 22,969 of P. paru, and 8,757 specimens of H. tricolor between 1995 and 2000 (Monteiro-Neto et al. 2003), although these values may be overestimated (Gasparini et al. 2005). Cleaner species play an important ecological role in reef habitats, and their removal may negatively affect other fish species, including commercially important ones (Sazima et al. 1999, Monteiro-Neto et al. 2003). Therefore, knowledge about the feeding behavior of juvenile angelfish is important for the conservation of angelfish species and of coral reef communities. In this study we tested the hypothesis that sponges are an important part of the diet of juvenile angelfish, and not only of the adults. We identified the sponge species found in the gut contents of juveniles of Pomacanthus paru, Holacanthus ciliaris and H. tricolor in Salvador, Bahia state, Brazil. We also estimated the frequency of each sponge species in the gut contents of juvenile angelfish, and tested the correlation between fish size and number of prey species. Our results may aid in the development of a better diet for the growth and reproduction of angelfish in captivity, thus helping to protect angelfish species which are currently threatened by the increase of marine ornamental fish trade in Brazil.

Fig. 1: Location of the study area.

Materials and methods


Sixteen specimens belonging to three species of angelfish were studied: Pomacanthus paru (n=5); Holacanthus ciliaris (n=6) and Holacanthus tricolor (n=5). Fish were collected in four locations in Salvador, Bahia state, Brazil (Fig. 1): Praia

da Ribeira, in Todos os Santos Bay (1254S3829W), Barra Grande, in Itaparica Island (1303S3838W), Rio Vermelho (1300S3830W), and Praia da Pituba (1300S 3830W). The specimens were collected from 18/II/2003 to 10/III/2004 by commercial ornamental fisheries (Ax Online Ltd.) in shallow reefs and rocky bottoms. Only juveniles were caught, easily recognized by their special color patterns, which change after sexual maturity is reached (Bhlke and Chaplin 1968, Thresher 1980). Specimen size varied between 5.0-14.5 cm in H. ciliaris, 5.0-14.0 cm in H. tricolor, and 3.0-14.5 cm in P. paru. The stomachs were removed and their contents were separated based on color, texture and consistency. Only the sponge fragments were identified to lower rank taxons, and no attempts were made to quantify the abundance of each species consumed in the gut contents of the 16 angelfish specimens analyzed. The sponge fragments were dehydrated in an alcohol series (50-100%) with a final xylene step and included in paraffin. Transverse sections were mounted on microscope slides for identification. Free spicules were not considered as evidence of predation, only fragments large enough to be sectioned and in which the skeleton could be observed. Sponges were deposited in the sponge collection of Museu Nacional, Universidade Federal do Rio de Janeiro, Brazil. A linear regression between fish size (standard length SL) and number of prey species was calculated online (http:// faculty.vassar.edu/lowry/VassarStats.htm).

133

Results
Sponges comprised more than 90% of the gut contents of juvenile angelfish in Salvador, together with a few unidentified filamentous algae (< 10%). The guts of two juveniles of H. ciliaris were empty. A total of 34 sponge species were found in the diet of angelfish in Salvador (Table 1). The greatest species richness was observed in gut contents of Holacanthus tricolor (22 sponge species), followed by H. ciliaris and Pomacanthus paru (15 and 14 sponge species, respectively). Twelve sponge species were only predated by H. tricolor: Acanthanchora sp., Coelosphaeridae unidentified, Desmapsamma anchorata, Lissodendoryx sp., Microcionidae unidentified 1, Microcionidae unidentified 2, Monanchora sp., Myxillina unidentified, Pachastrellidae unidentified, Phellodermidae unidentified, Plakinastrella sp., and Timea sp. Only three species were predated exclusively by Holacanthus ciliaris: Artemisina sp., Chalinidae unidentified, and Tethya sp. Six species were eaten exclusively by P. paru: Chondrilla nucula complex, Cyamon sp., Mycale laxissima, Niphatidae unidentified, Raspaciona sp., and Tetillidae unidentified (Table 1). As a whole, the sponge species most frequently predated

by pomacanthids in Salvador were Tedania ignis, Mycale sp., and Spirastrella sp., all present in 37.5% of the 16 specimens analyzed. Although Tedania ignis was not consumed by H. ciliaris, it was very frequent in the gut contents of P. paru (80% of the specimens examined) and common in H. tricolor (40%; Table 1). The number of sponge species per stomach varied from 4-9 in H. tricolor, from 1-7 in P. paru, and from 0-8 in H. ciliaris. The curves of accumulated richness of sponge species in angelfish diet did not reach stabilization with the small sample sizes studied here, neither with each fish species considered separately nor when they were considered together (Fig. 2). This indicates that the real number of sponge species in the diet of juvenile angelfish in Salvador is probably much higher than that shown by our results. There was a significant positive correlation (p<0.05) between fish size and number of sponge species in gut contents, although with a low coefficient of determination (r2 = 0.465; Fig. 3). The single smallest fish (3 cm SL) had only one sponge species in its stomach, and three of the five smallest fishes (3.0-5.5 cm SL) fed on few sponge species (15 species per stomach).

Table 1: Frequency of occurrence of sponge species in gut contents of juvenile angelfish in Salvador, Brazil (in %). Combined = frequency in all species pooled together (n=16). Sponge species Acanthanchora sp. Acervochalina sp. Artemisina sp. Chalinidae unidentified Chondrilla nucula complex Clathria sp. 1 Clathria sp. 2 Coelosphaeridae unidentified Cyamon sp. Desmapsamma anchorata Halichondriidae unidentified Haplosclerida unidentified Lissodendoryx sp. Microcionidae unidentified 1 Microcionidae unidentified 2 Mycale laxissima Mycale sp. Mycalidae unidentified Myxillina unidentified Monanchora sp. Niphatidae unidentified Pachastrellidae unidentified Phellodermidae unidentified Plakinastrella sp. Raspaciona sp. Spirastrella sp. Stelleta sp. 1 Stelleta sp. 2 Strongylacidon sp. Suberitidae unidentified Tedania ignis Tethya sp. Tetillidae unidentified Timea sp. Species richness P. paru 20 20 40 20 20 40 H. tricolor 20 H. ciliaris 17 17 17 33 17 Combined 6.25 12.50 6.25 6.25 6.25 25.00 25.00 6.25 6.25 12.50 12.50 31.25 6.25 12.50 6.25 6.25 37.50 25.00 12.50 12.50 6.25 6.25 6.25 6.25 6.25 37.50 12.50 12.50 12.50 12.50 37.50 6.25 6.25 6.25 34

40 20 20 40 20 20 20 20 20 60 40 40 20 20 20 20 20 20 20 40 20 22

50 17 50 17

20 40

20

20 60 20

80 20 14

33 17 17 17 17 17

15

134

Fig. 2: Curves of accumulated richness of sponge prey species in juvenile angelfish gut contents. A, Holacanthus tricolor; B, Holacanthus ciliaris; C, Pomacanthus paru; D, all species pooled together.

Discussion
Juvenile angelfish fed largely on sponges in Salvador (> 90%), and only a few filamentous algae were also found in their gut contents. These figures are similar to those of adult angelfish diet in the Caribbean, where sponges comprised 74.8% to 97.1% of the diet of Pomacanthus paru and Holacanthus ciliaris, respectively (Randall and Hartman 1968). Algae were also the second most common item in the diet of adult pomacanthids in the Caribbean, varying from 0.8% to 13.4% of the diet of H. tricolor and P. paru, respectively (Randall and Hartman 1968). Other food items common in Caribbean adults such as tunicates, hydroids, zoantharians, and bryozoans were not found in juveniles from Salvador. Fourteen out of 16 fishes (87.5%) collected in one year had sponge remains in their stomachs, indicating that spongivory is not occasional, but frequent and widespread in the juvenile angelfish population in Salvador. Adult angelfish appear to have a more varied diet, at least in the Caribbean, where sponges were found in only 12 of 23 stomachs of P. paru (52.1%) and 18 out of 34 specimens of P. arcuatus (52.2%). Adults of H. tricolor are more specialized in sponges, with sponge remains in 22 of 24 specimens (91.7%; Randall and Hartman 1968). The diet of juvenile angelfish in Salvador includes at least 34 sponge species. All three angelfish species studied here, Pomacanthus paru, Holacanthus ciliaris, and H. tricolor

were generalists in the consumption of sponges, eating 14 to 22 different species each. The real number of sponge species consumed by these angelfish is probably higher than that, as indicated by the shape of the accumulated richness curves (Fig. 2). Although there was a significant positive correlation between fish size and number of preyed sponge species (Fig. 3), the coefficient of determination was low and even the smallest fishes had sponges in their gut contents. Part of this correlation may reflect greater gut volume in larger fish individuals, allowing them to have more bites (and therefore more species) in their guts at the time they were collected. The diet of pomacanthid juveniles in Salvador is very similar to that of the adults, both in the Caribbean and in Brazil: they are smorgasbord-feeding sponge specialists, consuming a variety of benthic invertebrates and algae, apparently chosen according partly to prey palatability and partly to prey availability in the environment (Randall and Hartman 1968, Hourigan et al. 1989, Wulff 2006a, Batista 2006). This is at odds with some studies which suggest that juveniles of H. ciliaris and P. paru are cleaners until they have approximately 10-15 cm in total length, when they change to benthic feeding of a mainly sponge and algae diet (Feder 1966, Bhlke and Chaplin 1968, Hourigan et al. 1989, Sazima et al. 1999). Other studies however found little cleaning activity in juveniles of P. paru (Wicksten 1995, 1998). Among Tropical Western Atlantic angelfish, juveniles of P. paru seem to be the most specialized as cleaners, with P. arcuatus and H. ciliaris having a mixed diet composed mostly of algae and detritus

135

Fig. 3: Linear regression between fish size (standard length in cm) and the number of sponge species in gut contents of juvenile angelfish. Slope = 1.1173, intercept = 5.0347, Standard error of the estimate = 3.169.

and being only occasional cleaners (Thresher 1980, Deloach 1999). The diet of H. tricolor juveniles was hitherto poorly known, although cleaning activity has been reported in a few other studies (Thresher 1979, 1980, Gasparini and Floeter 2001). The method of study employed by each author is important to explain such disagreements. When only direct underwater observation is considered (e.g., counting the bites given in each prey species), there is a trend to increase the relative importance of algae and of cleaning activity in the diet of pomacanthids (Bhlke and Chaplin 1968, Thresher 1980, Hourigan et al. 1989, Sazima et al. 1999, Batista 2006). Juveniles of angelfish such as H. tricolor dwell mostly in cryptic habitats inaccessible for divers (Thresher 1980). These habitats are often dominated by sponges (Sar and Vacelet 1973). Other species of angelfish, including adult specimens, also use small caves and reef crevices for sheltering and foraging, biasing the direct observation of preyed items towards more exposed organisms such as algae and gorgonians (Thresher 1980, Batista 2006). When only gut contents are analyzed, the relative importance of spongivory seems to increase, both in proportion to algae and benthic invertebrates and in number of species (e.g., Randall and Hartman 1968, Deloach 1999). It is possible that sponge fragments remain recognizable in the guts of angelfish longer than copepods and filamentous algae, due to their high collagen and spicule content (Chanas and Pawlik 1995). This would artificially increase the proportion of spongivory in studies based only in gut contents and without direct observations, such as the present one (see also Randall and Hartman 1968). The complementary use of both field observations and gut content analysis allows a better understanding of pomacanthid feeding habits (Hourigan et al. 1989, Sazima et al. 1999, Batista 2006, Wulff 1994, 2006a). It is possible therefore that juvenile angelfish in Salvador are not almost exclusively sponge-eaters as shown by our results,

but might also consume other benthic organisms and act as cleaners occasionally. This issue can only be solved through systematic direct observations on juvenile angelfish, which could not be carried out in this study. Another explanation for the variation in the diet of pomacanthid juveniles in different studies is a high dietary plasticity of pomacanthid species. Allopatric angelfish populations may specialize to use different resources, depending on their availability in each locality. For example, the abundance of reef fish in general is greater in the Abrolhos National Marine Park, which is a protected area, than in Salvador, which has been subject to heavy fishing (Gasparini et al. 2005, Floeter et al. 2006). It may thus be easier for juveniles of pomacanthids such as P. paru to establish a cleaning station in Abrolhos than in Salvador, where they have to adapt their diet to the available resources, mainly sponges and algae. The presence of a suitable habitat for juveniles close to a reef fish community also helps to explain the intense cleaning activity of P. paru juveniles in Abrolhos reefs (Sazima et al. 1999). Although always composed mostly of sponges, the specific composition of the diet of adult angelfish varied strongly in different localities such as the Bahamas, Virgin Islands, and Panama in the Caribbean (Randall and Hartman 1968, Feddern 1968, Hourigan et al. 1989, Wulff 1994), and Atol das Rocas, Abrolhos and Ilha Grande in Brazil (Batista 2006), irrespective of the study method. In all these locations and in Salvador, each species of angelfish fed on a diverse array of sponge species, ranging from 12 in P. paru to 40 in H. ciliaris, both in the U.S. Virgin Islands (Randall and Hartman 1968, Hourigan et al. 1989). Together, Pomacanthus paru and P. arcuatus ate 64 sponge species in Panama (Wulff 1994). The only exception was H. tricolor, which was observed feeding on a single sponge species in Panama and was therefore considered a possible specialist (Wulff 1994). However, adults of H. tricolor are known to fed on 14 to 28 different sponge species in the U.S. Virgin Islands and Puerto Rico (Randall and Hartman 1968, Hourigan et al. 1989), and juveniles in Salvador fed on 22 sponge species. This indicates that H. tricolor is also a generalist in the consumption of sponges, like most other pomacanthids. The number of sponge species eaten by each individual fish in Salvador varied from 0-9 (average 4.5). This tolerance to predate upon diverse sponge species may be a strategy of angelfish to overcome the effects of sponge toxins by eating small amounts of each toxic substance from many different sponge species (Wulff 1994). It has also be suggested as an adaptation of benthic fish species with pelagic larvae such as angelfish to increase their chances of survival in areas with different availability of specific food items (Hourigan et al. 1989). The similarity between gut contents of different but co-specific specimens was low in Salvador (maximum 0.273 between two specimens of H. tricolor; data not shown), indicating that individual fish choose their food independently. This may be related to the very small home range of angelfish juveniles (around 1-2 m2), much smaller than that of the adults (up to 2,300 m2 in P. paru; Thresher 1980, Hourigan et al. 1989). All specimens studied here came from the same general region in Salvador, but dietary similarity between the three species was low (maximum 0.296 between H. ciliaris and H.

136

tricolor; data not shown) and in the same range of intraspecific similarity. Most of the interspecific variation found in angelfish diet in Salvador thus appears to be random, and due to the sum of independent choices of individual fishes of the three different species studied. As most fishes, spongivores have great plasticity in their feeding behavior. This variability could have influence from food availability, fish size, species and other temporal and spatial factors. However, part of the differences in dietary composition between sympatric species is probably due to an active choice of preys, possibly restrained by the physiological tolerance of each fish species to the preys toxins. A certain degree of choice in pomacanthids is supported by observations of predation upon relatively rare sponge species (Hourigan et al. 1989, Wulff 1994). Holacanthus passer often feeds on plankton and fish faeces in the water column in the eastern Pacific (Aburto-Oropeza et al. 2000); there are few exposed sponges on coral reefs there, but when cryptic sponges were exposed the angelfish quickly swam down out of the water column to feed on them (Wulff 1997). Caribbean angelfish also prefer to eat mangrove or cryptic sponge species whenever they are made available in the reefs (Dunlap and Pawlik 1996, Wulff 2005). In general, spongivorous fish tend to choose the most palatable or undefended prey species available in a given locality (Dunlap and Pawlik 1996), although which prey is more palatable varies according to the predator species (Wulff 2006a). For instance, the fire-sponge Tedania ignis is commonly eaten by H. tricolor and P. paru, but apparently not by H. ciliaris, both in Brazil and in the Caribbean (Table 1; see also Randall and Hartman 1968). The starfish Oreaster reticulatus also feeds on Tedania ignis in Belize, but not on the sibling species T. klausi (Wulff 2006b). Also, the dietary similarity between congeneric species of both Pomacanthus and Holacanthus is greater than between species from different genera (Table 1; see also Hourigan et al. 1989). Whether this truly represents a species-specific choice of prey by pomacanthids remains to be experimentally demonstrated. Both adult and juvenile angelfish present high plasticity, tolerance, and a certain degree of choice in their smorgasbord sponge-feeding specialized strategy. This appears to be a highly derived feeding strategy, which is coupled with morpho-functional modifications in the teeth, jaws, and gill rakers that make pomacanthids (especially Holacanthus and Pomacanthus) more apt to feed on structurally resilient and firmly attached benthic prey such as sponges, gorgonians and tunicates (Hourigan et al. 1989, Bellwood et al. 2004; Konow and Bellwood 2005). Sponges have been evolving during at least 580 MY (Li et al. 1998), and they developed chemical and physical defenses so effective that only very specialized predators such as angelfish and opisthobranchs are able to feed on them. This may help to explain why there are so few predators of sponges, despite their great abundance in coral reefs. Angelfish are threatened by marine ornamental fish trade in Brazil, and juveniles are the targets preferred by aquarists (Gasparini et al. 2005, Floeter et al. 2006). The maintenance of angelfish in aquaria is difficult due to their aggressiveness and their diet made up mostly of sponges (Thresher 1980). The development of artificial food based on the sponge species found in this study to be eaten by angelfish juveniles in the

field might help their reproduction and survival in captivity, allowing aquarists to enjoy angelfish in their saltwater tanks without eliminating them from coral reefs, where they play important ecological roles.

Acknowledgements
We thank Jos de Anchieta Nunes, Camilo Ferreira and Ericka Coni for laboratory assistance. We are also grateful to Samuele Clerici of Ax Online Fishes for the kind donation of specimens for study. We thank the two anonymous reviewers for their comments, which greatly improved the manuscript. This study was supported by grants and fellowships from Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico (CNPq) and Fundao Carlos Chagas Filho de Amparo Pesquisa do Estado do Rio de Janeiro (FAPERJ).

References
Aburto-Oropeza O, Sala E, Snchez-Ortiz C (2000) Feeding behaviour, habitat use, and abundance of the angelfish Holacanthus passer (Pomacanthidae) in the southern Sea of Corts. Environ Biol Fish 57: 435-442 Allen GR, Steene R, Allen M (1998) A guide to angelfishes and butterflyfishes. Odyssey Publishing, Perth Batista D (2006) Espongivoria por Pomacanthus paru Bloch, 1787 (Perciformes: Pomacanthidae) na costa brasileira. MSc Thesis, Universidade Federal Fluminense, Niteri Becerro MA, Paul VJ (1998) Intra-colonial variation in chemical defenses of the sponge Cacospongia sp. and its consequences on generalist fish predators and the specialist nudibranch predators Glossodoris pallida. Mar Ecol Prog Ser 168: 187-196 Bellwood DR (1988) Ontogenetic changes in the diet of early postsettlement Scarus species (Pisces: Scaridae). J Fish Biol 33(2): 213-219 Bellwood DR, Herwerden LV, Konow N (2004) Evolution and biogeography of marine angelfishes (Pisces: Pomacanthidae). Mol Phylog Evol 33: 140-155 Bhlke JE, Chaplin CCG (1968) Fishes of the Bahamas and adjacent tropical waters. Livingston Publications, Wynnewood Chanas B, Pawlik JR (1995) Defenses of Caribbean sponges against predatory reef fish. II. Spicules, tissue toughness, and nutritional quality. Mar Ecol Prog Ser 127: 195-211 Debelius H, Tanaka H, Kuiter RH (2003) Angelfishes, a comprehensive guide to Pomacanthidae. TMC Publishing, Chorley Deloach N (1999) Reef fish behavior Florida, Caribbean, Bahamas. New World Publications, Jacksonville Diaz MC, Rtzler K (2001) Sponges: an essential component of Caribbean coral reefs. Bull Mar Sci 69(2): 535-546 Dunlap M, Pawlik JR (1996) Video-monitored predation by Caribbean reef fishes on an array of mangrove and reef sponges. Mar Biol 126: 117-123 Feddern HA (1968) Systematics and ecology of Western Atlantic angelfishes, family Chaetodontidae, with an analysis of hybridization in Holacanthus. PhD Thesis, University of Miami, Coral Gables Feder HM (1966) Cleaning symbiosis in the marine environment. In: Henry SM (ed). Symbiosis. Vol. 1. Academic Press, New York. pp. 327-380 Floeter SR, Halpern BS, Ferreira CEL (2006) Effects of fishing and protection on Brazilian reef fishes. Biol Conserv 128: 391-402

137

Gasparini JL, Floeter S R (2001) The shore fishes of Trindade Island, southwestern Atlantic. J Nat Hist 35 (11): 1639-1656 Gasparini JL, Ferreira CEL, Floeter SR, Sazima I (2005) Marine ornamental trade in Brazil. Biodiv Conserv 14: 2883-2899 Gemballa S, Schermutzki F (2004) Cytotoxic haplosclerid sponges preferred: a field study on the diet of the dotted sea slug Peltodoris atromaculata (Doroidoidea: Nudibranchia). Mar Biol 144: 12131222 Hill MS, Lopez NA, Young KA (2005) Anti-predator defences in Western North Atlantic sponges with evidence of enhanced defence through interactions between spicules and chemicals. Mar Ecol Prog Ser 291: 93-102 Hourigan TF, Stantion FG, Motta PJ, Kelley CD, Carlson B (1989) The feeding ecology of three species of Caribbean angelfishes. Environ Biol Fishes 24(2): 105-116 Konow N, Bellwood DR (2005) Prey-capture in Pomacanthus semicirculatus (Teleostei, Pomacanthidae): functional implications of intramandibular joints in marine angelfishes. J Exp Biol 208: 1421-1433 Li CW, Chen JY, Hua TE (1998) Precambrian sponges with cellular structures. Science 279: 879-882 Mariani S, Uriz MJ (2001) Copepods of the genus Asterocheres (Copepoda: Siphonostomatoida) feeding on sponges: behavioral and ecological traits. Invertebr Biol 120: 269-277 Meyland A (1988) Spongivory in hawksbill turtles: a diet of glass. Science 239: 393-395 Monteiro-Neto C, Cunha FEA, Nottingham MC, Arajo ME, Rosa IL, Barros GML (2003) Analysis of the marine ornamental fish trade at Cear sate, northeast Brazil. Biol Conserv 12: 1287-1295 Muoz AA, Ojeda FP (2000) Ontogenetic changes in the diet of the herbivorous Scartichthys viridis in a rocky intertidal zone in central Chile. J Fish Biol 56: 986-998 Pawlik JR (1983) A sponge-eating worm from Bermuda: Branchiosyllis oculata (Polychaeta, Syllidae). Mar Ecol 4: 65-79 Pawlik JR, Chanas B, Toonen RJ, Fenical W (1995) Defenses of Caribbean sponges against predatory reef fish I. Chemical deterrency. Mar Ecol Prog Ser 127: 183-194 Randall JE, Hartman WD (1968) Sponge-feeding fishes of the West Indies. Mar Biol 1(3): 216-225 Reiswig HM (1973) Population dynamics of three Jamaican Demospongiae. Bull Mar Sci 23: 191-226 Ros R, Duffy JE (1999) Description of Synalpheus williamsi: a new species of sponge-dwelling shrimp (Crustacea: Decapoda: Alpheidae), with remarks on its first larval stage. Proc Biol Soc Wash 112: 541-552

Santos CP, Coutinho AB, Hajdu E (2002) Spongivory by Eucidaris tribuloides from Salvador, Bahia. J Mar Biol Assoc UK 82: 295297 Sar M, Vacelet J (1973) cologie des dmosponges. In Grass PP (ed). Trait de Zoologie, vol. 3, Spongiaires. Masson, Paris. pp. 462-576 Sazima I, Moura RL, Sazima C (1999) Cleaning activity of juvenile angelfish, Pomacanthus paru, on the reef of the Abrolhos Archipelago, western South Atlantic. Environ Biol Fishes 56: 399407 Sturm EA, Horn MH (1998) Food habits, gut morphology and pH, and assimilation efficiency of the zebra-perch Hermosilla azurea, an herbivorous kyphosid fish of temperate marine waters. Mar Biol 132: 515-522 Thresher RE (1979) Possible mucophagy by juvenile Holacanthus tricolor (Pisces: Pomacanthidae). Copeia 1979: 160-162 Thresher RE (1980) Reef fish: behavior and ecology on the reef and in the aquarium. John Bartholomew and Sons, Edinburg Waddell B, Pawlik JR (2000) Defenses of Caribbean sponges against invertebrate predators. II. Assays with sea stars. Mar Ecol Prog Ser 195: 133-144 Wicksten MK (1995) Associations of fishes and their cleaners on coral reefs of Bonaire, Netherland Antilles. Copeia 1995: 477481. Wicksten MK (1998) Behavior of cleaners and their client fishes at Bonaire, Netherland Antilles. J Nat Hist 32: 13-30 Wulff JL (1994) Sponge feeding by Caribbean angelfishes, trunkfishes and filefishes. In: van Soest RWM, van Kempen TMG, Braekman JC (eds). Sponges in time and space: biology, chemistry, paleontology. Balkema, Rotterdam. pp. 265-271 Wulff JL (1995) Sponge-feeding by the starfish Oreaster reticulatus. Mar Biol 123: 313-325 Wulff JL (1997) Causes and consequences of differences in sponge diversity and abundance between the Caribbean and eastern Pacific at Panama. Proc 8th Int Coral Reef Symp, Balboa 2: 1377-1382 Wulff JL (2001). Assessing and monitoring coral reef sponges: why and how? Bull Mar Sci 69(2): 831-846 Wulff JL (2005) Trade-offs in resistance to competitors and predators, and their effects on the diversity of tropical marine sponges. J Anim Ecol 74: 313-321 Wulff JL (2006a). Ecological interactions of marine sponges. Can J Zool 84: 146-166 Wulff JL (2006b) Sponge systematics by starfish: predators distinguish cryptic sympatric species of Caribbean fire sponges, Tedania ignis and Tedania klausi n. sp. (Demospongiae, Poecilosclerida). Biol Bull 211: 83-94

Porifera research: Biodiversity, innovation and sustainaBility - 2007

139

Growth and morphology of a reef-forming glass sponge, Aphrocallistes vastus (Hexactinellida), and implications for recovery from widespread trawl damage
William C. Austin(1*), Kim W. Conway(2), J. Vaughn Barrie(2), Manfred Krautter(3)
Marine Ecology Centre & Khoyatan Marine Laboratory, 9835 Seaport Place, Sidney, B.C., Canada. baustin@mareco.org Pacific Geoscience Centre, Geology Survey of Canada, P.O. Box 6000, Sidney, B.C., Canada. KConway@nrcan.gc.ca, vbarrie@nrcan.gc.ca (3) Institut fr Geologie und Palontologie der Universitt Stuttgart, Herdweg 51, D-70174, Stuttgart, Germany. Manfred.krautter@geologie.uni-stuttgart.de
(1) (2)

Abstract: Living hexactinellid reefs, known only on the western Canadian shelf, are being damaged by dredging and trawling. Recovery of damaged or destroyed hexactinellid reefs depend on many interrelated factors including sponge larval settlement and survival, sponge growth rates and the balance between suspended sediment trapping by the sponges and smothering by sedimentation. In this paper we present our work on one species found on the reefs, Aphrocallistes vastus Schulze 1887, measuring growth rates (approximately 300 cm2 yr-1 in surface area), the sizes of larger sponges (up to 3.4 m long x 1.1 m high x 0.5 m wide), indicators of successful recruitment (low based on occurrence of only one small individual in study site), and the form sponges can take under various environmental conditions. A. vastus sponges are very fragile and one was observed to die after breaking in two. Broken sponges have been observed on trawled reefs. Keywords: Aphrocallistes, form, growth, Hexactinellida

Introduction
Hexactinellid sponge reefs are found widely distributed on the western Canadian shelf in both Georgia and Queen Charlotte Basins of British Columbia. Multibeam surveying has provided accurate maps of sponge reef distribution in water depths of 100 to 240 m. The largest reef complex is roughly 40 kilometers long and has been growing for about 9,000 years with bioherms commonly up to 15 m in height (Conway et al. 1991). These reefs form a stable and complex habitat for many species of invertebrates and fish. However, bottom trawling has damaged reefs in most areas (Conway et al. 2000, Krautter et al. 2001). Aphrocallistes vastus is a dominant species in all sponge reefs surveyed (Conway et al. 1991, Conway et al. 2000). However, it also regularly occurs in BC fjords at depths as shallow as 25 m (Leys et al. 2004) and in one location at 5 m (Austin 1999, 2003). Determination of predicted tide levels has shown that some of these sponges actually occurred at only 2 m below 0 datum (Austin 2007). A. vastus together with two other species (Heterochone calyx, Farrea occa) can be considered as foundation species in the sense that they are the reef formers and generate a complex hard substrate habitat supporting a diverse biota in contrast to the surrounding soft level bottom substrate (Conway et al. 1991, Austin pers. observ.). Given their role as foundation species, and the demonstrated impacts of

trawling over the reefs (Conway et al. 2001), there has been much interest in assessing growth rates and recruitment as important determinants of recovery rates. However, estimates of minimum growth rates have only been inferred from observed sizes of A. vastus on a pipeline, cables, and a sunken vessel available for settlement for a known number of years. (see discussion for details). Shallow and accessible populations of Aphrocallistes vastus are present in Saanich Inlet, a fjord near Victoria, BC (Austin 1984, Leys et al. 2004) allowing these sponges to be studied as proxies for the sponges of the vast, but remote, northern reefs. Divers could directly observe, measure and photograph sponges over time. Here we present the results to date on growth, form, size, and recruitment at Senanus Reef in Saanich Inlet.

Material and methods Growth


Increase in mass of the sponge wall would be the most direct measure of growth but is not feasible for in situ measurements. However, the wall thickness is nearly constant for most of the sponge so that increase of surface area can approximate growth. Weighted, numbered floats were placed adjacent to sponges selected for subsequent growth measurements.

140

Initially five sponges were selected with a surveyors meter rod over time to assess growth; the very irregular form of four of these precluded accurate measurements of surface area. One small individual was ideal for measurement. It included a solid non-growing phallus-like cylinder and a growing fairly flat lobe. Multiple photos were taken while looking at a photo taken previously so that photos could be taken from the same position and distance relative to the sponge. A surveyors rod was held at the same position adjacent to the sponge. The sponge was photographed on eight dates over a period of four years. The outline of the sponge was drawn on transparent acetate which was slightly enlarged or reduced as necessary to fit the outline of the non-growing phallus portion as well as the decimeter spacing on the surveyors rod. The acetate was overlain on graph paper and the area was calculated by summing the squares within the outline. The area was also calculated from digitized images.

total surface area is, therefore, approximately 33,840 cm2 or 3.38 m2 ([948.4 g/0.55 g] x 19.625 cm2). Divers measured some of the larger sponges (Fig. 4, Table 1). Most of the sponges in the Senanus Reef population are on the order of one meter in size and typically have a very convoluted surface which amplifies the surface area considerably. Between 30 and 60 such sponges would be seen on a dive. Divers also looked for sponges smaller than approximately 5 cm in height or width. Six divers were able to find only one small sponge over the course of their dives (about 20 minutes) in March and September 2003.

Form
Aphrocallistes vastus individuals can take many forms in addition to the moderately dense mitten form dominating Senanus Reef (Fig. 5A). In an area of very high currents (Seymour Narrows) A. vastus has a very dense compact form (Fig. 5B). On Senanus Reef a few sponges form a tall narrow cylinder with sparse lateral mittens (Fig. 5C). This growth form dominates on some sponge reefs along with chalices (Fig 5D). Tubes with no mittens (Fig 5E) infrequently occur in fjords. Divers made counts of the number of sponges with living and dead bases. Seventy nine percent of 28 sponges over 30 cm in height had dead bases (Fig. 6).

Surface area of large sponge


Divers harvested one moderate size sponge to determine surface area of the sponge wall. In A. vastus the wall thickness remains constant throughout the body except for the edges of the mittens and of the oscula. Given that the thickness of the sponge wall remains constant, the ratio of surface area to weight should be approximately the same throughout the sponge. This sponge measured 0.65 m long x 0.48 m in diameter and occupied a space of about 0.12 m3. The sponge was air dried for 4 months.

Discussion Growth
We consider that surface area increases are a good measure of biomass changes as the thickness of the fused sponge wall is approximately the same throughout the sponge. The soft parts where growth occurred were limited to the ends of branches or of the main tube. Wulff (1990) reported that growth was only at branch apices in 3 demosponges she studied. The measurements are a relatively small underestimate as the rounded edges would be directed obliquely at the camera. An additional underestimate is likely for the surface area of the sponge taken at year 3 as there are some slight bulbous lateral expansions on the edges. The limited data, if reasonably accurate, indicate a modest increase in growth rate with increased size. If the growth rate prior to year 0 approximates that measured for year 0 to year 1, then extrapolating back (dotted line in Fig. 3) the sponge first settled about 1 years earlier. The straight lines on the graph are for visualization of overall growth rate. The lines would actually be slightly curved upward. At this time we only have growth rates for one sponge and for the first 5 years of age. Changes in a linear dimension (height or width) are often used as a measure of growth. Given that this dimension may bear little relation to increase in mass (see e.g. the different growth forms in Fig. 5), for purposes of comparison we included the length along the greatest axis when we first measured the sponge on year 0 and when we measured it again 4 years later. This was a difference of 39.8 cm or an average increase in size along one dimension of 10 cm year-1. Austin (2003) reported on the height of A. vastus sponges that had settled on a gas pipeline that had been in place for 9 years in the Strait of

Form
The forms of A. vastus sponges were observed and photographed from a range of habitats.

Results Growth
The appearance of the measured sponge over a 4 year period is shown in Fig. 1. The area of the A. vastus sponge when first measured, with additions one year later and 3 years later are shown in Fig. 2. In A. vastus the framework spicules are fused together along the main stem and at least on the sides of mitten shaped lateral projections. The edges of the mittens may be soft as are the edges of the oscula. Increase in surface area only occurred in the soft unfused areas of the sponge. The surface area (2 sides) was 369 cm2 when first measured; 661 cm2 one year later and 1,631 cm2 3 years later (Fig. 3). No attempt was made to assess surface area in the sponge photographed after 4 years as much of the surface was directed obliquely at the camera. However, the greatest linear dimension increased from 10.2 cm in year 0 to 50.0 cm in year 4

Surface area of large sponge


The large sponge collected by the divers weighed 948.4 g. Six 5 cm diam. wall plugs taken from different regions of the sponge were weighed. Weights ranged from 0.44 g to 0.74 g (mean 0.55 g for plugs with a surface area of 19.625 cm2. The

141

Fig. 1: Size of phallus form of A. vastus Sept. 12, 2002 (A), Sept. 13, 2003 (B), Feb. 18, 2006 (C), and Sept. 10, 2006 (D).

Georgia (Secret Cove). The largest was 64 cm tall, equivalent to an average growth rate of 7 cm year-1 if the sponge settled on the pipe when it was first installed. If this sponge had a linear growth rate comparable to that measured in our tagged sponge (10 cm year-1) and our tagged sponge was 1 year old, then the largest pipeline sponge could have settled 3 years after the pipeline was installed, and was 6 years old when measured. Leys et al. (2007) report the linear growth rate of a small Aphrocallistes vastus of approximately 1-3 cm year-1 based on photographs of the same sponge taken 1 year apart. They report massive changes in shape with growth including the flanges or projections and the location of the osculum. Our observations as noted above are that no changes occurred in the fused portions except for repair of damage to small areas of the surface. We suggest that some of the changes seen between the two images in Leys et al. (2007) are due to differences in perspective as the two images appear to be rotated about 45o apart when viewed from above the sponges.

Fig. 2: Area of A. vastus sponge when first measured, with additions one year later and 3 years later.

142

Fig. 3: Growth rate of A. vastus sponge over 3 years (solid line) with estimate of time when first settled (dotted line).

Table 1: Sizes of large A. vastus Length 3.4m 2.7m 2.1m 1.4m 1.4m Height 1.1m 1.0m 1.0m 2.0m 0.8m Width 0.5m 1.4m 0.4m 0.6m 0.8m

Fig. 4: Typical form of a giant A. vastus sponge.

There have been a few studies reported on growth rates in other species of hexactinellids. Dayton (1979) monitored Antarctic populations of 3 hexactinellid species over periods of 3 to 10 years. Scolymastra joubini and Rosella nuda showed no evidence of growth except for slight growth of 2-3 cm over 3-10 years in 3 individuals. However, small individuals of Rossella racovitzae increased in volume up to 292% over 3 years and larger individuals of the same species increased in length by 11 to 16 cm over 10 years. Marliave (1992) measured the length of Rhabdocalyptus dawsoni (an approximately cylindrical hexactinellid) over a period of about 6 months at the entrance to a British Columbia fjord (Howe Sound). He found that large (30-100 cm) sponges grew less than 20% in length while small (2-3.5 cm) sponges grew up to 71% in length. Leys and Lauzon (1998) measured the change in length and in volume of Rhabdocalyptus dawsoni in the same fjord as that used in the present study. They found the average growth in length over 3 years was 1.98 cm year-1 with a minimum of 0.76 cm year-1 and a maximum of 5.7 cm

year-1. Gatti (2002) estimated the age of Rossella spp. based on respiration rates. She used these rates to model growth in AMIGO [Advanced modeling of invertebrate growth from Oxygen consumption]. The average size Rossella spp. were 186 years old and the largest was 1515 years old based on the model. The hexactinellid sponges studied by the above authors belong to a different order from that of Aphrocallistes, have a quite different type of skeleton and generally have a cylindrical rather than a branching growth form. Given these differences, the growth rate in greatest dimension for our individual Aphrocallistes vastus averaging 10 cm year -1 is significantly greater than that found in other hexactinellids to date. The total surface area (3.68 m2) in the harvested sponge is about 20 times that of our measured sponge when estimated to be 5 years old. If the growth rate increase in our measured sponges holds true for large individuals, the harvested sponge would be about 1 century old. The largest sponge measured was 30 times the size of our harvested sponge based on overall height, width and depth. We will not speculate on the age of this sponge, but hope to obtain some age estimates in the coming year. A 1 m high Rhabdocalyptus dawsoni, in the same inlet was estimated to be 220 years old based on

143

Fig. 5: Various forms of A. vastus sponge. Typical in fjord (A); in area of high water currents (B); atypical skeleton sponge (C); on sponge reef (D); atypical form in fjord (E).

144

Fig. 6: Living A. vastus sponge with dead base.

measured volume increases for small individuals (Leys and Lauzon 1998). Given that divers were able to find only one small (< 5cm) sponges after a total of 2 hours searching, we conclude that recruitment was nil or very low over the period of the study. Divers report large numbers of small individuals at another site about 9 km away. Leys et al. (2007) report that of the many specimens of NE Pacific Aphrocallistes vastus collected since the early 1980s, developing embryos were found only once.

above the substrate so is less subject to clogging from buildup or re-suspension of sediment. We speculate that, perhaps, rather than living material dying at the base, the syncytium might move apically. Syncytial streaming has been demonstrated in microscopic preparations of Rhabdocalyptus dawsoni tissue sandwiched between slide and coverslip (Leys and Mackie 1994). The rate of streaming was 2 m sec-1, which is equivalent to 7 mm hour-1. Cytoplasmic streaming at a macroscopic level might be inferred from the opening and closing of spaces in the soft portions of the osculum noted by Austin (2003). The rate or frequency of apical streaming could be in response to impacts from sedimentation. Hence, sponge reefs subject to moderate sedimentation might grow up more slowly than those with high sedimentation. Such a mechanism could help explain how sponges avoid burial but also do not grow high so fast that they become unstable. For such a strategy to be successful the base and its attachment must be structurally sound during the life of the sponge. A. vastus may live, at least, many decades. Maldonado et al. (2005) have demonstrated that unlike diatoms, the silica in sponge spicules (including one hexactinellid species) showed little or no dissolution after acid cleaning and submersing in water low in silicates over an 8 month period. There is some support for very slow dissolution in the field from observations of hexactinellid sponge bases attached to the wall of Saanich Inlet. These are at a depth where the water has been anoxic for at least many decades (Levings et al. 1983).

Form
Aphrocallistes vastus is very fragile. Most of the wall has a texture and friability of a thin slice of toast. An erect growth form is suitable in the deep sea, and in most areas of a fjord where currents are minimal. The low compact form shown in Fig. 5B is likely an adaptation to the strong currents in the area. The mittens in Fig. 5A amplify the surface area and their largely vertical orientation minimizes clogging by sediment as discussed elsewhere (Austin 2003). The tall thin sponge (Fig. 5C) has been dubbed the skeleton sponge by divers. It occurs in the same habitat as the more typical sponges. Simple tubes (Fig. 5E) and chalices (Fig. 5D) may be a response to some environmental factor. For example, the entire inhalant surface would be facing obliquely down in a chalice form where sediment would not accumulate. We can only surmise that sediment would be blown off the upper exhalent surface. Some demosponges vary their form during growth. Halichondria panicea, for example has a low encrusting form which becomes stiffer and stronger in high wave/current energy environments, while in low current energy environments it has an erect ramose form and a more pliant, weaker skeleton (Palumbi 1986, Barthel 1991). The number of moderate sized sponges with dead bases (79%) was surprising. One interpretation of the data is that those with dead bases are dying which, if correct, indicates a major die-off of sponges at our study site. Another interpretation is that concentrating growth above a dead base has adaptive value. The filtering portion of the sponge is

Conclusions
What are the implications of our observations on impacts of trawling over A. vastus populations? The fragile nature of these sponges and their high profile dictates that they would most certainly be broken by a trawl and such damage has been observed on the reefs (e.g., Conway et al. 2001). Once broken, several lines of evidence indicate that they would likely not survive. When small areas are broken or removed by a hole saw, regeneration repairs the damage (Austin 2003). However, divers found a sponge sliced in two (likely by fishing line) (Austin et al. unpublished), and it subsequently died. If a sponge were knocked on its side by a trawl the broad surface of the mittens would be horizontal resulting in accumulation of sediment on the upper surface. Similarly, the tubular form common in sponge reefs, if knocked over, would be buried in sediment on its lower side and subject to sedimentation on its upper side. If the growth rate measured in our study is representative of growth rates on a sponge reef, then juveniles could reach a moderate size in a decade or two. However, many decades to perhaps a century or more would elapse before an old growth reef was fully developed. Recruitment of juveniles is also a key factor. No small (<5 cm) sponges have been found on Senanus reef while they may be common at a site about 9 km away. Finally, A. vastus is one of two (Strait of Georgia reefs) or three (northern reefs) reef forming sponges. Nothing is known about the growth of the other species. However, their fragility is comparable to that of A. vastus (Austin pers. observ).

145

Acknowledgements
Our thanks to Paula Romagosa, who worked on the graphics and who with Katya Austin reviewed the manuscript for spelling, grammatical and typographical errors, Sherry Ward, Coastal & Ocean Resources Inc., who helped on digitizing images, Jonathan Grant & Fred Holmes vessel operators, and the key contributions by members of the Victoria Dive Club: Mike Miles, Neil Lake, Tom Dakin, Doug Bifford, Parris Champoise, Joe Doiron, Carole Valkenier Pope, Ian Pope, Al Truby, Andy Murch, Sandie Hankewich, Doug Campbell, Mike Kalina, James Dranchuk, Mark Gottfried, Fred Peters, and David Willis. Also, our thanks to two anonymous reviewers for their helpful suggestions.

References
Austin WC (1984) Underwater birdwatching. In: Juniper SK, Brinkurst RO (eds). Proceedings of a multidisciplinary symposium on Saanich Inlet. Canadian Tech Rept Hydrogr Ocean Science 38. pp 104 Austin WC (1999) The relationship of silicate levels to the shallow water distribution of hexactinellids in British Columbia. Memoir Queensl Mus (abstract) 44: 44 Austin WC (2003) Sponge gardens. A hidden treasure in British Columbia. http://mareco.org/khoyatan/spongegardens (accessed on September 23, 2006) Austin WC (2007) Sponge gardens update. A hidden treasure in British Columbia. http://mareco.org/khoyatan/spongegardens Barthel D (1991) Influence of different current regimes on the growth form of Halilchondria panicea Pallas. In: Reitner J, Keupp H (eds). Fossil and recent sponges. Springer-Verlag, Berlin. pp. 387-394 Conway KW, Barrie JV, Austin WC, Luternauer JL (1991) Holocene sponge bioherms on the western Canadian continental shelf. Cont Shelf Res 11: 771-790 Conway KW, Krautter M, Barrie JV, Austin WC, Neuweiler M (2000) Extant hexactinellid sponge reefs: our endangered seafloor heritage. Abstracts GeoCanada, May 29-June 2, 2000. Calgary Conway KW, Krautter M, Barrie JV, Neuweiler M (2001) Hexactinellid sponge reefs on the Canadian continental shelf: a unique living fossil. Geoscience Canada 28(2): 71-78

Dayton PK (1979) Observations of growth, dispersal and population dynamics of some sponges in McMurdo Sound, Antarctica. In: Lvi C, Boury-Esnault N (eds). Biologie des spongiaires. Colloques Internationaux du CNRS, vol. 291. ditions du CNRS, Paris. pp. 271-282 Gatti S. (2002) The role of sponges in high-Antarctic carbon and silicon cycling a modeling approach. Ber Polarforsch Meeresforsch 434: 1-102 Krautter M, Conway KW, Barrie JV, Neuweiler M (2001) Discovery of a living dinosaur; globally unique modern hexactinellid sponge reefs off British Columbia, Canada. Facies 44: 265-282 Levings CD, Foreman RE, Tunnicliffe VJ (1983) Review of the benthos of the Strait of Georgia and contiguous fjords. Can J Fish Aq Sci 40: 1120-1141 Leys SP, Lauzon NRJ (1998) Hexactinellid sponge ecology: growth rates and seasonality in deep water sponges. J Exp Mar Biol Ecol 230: 111-129 Leys SP, Mackie GO (1994) Cytoplasmic streaming in the hexactinellid sponge Rhabdocalyptus dawsoni (Lambe 1873). In: van Soest RWM, van Kempen TMG, Braeckman J (eds). Sponges in time and space: biology, chemistry, paleontology. Balkema, Rotterdam. pp. 417-423 Leys SP, Wilson K, Holeton C, Reiswig HM, Austin WC, Tunnicliffe VJ (2004) Patterns of glass sponge (Porifera, Hexactinellida) distribution in coastal waters of British Columbia. Canada. Mar Eco Prog Ser 283: 133-149 Leys SP, Mackie GO, Reiswig HM (2007) The biology of glass sponges. Adv Mar Biol 52: 1-145 Marliave JB (1992) Environmental monitoring through natural history research. Canadian Tech Rpt Fish Aq Sci 1879: 199-209 Maldonado M, Carmona M, Velsquez Z, Puig A, Cruzado A, Lpez A, Young CM (2005) Siliceous sponges as a silicon sink: an overlooked aspect of benthopelagic coupling in the marine silicon cycle. Limnol Oceanogr 50(3): 799-809 Palumbi SR (1986) How body plans limit acclimation responses of a demosponge to wave force. Ecology 67(1): 208-214 Wulff JL (1990) Patterns and processes of size change in Caribbean demosponges of branching morphology. In: Rtzler K (ed). New perspectives in sponge biology. Smithsonian Institution Press, Washington. pp. 425-434

Porifera research: Biodiversity, innovation and sustainaBility - 2007

147

Symbiotic relationships between sponges and other organisms from the Sea of Cortes (Mexican Pacific coast): same problems, same solutions
Enrique vila(1,2*), Jos Lus Carballo(1), Jos Antonio Cruz-Barraza(1,2)
Laboratorio de Ecologa del Bentos, Instituto de Ciencias del Mar y Limnologa. Universidad Nacional Autnoma de Mxico, Estacin Mazatln, Avenida Joel Montes Camarena S/N, Apartado Postal 811, Mazatln 82000, Mxico. Tel. +52 669 985 28 45. Fax: +52 669 982 61 33. kike@ola.icmyl.unam.mx (2) Posgrado en Ciencias del Mar y Limnologa, UNAM, Mazatln, Mxico
(1)

Abstract: This study provides a morphological description of three symbiotic associations between sponges (Haplosclerida) and other macroorganisms from the Sea of Cortes (Mexican Pacific Ocean). These associations include: (1) a two-sponge association (Haliclona sonorensis/Geodia media), (2) a sponge-red macroalga association (Haliclona caerulea/Jania adherens) and (3), a sponge-coral association (Chalinula nematifera/Pocillopora spp.). So far these interactions seem to be obligatory for the sponges (Haliclona spp. and C. nematifera), since we have never found them living in isolation. Interestingly, similar associations have been described from other places around the world. Associations quite similar to (1) have been described from the Caribbean, and associations (2) and (3) are comparable to others described from the Western Pacific. Instead of comparing these associations with their sibling associations worldwide, we discussed the ability of haplosclerid sponges to form symbiotic associations with other organisms, since these sponges pertain to the group of which the most associations have been described. Keywords: Sea of Cortes, sponge-alga, sponge-coral, symbiotic associations, two-sponge

Introduction
Sponges are one of the major phyla found in the hardsubstrate marine benthos (Sar and Vacelet 1973). One of their more interesting characteristics is that they are able to establish a great diversity of relationships (mutualism, commensalism and parasitism) with unicellular and multicellular organisms (Palumbi 1985, Rtzler 1990, Magnino and Gaino 1998, Ilan et al. 1999, Wulff 1999, Thakur and Mller 2004). Most of these relationships have been reported in the Caribbean (West 1979, Rtzler 1990, Wulff 1997a, 1997b, 1999, Wilcox et al. 2002), Mediterranean Sea (Uriz et al. 1992, Gaino and Sar 1994), Red Sea (Meroz and Ilan 1995, Ilan et al. 1999), Indo-Pacific region (Steindler et al. 2002, Calcinai et al. 2004) and Western Pacific (Vacelet 1981, Trautman et al. 2000). Surprisingly, in a large number of cases, the same species are involved in similar interactions in different oceans (Ilan et al. 1999, Wulff 2006). For example, the sponges Haliclona caerulea Hechtel, 1965, Haliclona cymaeformis Esper, 1794 and Dysidea janiae (Duchassaing and Michelotti, 1864) are species found to establish similar interactions with red macroalgae (Vacelet 1981, Rtzler 1990, Trautman et al. 2000, Carballo and vila 2004). However, it is unknown whether this group of cosmopolitan associations occurs in the East Pacific Ocean.

The order Haplosclerida (Demospongiae) is the group in which the most of the symbiotic relationships have been registered with the family Chalinidae Gray, 1867 accounting for more than 50% of the associations described in this order (see discussion). Sponges of this order can establish associations with microorganisms such as bacteria (Vacelet et al. 2001), cyanobacteria (Steindler et al. 2002), dinoflagellates (Garson et al. 1998) and zoochlorellae (Frost and Williamson 1980), and with macroorganisms such as polychaetes (Dauer 1974), macroalgae (Vacelet 1981), mangroves (Ellison et al. 1996), barnacles (Ilan et al. 1999), hydrozoans (Schuchert 2003), anthozoans (West 1979), ophiurids (Henkel and Pawlik 2005), insects (Gaino et al. 2004) and other sponges (Wilcox et al. 2002). In the present study, we describe three interactions involving haplosclerid sponges from the Sea of Cortes (Mexican Pacific Ocean). They are a two-sponge association (Haliclona sonorensis-Geodia media), a sponge-alga association (Haliclona caerulea-Jania adherens) and a sponge-coral association (Chalinula nematifera-Pocillopora spp.). We discuss the surprising parallelism that exists worldwide, given that very similar interactions occur in different oceans. In addition, we comment on the characteristics that this group in which more symbiotic relationships have been registered has in order to establish these symbiotic associations.

148

Material and methods


Specimens of the three associations were collected by SCUBA diving from 2 to 12 m depth, in four localities from the Sea of Cortes (Eastern Pacific Ocean, Mexico) (Fig. 1). We followed the techniques described by Rtzler (1974) for spicule and tissue preparations for light microscopy. Crosssections of the specimens were washed in distilled water and then dried on a cover glass and coated with gold for scanning electron microscope (SEM) observations. A number of 20 to 50 spicules chosen randomly were measured (length x width) from each of the specimens studied. The number between brackets in each description is the average. After the description, the specimens were fixed in formaldehyde 4% and after 24 h they were transferred to 70% alcohol for their preservation. All the specimens were deposited in the Coleccin de Esponjas of the Instituto de Ciencias del Mar y Limnologa, UNAM (LEB-ICML-UNAM), in Mazatln (Mexico). In the Haliclona sonorensis/Geodia media association, the surface of G. media covered by H. sonorensis was estimated. First we took photographs of the specimens, then we determined the covered area (%) using the computer program Coral Point Count with Excel extensions (CPCe) (Kohler and Gill 2006). The frequency of the C. nematifera/Pocillopora spp. association was determined in three transects of 50 m length at a depth between 4 to 6 m. In each one of these transects we chose 20 colonies of coral at random, and determined the percentage of these containing sponge. In the same area, we also checked if the sponge was on another type of substratum. For each sample of the association we determined the species of coral and estimated the percentage of the colony overgrown by the sponge as number of branches with sponge of the total of branches.

Results
Association Haliclona sonorensis Cruz-Barraza and Carballo, 2006 Geodia media Bowerbank, 1873 Material examined. Eleven specimens of the association were collected between 2 and 5 m depth in two localities from the northern Sea of Cortes: Punta Cazn (Baha Kino, Sonora, 285220 N, 1120201 W), and Punta Pinta (La Choya, Puerto Peasco, Sonora, 311805 N, 1135911 W) (Fig. 1), from August 2000 to April 2005. Description of the species involved in the association. The epizoic sponge was identified as Haliclona sonorensis CruzBarraza and Carballo, 2006, which is a cushion-shaped sponge from 2 to 5 mm in thickness (Fig. 2A). Consistency is soft, compressible, but fragile and brittle. The surface is smooth and the ectosomic layer is not easily detachable. The color is pinkish violet in life and ocre or light brown in alcohol. The oscules are scarce, and circular or oval-shaped (from 0.5 to 1 mm in diameter), situated at the summits of volcano-shaped elevations. The skeletal material is constituted by oxeas that measure: 77-(112)-150 x 2-(5.6)-10 m. The supporting sponge was identified as Geodia media Bowerbank, 1873. This is a massive-incrusting to massive amorphous sponge (from 3.5 to 8 cm thick). The surface is

irregular, smooth to the naked eye, but finely rough to the touch. The natural color of the surface is from dark-brown to white. The choanosome is yellowish or beige. Small ostialpores from 150 to 300 m are regularly distributed on the surface. The oscules are contained in several small, circular or oval-shaped well-defined flattened sieves (containing from 7 to more than 100 oscules). The oscules measure from 0.22 to 2.5 mm in diameter. Consistency of the ectosome is very hard due to the cortex of sterrasters. The choanosome is cavernous and very densely spiculated, with a firm and slightly compressible consistency. The skeletal material is constituted by megascleres: oxeas, 620-(1430)-1950 x 10(31)-42 m; large styles, 620-(1077)-1260 x 22-(36)-45 m; strongyloxeas, 150-(197.2)-292 x 2.5-(4.9)-7.5 m; plagiotriaenes, 550-(1078)-1700 m rabdome length; and anatriaenes, 1120-(1410)-2040 m rabdome length, and microscleres: sterrasters, 25-(62.8)-90 m oxyasters, 20(27.2)-45 m; oxyspherasters, 6.3-(9.5)-13 m. Description of the association. The specimens of the association were found attached to the rocky substrata, covering areas from 8 x 6.5 to 20 x 15 cm, approximately. H. sonorensis forms a thin layer that covers up to 57 % of the surface of G. media, while the surface that is not covered by the sponge is occupied by other epibionts (green and red algae, bryozoans, polychaetes and bivalves). Only the oscular areas (from 1 to 4 cm in diameter) are free of these epibionts (Fig. 2A). In some cases, more than one individual of H. sonorensis was observed on a same specimen of Geodia, which was evident by their different tonality of coloration. Despite being interwoven, Haliclona was unattached in some areas, where we observed that the external tissue of Geodia did not seem to be damaged by the epizoic sponge (Fig. 2B, C). The area of G. media lacking epibionts has a rough texture due to the external layer of sterrasters (Fig. 2C, D), but the SEM showed that the megascleres (triaenes and oxeas) of G. media protrude the surface in the areas covered by Haliclona (Fig. 2C, F) penetrating in the Haliclona sonorensis tissue (Fig. 2D, E). There were spicules (oxeas) of H. sonorensis inside the ostias and embedded in the choanosome of G. media, and there were also sterrasters of G. media in the choanosome of H. sonorensis. Haliclona sonorensis has been invariably found living on the surface of G. media which suggests that this species needs to live in association with G. media. Association Chalinula nematifera (de Laubenfels, 1954) Pocillopora spp. Lamarck, 1816 Material examined. A total of ten specimens of this association were collected between 3 and 12 m depth in three sites from Isabel Island (Baha Tiburones, Playa Las Monas and Playa Iguanas), Nayarit, Mexico (285220 N, 1120201 W) (Fig. 1), from December 2003 to July 2006. Morphological description of the species in the association. The epizootic sponge has been identified as Chalinula nematifera (de Laubenfels, 1954). This is an encrusting sponge of violet color (1-4 mm thickness). This sponge grows only on live corals found in Isabel Island (Fig. 3). The surface is smooth to the naked eye, but it is punctated and shaggy in some places. Oscules are abundant, circular, from 4 to 6 mm

149

Fig. 1: Sampling localities (letters). The numbers indicate the site where specimens of each sponge association were collected: (1) two-sponge association, (2) sponge-alga association and (3) sponge-coral association.

in diameter, and regularly distributed on the surface. They are situated at the summits of volcano-shaped elevations. Consistency is soft and spongy, somewhat elastic and slimy. The skeleton consists of oxeas: 87-(98)-112.5 x 2.5-(4.4)-5 m. Our specimens also show the characteristic pale threads through the body as described by de Laubenfels (1954), which presumably is a symbiotic fungus (WF Prudhomme van Reine, comments in de Weerdt 2002). The coral species on which C. nematifera was found were identified as Pocillopora damicornis Linnaeus, 1758, P. meandrina Dana, 1846, P. capitata Verrill, 1864 and P. verrucosa Ellis and Solander, 1786. In general, these coral species have characteristic shape because they form densely ramified colonies. They have calices crowded together over regularly-spaced wart-like projections (verrucae) (P. capitata and P. verrucosa). Description of the association. C. nematifera was found always on live ramified corals that live in areas exposed to light, most frequently with P. verrucosa (67%), and never on another type of substratum. Nevertheless, it is possible to find these species of coral (mentioned above) without the sponge.

Approximately 17% of the Pocillopora colonies studied had C. nematifera in association. In these sponge/coral interactions, C. nematifera can cover branches partially or totally (55.12% of the total of branches of the colony), and in all these cases we observed that the surface of the covered coral has no polyps. This sponge adhered firmly to the coral and is not easy to detach it from the substratum without breaking it. In fact, through the SEM images, we observed that the skeletal structure of the sponge seems to be cemented to the coral septa by spongin layers (Fig. 3C). Association Haliclona caerulea Hechtel, 1965 Jania adherens Lamouroux, 1816 Material examined. Sixteen specimens of the sponge-alga association were collected from ten sites from the Mazatln Bay (231349 N, 1062743 W), Sinaloa, Mexico (Fig. 1), between 2 to 6 m depth, from November 1997 until October 2003. Morphological description of the species in the association. Haliclona (Gellius) caerulea is a massive sponge (from

150

Fig. 2: Haliclona sonorensis Geodia media association. A. two-sponge association containing several epibionts on its surface except on the osculate area. B. cross section of a specimen showing the Geodia surface almost totally covered by Haliclona sonorensis. D. SEM image showing the surface contact of the two interacting sponges, and C, E, F. the megascleres of Geodia protruding its ectosome, which are used as anchorage for the external sponge. The arrow in F shows an ostium of the internal sponge. Scale bars: A and B= 2 cm, C= 5 mm, D= 500 m, E= 200 m, F= 500 m.

3 to 13 cm high), white or beige in life and very brittle. The skeleton is constituted by oxeas (82.5-(177.3)-210 m) and sigmas (17.5-(21.6)-30 m). The sponge has an unispicular ectosomal skeleton, formed by an isotropic tangential reticulation of oxeas, and the choanosomal skeleton is a somewhat confused reticulation of uni-multispicular primary and secondary lines that are difficult to appreciate because of their association with the alga (Fig. 4B). The alga was identified as Jania adherens Lamouroux, 1816, which is an articulated erect red macroalga. The alga is

pink with white joints, repeatedly branched, with a calcified thallus (from 0.4 to 0.5 mm diameter) except at the genicula. Description of the association. This sponge lives in intimate association with the red calcareous alga Jania adherens. The association consists of a massive and compact form where the sponge completely fills the spaces between the algal branches (Fig. 4A). The sponge generally covers the alga, and the algal branches very rarely protrude beyond the association surface. The morphology of the association is derived from the growth form of both organisms. Specimens of this association are lo-

151

Fig. 3: A. Chalinula nematifera Pocillopora spp. association. B. C. nematifera tissue in close contact with the coral polyps (arrows). C. SEM image showing the skeletal structure of the sponge cemented to the coral septae (arrows). Scale bars A= 1 cm, B= 2000 m, C = 100 m.

cally abundant in rocky substrate environments (2-6 m depth), in areas of strong wave force. However, in these places, it is not possible to find the two species living in isolation, even though J. adherens lives in isolation in the intertidal zone. Jania adherens forms compact turfs approximately 2 cm high in the intertidal zone. However, in the subtidal zone (in association with H. caerulea) it reaches up to 13 cm in height. In the images obtained by SEM, we observed that the spicules of H. caerulea adhere firmly to the stalks of J. adherens by means of a fine spongin layer (Fig. 4C). We also observed that the primary lines of the sponge are partially replaced by the macroalgal thallus.

Discussion Two-sponge association


Interactions among two or more sponges of different species, including cases of mutualism (Wilcox et al. 2002), parasitism (Wulff 1999) or space competition (Rtzler 1970, Wulff 2006) have been previously recorded. A two-sponge association quite similar to ours was described recently from the Florida Keys (Wilcox et al. 2002). In both cases, as it has also been documented for other two-sponge associations (Sim 1998, Wilcox et al. 2002),

152

Fig. 4: A. Haliclona caerulea Jania adherens association. B. SEM images showing the skeletal structure of H. caerulea, which are partially substituted by the J. adherens branches in the association, and C. a close up of a spicule secondary line adhered to the algal thallus by means of a fine spongine layer. Scale bars A= 1 cm, B= 300 m, C= 60 m.

Haliclona sp. seems to be anchored on the protruding spicules of Geodia spp. There are studies that suggest that growth and survival increases when certain sponges of different species live in association, because they have different susceptibility to factors such as burial by sediment, fragmentation, predation and pathogens (Wulff 1997a, 1999, Engel and Pawlik 2005, Wulff 2006). However, in the association from the Florida Keys, it was suggested that it could be a mutualistic and obligatory symbiosis, where the external sponge protects its host from the predation, while Geodia sp. offers it a sure substratum for its survival (Wilcox et al. 2002). However, it is interesting to comment that the Caribbean sponge Geodia gibberosa is chemically undefended against turtle and fish predation (Dunlap and Pawlik 1996, 1998), and uses its chemical products to attract fouling organisms. This could resemble Geodia sp. in association with Haliclona sp. (Wilcox et al. 2002), since one of the benefits acquired by the sponge Geodia cydonium fouled by the red alga Rytyphlea tinctoria is the protection against the adverse effects of ultraviolet radiation, allowing the specimens to live under illuminated habitats (Mercurio et al. 2006). It is possible that an obligatory relationship also exists between H. sonorensis and G. media, at least for H. sonorensis, since it has not been found in isolation within the environment where the associations are encountered. In fact, the high specificity of Haliclona to live with Geodia is very interesting because this association does not originate in a highly space-competitive environment like reef ecosystems, and therefore it could not be a simple case of epizoism due to space limitation (Rtzler 1970). In addition, it is important to mention that there is no evidence of tissue damage on the surface of G. media covered by H. sonorensis, as it has been documented in other two-sponge interactions (Thacker et al.

1998). Although we did not make experiments to test whether a benefit exists between G. media and H. sonorensis, bioactive natural products have been described in G. media from the Sea of Cortes (Pettit et al. 1981, Pettit et al. 1990), that could attract fouling organisms, similar to Geodia gibberosa (Engel and Pawlik 2000, Engel and Pawlik 2005). In the association described here, one or more specimens of H. sonorensis are found attached to the same G. media specimen, establishing contact but without fusing. These observations support the idea that this sponge most likely colonizes G. media through larval settlement, in order to obtain an appropriate substratum for its survival (Wulff 1997b, Wilcox et al. 2002).

Sponge-coral association
Sponge-coral interactions are common in coral reefs where strong competition for space exists and where it is frequent that aggressive sponges overgrow the coral (Aerts 1998). The sponge Chalinula nematifera has been described previously overgrowing hard corals in reefs from the WestCentral Pacific (de Laubenfels 1954). Surprisingly, this species also seems to be specifically attracted to pocilloporid corals from the Sea of Cortes, since it has not been found colonizing other types of substratum. Nevertheless, the species of coral associated with this sponge can be found in an isolated form. This suggests that it could be an obligatory relationship for the sponge, and facultative for the host (Pocillopora spp.). We also suggest that this relationship can have negative outcome for the coral, since the sponge seems to be killing the coral tissue, similar to what has been documented for several sponge/coral interactions (Jackson and Buss 1975, PlucerRosario 1987, Macintyre et al. 2000, Aerts 2000, Rtzler 2002, de Voogd et al. 2004, Coles and Bolick 2006, Gochfeld

153

et al. 2006). However, we do not know if C. nematifera uses chemical products to kill the coral polyps (Nishiyama and Bakus 1999) or if it simply smothers them (Wulff 1999). Some authors have suggested that the ability of sponges to overgrow corals appears to depend on their growth rate and form (Aerts 2000). For example, in the sponge Terpios hoshinota, it was suggested that its success to overgrow large extensions of live corals is due to a fast spreading rate, aided by fast asexual propagation (fragmentation) (Plucer-Rosario 1987, Rtzler and Muzik 1993). Chalinula nematifera possibly finds a substratum that offers multiple protected microhabitats in pocilloporid corals, since they are densely ramified species which are often used as microhabitats by several organisms (Patton 1974). Thus, C. nematifera could likely benefit by finding an appropriate substratum that offers protection against environmental factors (e.g. UV radiation, sedimentation and/or predation). This is an advantage that it could not find with the other non-branched coral species (Porites, Psammocora, Pavona and Fungia) that inhabit the same site as the C. nematifera/ Pocillopora spp. association, most likely because they do not offer this kind of physical protection. Although in most of the sponge-coral interactions that have been described, it has been found that these relationships seem to be negative for the coral (de Voogd et al. 2004, Coles and Bolick 2006, Gochfeld et al. 2006, Lpez-Victoria et al. 2006), there are also documented cases of mutualisms; for example, the sponge Mycale laevis Carter 1882 which is found encrusting on the lower surfaces of flattened reef corals (mainly on Montastrea annularis) in the Caribbean Sea. M. laevis benefits by colonizing a substrate that is free from other sessile organisms. In turn, the coral benefits from an increased feeding efficiency as a result of water currents produced by the sponge and it is protected from invasion by boring sponges (Goreau and Hartman 1966). In the case of sponge/octocoral associations, the sponge generally obtains structural support from its partner and the octocoral obtains protection against predation (van Soest 1987, Calcinai et al. 2004). In addition, it has been documented that both organisms also benefit by increasing their dispersal capacity (Calcinai et al. 2004).

and vila 2004, Carballo et al. 2006). However, in spite of the similarities of these two interactions, the H. caerulea/ J. adherens association does not live in an oligotrophic environment in the Bay of Mazatln (see Carballo 2006). Our investigations into the H. caerulea-Jania adherens association suggests that this is an obligatory and mutualistic association, where both organisms benefit by increased growth, widespread their distribution area toward an environment where none can inhabit by itself, and by the acquisition of a structural support that gives them bigger rigidity than their free-living form (vila and Carballo 2004, Carballo and vila 2004, Carballo et al. 2006). H. caerulea can also substitute part of its skeletal structure (mainly primary lines) with the branches of J. adherens, reducing the investment in spicule production (Carballo et al. 2006), as it has been documented in other sponge symbioses (de Laubenfels 1950, Vacelet 1981, Rtzler 1990, Uriz et al. 1992). In addition, it has been demonstrated that H. caerulea in association with J. adherens (from the Pacific coast of Panama) acquires the benefit of being protected against fish predation (Wulff 1997a). On the other hand, H. caerulea has never been found in isolated form at the depth range where the sponge-alga association lives in the Bay of Mazatln, because it has a very fragile structure, and it is unable to live in that environment, which is characterized by strong water movement (Carballo et al. 2006). Although this association reproduces mainly by fragmentation, larval settlements of H. caerulea have been registered in the intertidal zone where J. adherens inhabits in isolated form (vila and Carballo 2006). In fact, laboratory experiments demonstrated that the larvae of H. caerulea can select J. adherens as substratum while rejecting others (vila and Carballo 2006), probably because the alga fronds offer a shady and tangled microrefuge, which could increase the post-settlement survival (Buss 1979, Maldonado and Uriz 1998). On the other hand, it is important to add that this association has also been found in other localities in Mexico, such as Tuxpan, Veracruz (in the Gulf of Mexico) and Manzanillo, Colima and Huatulco, Oaxaca (in the Mexican Pacific) (personal observations).

Sponge-alga association
Associations between sponges and photosynthetic organisms are important not only for the partners, but also for the ecosystem they inhabit, because they can contribute significantly to the primary productivity, mainly in oligothrophic ecosystems (Wilkinson 1983, Steindler et al. 2002). One of the most-studied sponge/macroalga symbioses is the Haliclona cymaeformis/Ceratodictyon spongiosum association (from the Great Barrier Reef, Australia) (Trautman et al. 2000, Trautman and Hinde 2002, Davy et al. 2002), which we could consider as a sibling association of the H. caerulea/J. adherens association described here. In both cases the mutualistic association derives similar benefits for the partners (such as protection against physical disturbances, structural support, high dispersal capacity through fragmentation), and in both cases the sponge is a species of Haliclona that lives associated with a red macroalga (Trautman et al. 2000, Trautman and Hinde 2002, Carballo

The diversity of associations among the haplosclerid sponges


The three symbiotic sponges (H. sonorensis, C. nematifera and H. caerulea) described in this study belong to the order Haplosclerida, which is one of the most diverse groups of the phylum Porifera (Hooper and van Soest 2002). Upon revising the literature, we have found that most of the sponges (21% of 248 cases) that have been previously recorded establishing associations with other taxa (see introduction), belong to the order Haplosclerida, and mostly to the family Chalinidae (50% of 51 registrations) (Fig. 5) (Dauer 1974, Vacelet 1981, Ellison et al. 1996, Garson et al. 1998, Ilan et al. 1999, Vacelet et al. 2001, Steindler et al. 2002, Wilcox et al. 2002, among others). These associations have been recorded mainly in shallow tropical and subtropical environments (such as coral reefs), which are characterized by a high competition for space and food by the organisms that inhabit there. It seems that some sponges have probably learned to associate with

154

Fig. 5: A. Symbiotic associations between sponges and other organisms registered in the different orders of the class Demospongiae and B. in the order Haplosclerida.

other organisms that offer them some kind of protection against environmental factors (such as fish predation, sedimentation, UV radiation, tissue resistance). For example, in the Geodia sp./Haliclona sp. association, it has been suggested that Geodia sp. is chemically defended against fish predation and protected from sedimentation by the sponge partner (Wilcox et al. 2002). In another haploclerid species such as Haliclona cymaeformis and Haliclona implexiformis that live associated with photosynthetic organisms (macroalga and mangrove respectively), it has been demonstrated that nutrient translocation also exists between the sponge and its host (Ellison et al. 1996, Davy et al. 2002). In the case of species that shelter cyanobacteria (e.g. Adocia atra and Haliclona debilis) in their tissue, it has been suggested that they can benefit by protecting their surface from UV-radiation and/or obtain an alternative source of food (Rtzler 1990, Steindler et al. 2002). It has also been suggested that they reinforce their skeletal structure using their partner to avoid being broken into fragments by the current, as is the case of H. caerulea (Carballo et al. 2006) and H. cymaeformis (Trautman and Hinde 2002). On the other hand, there are many bioactive compounds known in haplosclerid sponges (Frincke and Faulkner 1982, Isaacs and Kashman 1992, van Soest and Braekman 1999), which can also be used for different purposes such as antifoulants (Sera et al. 2002), as antimicrobial compounds (Xue et al. 2004, Ely et al. 2004) or in competition for space (Nishiyama and Bakus 1999).

Conclusions
In general, it seems that in the sponge/sponge and sponge/macroalga associations the relationships are usually mutualistic, or at least no type of damage has been evidenced among the associated organisms. In contrast, in most of the non-boring sponges associated to ramified corals that have been described (including C. nematifera/Pocillopora spp. association), the relationship seems to be negative for the coral species. Nevertheless, it is necessary to do more experimental and population dynamics studies of these associations in order to understand more about the complexity of their life history and their ecological importance in the ecosystem they inhabit.

Acknowledgements
We are grateful to the following sources of funding: CONABIO FB666/S019/99, CONABIO FB789/AA004/02, CONABIO DJ007/26 and CONACYT SEP-2003- C02-42550. This work was carried out under permission of SAGARPA (Permit numbers: DGOPA.02476.220306.0985 and DGOPA.06648.140807.3121). We thank Clara Ramrez, Pedro Allende and Victoria Montes for help with the literature and images, German Ramrez and Carlos Surez for their computer assistance, Cayetano Robles, Gonzalo Prez and Cristina Vega for their assistance in the field sampling, and the Director Parque Nacional Isla Isabel Jorge Castrejn for the availability and the permission conferred for the collection of the samples in Isabel island.

155

References
Aerts LAM (1998) Sponge/coral interactions in Caribbean reefs: analysis of overgrowth patterns in relation to species identity and cover. Mar Ecol Prog Ser 175:241-249 Aerts LAM (2000) Dynamics behind standoff interactions in three reef sponge species and the coral Montastraea cavernosa. PSZN Mar Ecol 21: 191-204 vila E, Carballo JL (2004) Growth and standing stock biomass of a mutualistic association between the sponge Haliclona caerulea and the red alga Jania adherens. Symbiosis 36(3): 225-244 vila E, Carballo JL (2006) Habitat selection by larvae of the symbiotic sponge Haliclona caerulea (Hechtel, 1965) (Demospongiae, Haplosclerida). Symbiosis 41: 21-29 Buss LW (1979) Bryozoan overgrowth interactions-the interdependence of competition for space and food. Nature 281: 475-477 Calcinai B, Bavestrello G, Cerrano C (2004) Dispersal and association of two alien species in the Indonesian coral reefs: the octocoral Carijoa riisei and the demosponge Desmapsamma anchorata. J Mar Biol Assoc UK 84: 937-941 Carballo JL (2006) Effect of natural sedimentation on the structure of tropical rocky sponge assemblages. Ecoscience 13: 119-130 Carballo JL, vila E (2004) Population dynamics of a mutualistic interaction between the sponge Haliclona caerulea, and the red alga Jania adherens. Mar Ecol Prog Ser 279: 93-104 Carballo JL, vila E, Enrquez S, Camacho L (2006) Phenotypic plasticity in a mutualistic association between the sponge Haliclona caerulea and the calcareous macroalga Jania adherens induced by transplanting experiments. I: morphological responses of the sponge. Mar Biol 148: 467-478 Coles SL, Bolick H (2006) Assessment of invasiveness of the orange keyhole sponge Mycale armata in Kneohe Bay, Oahu, Hawaii. 7th Int Sponge Symp (abstract) pp. 110 Cruz-Barraza JA, Carballo JL (2006) A new species of Haliclona (Demospongiae: Haplosclerida) living in association with Geodia media Bowerbank (Mexican Pacific coast). Zootaxa 1343: 43-54 Davy SK, Trautman D, Borowitzka MA, Hinde R (2002) Ammonium excretion by a sponge and its ecological importance in a spongerhodophyte symbiosis. J Exp Biol 205: 3505-3511 Dauer DM (1974) Polychaete fauna associated with Gulf of Mexico sponges. Florida Scientist 36: 192-196 de Laubenfels MW (1950) The sponges of Kaneohe Bay, Oahu. Pac Sci 4(1): 3-36 de Laubenfels MW (1954) The sponges of the west-central Pacific. Oreg St Monogr Zool 7: 1-306 de Voogd N, Becking LE, Hoeksema BW, Noor A, van Soest RWM (2004) Sponge interactions with spatial competitors in the Spermonde Archipelago. In: Pansini M, Pronzato R, Bavestrello G, Manconi R (eds). Sponge science in the new millennium. Boll Mus Ist Biol Univ Genova 68: 253-261 de Weerdt WH (2002) Family Chalinidae Gray. In: Hooper JNA, van Soest RWM (eds). Systema Porifera: a guide to the classification of sponges. Kluwer Academic/Plenum Publishers, New York. pp. 852-873 Dunlap M, Pawlik JR (1996) Video-monitored predation by Caribbean reef fishes on an array of mangrove and reef sponges. Mar Biol 126: 117-123 Dunlap M, Pawlik JR (1998) Spongivory by parrotfish in Florida mangrove and reef habitats. PSZN Mar Ecol 19: 325-337

Ellison AM, Farnsworth EJ, Twilley RR (1996) Facultative mutualism between red mangroves and root-fouling sponges in Belizean mangal. Ecology 77(8): 2431-2444 Ely R, Supriya T, Naik CG (2004) Antimicrobial activity of marine organisms collected off the coast of South East. India. J Exp Mar Biol Ecol 309: 121-127 Engel S, Pawlik JR (2000) Allelopathic activities of sponge extracts. Mar Ecol Prog Ser 207: 273-281 Engel S, Pawlik JR (2005) Interactions among Florida sponges. II. Mangrove habitats. Mar Ecol Prog Ser 303: 145-152 Frincke JM, Faulkner DJ (1982) Antimicrobial metabolites of the sponge Reniera sp. J Am Chem Soc 104: 265-269 Frost TM, Williamson CE (1980) In situ determination of the effect of symbiotic algae on the growth of the freshwater sponge Spongilla lacustris. Ecology 61: 1361-1370 Gaino E, Lancioni T, La Porta G, Todini B (2004) The consortium of the sponge Ephydatia fluviatilis (L.) living on the common reed Phragmites australis in Lake Piediluco (central Italy). Hydrobiologia 520: 175-178 Gaino E, Sar M (1994) Siliceous spicules of Tethya seychellensis (Porifera) support the growth of a green alga: A possible light conducting system. Mar Ecol Prog Ser 108: 147-151 Garson MJ, Flowers AE, Webb RI, Charan RD, McCaffrey EJ (1998) A sponge/dinoflagellate association in the haplosclerid sponge Haliclona sp.: cellular origin of cytotoxic alkaloids by Percoll density gradient fractionation. Cell Tissue Res 293: 365-373 Gochfeld D, Harrison L, Olson J, Lesser MP, Ankisetty S, Slattery M (2006) Allelopathic effects of the Caribbean sponge Svenzea zeai on the coral Montastrea annularis. In: Custdio MR, Lbo-Hajdu G, Hajdu E, Muricy G (eds). 7th International Sponge Symposium Book of Abstracts (Armao dos Bzios, Brazil). Museu Nacional, Srie Livros, vol. 16. pp. 116 Goreau TF, Hartman WD (1966) Sponge: effect on the form of reef corals. Science 151: 343-344 Henkel TO, Pawlik JR (2005) Habitat use by sponge-dwelling brittlestars. Mar Biol 146: 301-313 Hooper NA, van Soest RWM (2002) Systema Porifera: a guide to the classification of sponges. Kluwer Academic, Plenum Publishers, New York Ilan M, Loya Y, Kolbasov GA, Brickner I (1999) Sponge inhabiting barnacles on coral reefs. Mar Biol 133: 709-716 Isaacs S, Kasbman Y (1992) Shaagrockol B and C; two hexaprenylhydroquinone disulfates from the Red Sea sponge Toxiclona toxius. Tetrahedron Lett 33: 2227-2230 Jackson JBC, Buss L (1975) Allelopathy and spatial competition among coral reef invertebrates. Proc Natl Acad Sci 72: 5160-5163 Kohler KE, Gill SM (2006) Coral Point Count with Excel extensions (CPCe): A Visual Basic program for the determination of coral and substrate coverage using random point count methodology. Comp Geosci 32: 1259-1269 Lpez-Victoria M, Zea S, Weil E (2006) Competition for space between encrusting excavating Caribbean sponges and other coral reef organisms. Mar Ecol Prog Ser 312: 113-121 Macintyre IG, Precht WF, Aronson RB (2000) Origin of the Pelican Cays ponds, Belize. Atoll Res Bull 466: 1-11 Magnino G, Gaino E (1998) Haplosyllis spongicola (Grbe) (Polychaeta, Syllidae) associated with two species of sponges from East Africa (Tanzania, Indian Ocean). PSZN Mar Ecol 19: 77-87

156

Maldonado M, Uriz MJ (1998) Microrefuge exploitation by subtidal encrusting sponges: patterns of settlement and post-settlement survival. Mar Ecol Prog Ser 174: 141-150. Mercurio M, Corriero G, Gaino E (2006) Sessile and non-sessile morphs of Geodia cydonium (Jameson) (Porifera, Demospongiae) in two semi-enclosed Mediterranean bays. Mar Biol 148: 489-501 Meroz E, Ilan M (1995) Cohabitation of a coral reef sponge and a scyphozoan. Mar Biol 124: 453-459 Nishiyama GK, Bakus GK (1999) Release of allelochemicals by three tropical sponges (Demospongiae) and their toxic effects on coral substrate competitors. Memoir Queensl Mus 44: 411-417 Palumbi SR (1985) Spatial variation in an algal-sponge commensalism and the evolution of ecological interactions. Am Nat 126: 267-274 Patton WK (1974) Community structure among the animals inhabiting the coral Pocillopora damicornis at Heron Island, Australia. Bull Mar Sci 14(2): 212-243 Pettit GR, Rideout JA, Hasler JA (1981) Isolation of geodiastatins 1 and 2 from the marine sponge Geodia mesotriaena. J Nat Prod 44(5): 588-92 Pettit GR, Rideout JA, Hasler JA (1990) Isolation of geodiatoxins 2 and 4 from the marine sponge Geodia mesotriaena. Comp Bio Physiol Part C 96(2): 305-6 Plucer-Rosario G (1987) The effect of substratum on the growth of Terpios, an encrusting sponge which kills corals. Coral Reefs 5(4): 197-200 Rtzler K (1970) Spatial competition among Porifera: solution by epizoism. Oecologia 5: 85-95 Rtzler K (1974) The burrowing sponges of Bermuda. Smithson Contrib Zool 165:1-32 Rtzler K (1990) Associations between Caribbean sponges and photosynthetic organisms. In: Rtzler K (ed). New perspectives in sponge biology. Smithsonian Institution Press, Washington DC. pp. 455-466 Rtzler K (2002) Impact of crustose clionid sponges on Caribbean Reef corals. Acta Geol Hisp 37: 61-72 Rtzler K, Muzik K (1993) Terpios hoshinota, a new cyanobacteriosponge threatening Pacific reefs. In: Uriz MJ, Rtzler K (eds). Recent Advances in Ecology and Systematics of Sponges. Sci Mar 57(4): 395-403 Sar M, Vacelet (1973) cologie des Dmosponges. In: Grass PP (ed). Spongiaires. Anatomie, Physiologie, Systmatique, cologie. Masson de Cie, Paris. pp. 462-576 Schuchert P (2003) Hydroids (Cnidaria, Hydrozoa) of the Danish expedition to the Kei Islands. Steenstrupia 27: 137-256 Sera Y, Adachi K, Fujii K, Shizuri Y (2002) Isolation of Haliclonamides: New peptides as antifouling substances from a marine sponge species, Haliclona. Mar Biotechnol 3: 441-446

Sim CJ (1998) Two-sponge association from Komun Island, Korea. In: Watanabe Y, Fusetani, N (eds). Sponge sciences: multidisciplinary perspectives. Springer-Verlag, Tokyo. pp 109117 Steindler L, Beer S, Ilan M (2002) Photosymbiosis in intertidal and subtidal tropical sponges. Symbiosis 33:263-273 Thacker RW, Becerro MA, Lumbang WA, Paula VJ (1998) Allelopathic interactions between sponges on a tropical reef. Ecology 79(5): 1740-1750 Thakur NL, Mller WEG (2004) Biotechnological potential of marine sponges. Curr Sci 86(11): 1506-1512 Trautman DA, Hinde R, Borowitzka MA (2000) Population dynamics of an association between a coral reef sponge and a red macroalga. J Exp Mar Biol Ecol 244: 87-105 Trautman DA, Hinde R (2002) Sponge/algal symbioses: a diversity of associations. In: Seckbach J (ed). Symbiosis: Mechanisms and Model Systems. Kluwer Academic Publishers, Netherlands, 796 pp. Uriz MJ, Rosell D, Maldonado M (1992) Parasitism, comensalism or mutualism? The case of Scyphozoa (Coronatae) and horny sponges. Mar Ecol Prog Ser 81: 247-255 Vacelet J (1981) Algal-sponge symbioses in the coral reefs of New Caledonia: a morphological study. Proc 4th Int Coral Reef Symp, Manila 2: 713-719 Vacelet J, Sofyani AA, Lihaibi SA, Kornprobst JM (2001) A new haplosclerid sponge species from the Red Sea. J Mar Biol Assoc UK 81: 943-948 van Soest RWM, Braekman JC (1999) Chemosystematics of Porifera: a review. Memoir Queensl Mus 44: 569-589 West DA (1979) Symbiotic zoanthids (Anthozoa: Cnidaria) of Puerto Rico. Bull mar Sci 29: 253-271. Wilcox TP, Hill M, DeMeo K (2002) Observations on a new twosponge symbiosis from the Florida Keys. Coral Reefs 21: 198204 Wilkinson CR (1983) Net primary productivity of coral reef sponges. Science 219: 410-412 Wulff JL (1997a) Causes and consequences of differences in sponges diversity and abundance between the Caribbean and eastern Pacific at Panama. Proc 8th Int Coral Reef Symp, Panama 2: 1377-1382 Wulff JL (1997b) Mutualisms among species of coral reef sponges. Ecology 78: 146-159 Wulff JL (1999) A sponge that cheats on diffuse mutualism among other sponge species. Mem Qld Mus 44: 686 Wulff JL (2006) Ecological interactions of marine sponges. Can J Zool 84: 146-166 Xue S, Zhanga HT, Wua PC, Zhanga W, Yuan Q (2004) Study on bioactivity of extracts from marine sponges in Chinese Sea. J Exp Mar Biol Ecol 298: 71-78

Porifera research: Biodiversity, innovation and sustainaBility - 2007

157

Sponges of the marine karst lakes and of the coast of the islands of Ha Long Bay (North Vietnam)
Francesca Azzini(1), Barbara Calcinai(1), Carlo Cerrano(2), Giorgio Bavestrello(1), Maurizio Pansini(2*)
Dipartimento di Scienze del Mare, Universit Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy (2) Dip.Te.Ris. Universit di Genova, Corso Europa 26, 16132 Genova, Italy. mpansini@dipteris.unige.it
(1)

Abstract: The purpose of this paper is to describe the sponge assemblages of the marine karst lakes and some coastal sites in the islands of Ha Long Bay (Vietnam). These lakes are very shallow basins with a bottom covered by mud and vegetable debris. Patches of mangroves were seldom observed, while isolated colonies of massive corals are frequently found. Some lakes show a connection with the surrounding sea, evidenced by the flow of tidal streams; others are apparently closed but connected to the sea through the cavities of the karst system. The coast of Ha Long Bay islands are characterized by shallow depth, murky waters, patchy and low diversity reefs, and very low water-movement. A total of 63 demosponge species were identified: 46 species were recorded in the marine lakes, while 23 of them were never found outside the lakes. Extreme variations in environmental conditions occur yearly in the lakes, due to heavy monsoon rains that cause a stratification of the water column. A thermal crisis was recorded at the end of the summer in the Hang Du I lake, with bottom temperatures as high as 36C. Sponges can withstand these conditions but undergo important rearrangements, with a late summer degeneration followed by a very quick fall and winter growth. Keywords: Porifera, anchialine lakes, karst islands, environmental stress, Tonkin Gulf

Introduction
The Ha Long Bay (Vietnam) is located in the northern part of the Tonkin Gulf, in a shallow water area of the South China Sea where more than 3000 karst islands of different sizes are present. Karstic processes, enhanced by the tropical conditions, carved out of the limestone many depressions, leading to the formation of shallow salt water lakes similar to the meromictic marine lakes of Palau (Hamner and Hamner 1998) and to the anchialine lakes of East Kalimantan (Indonesia), where the only specific study on the sponge fauna of these special environments is in progress (de Voogd et al. 2006). The scope of this study was to investigate the species composition of the sponge fauna of the lakes compared with that of the coastal areas of the isles of the bay. The sponges of the coast of Vietnam are still scarcely studied. In fact one can find only a paper by Lindgren (1898), an inventory by Dawydoff (1952), a study of a collection from Nha Trang (Lvi 1961) and a recent paper devoted to boring sponges (Calcinai et al. 2006) among the literature. A checklist of sponges recorded from the South China Sea by Hooper et al. (2000) reports as many as 161 demosponges (106 of which identified at species level) from the coast of Vietnam.

Materials and methods Description of the study area


The climatic conditions of the study area are tropical, with a mean annual air temperature of 25C, warm and wet summer monsoons (up to 2000 mm/year of rain) from May to October and relatively cold and dry winter monsoons from November to April (Tang 2001). The marine lakes are remarkably different from one another in the degree of isolation from the surrounding sea. In some lakes, connection is detectable by the flow of tidal streams. Sometimes artificial canals have been built to enhance this water exchange. However, some lakes are apparently closed, being connected to the sea only through the cavities of the karst system. They are no more in pristine conditions, having been exploited for fishery, mollusc harvesting and aquaculture by people dwelling in boat villages located around the islands. In the surrounding sea, owing to the presence of thousands of islands, water is calm and turbid. The mean tidal excursion ranges between 3 and 4 m, salinity ranges around 32, and sea-water temperature varies between a minimum of 23C at the end of winter and 29-30C in late summer (Tang 2001).

Sampling
Three field campaigns were performed in April 2003, September 2003 and April 2004, in a joint venture between

158

the Universit Politecnica delle Marche and the Hai Phong Institute of Oceanology (HIO) for the study of biodiversity and conservation in a coastal area of Vietnam. Samples were collected either through SCUBA diving or snorkelling in 15 sites located in Ha Long Bay Islands (Fig. 1). Eight of these sites are salt lakes, while seven are located along the coast surrounding the islands. The latter were chosen among those periodically surveyed for biodiversity assessments by the team of HIO. Sponges were collected from the rocky shores of both the lakes and the islands down to a depth of about 3 m and from the small reefs along the islands, which are only slightly deeper (7-8 m). Lakes were considered as open (Hang Du II, Hang Tham, Hang Luong), semi-enclosed (Dau Be, Cat Ba, Me Cung) and enclosed (Hang Du I, Bui Xam) when they were connected to the sea by large canals, small conduits or through the karst system, respectively. No quantitative sampling was taken, but in each sampling station all the discrete sponge species were collected by three divers during 45 minutes of dive. The small-encrusting and cryptic species were not collected. Whenever possible, sponge specimens were photographed

in the field, or on board after collection; they were fixed in 4% formaldehyde solution in sea water and preserved in 60% ethanol. Some specimens were dried.

Results Distribution
Sixty three demosponges have been identified (36 to species level), out of 182 specimens collected from the marine lakes and the coasts of the isles of Ha Long Bay (Table 1). Forty six species were found in the lakes and 40 in the bay; 23 species were found only in the lakes and 17 only in the bay (Table 1). According to presence/absence data, the most common species in the studied area, including lakes and coastal sites, were Dysidea cinerea Keller, 1889 and Haliclona (Haliclona) sp. 2 (present in 60% of the collecting stations), Tethya seychellensis (Wright, 1881) (53%), Haliclona (Gellius) cymaeformis (Esper, 1794) and Spheciospongia tentorioides (Dendy, 1905) (46.6%), and Mycale philippensis (Dendy, 1896) (40%). However, in the absence of quantitative

Fig. 1: Location of the marine lakes and coastal sites surveyed in six island groups (Bo Hon, Hang Trai, Dau Be, Cat Ba, Conf and Cong Do) in the western part of Ha Long Bay (Vietnam): 1-Hang Luong Lake; 2-Me Cung Lake; 3-Bui Xam Lake; 4-Hang Du I Lake; 5-Hang Du II Lake; 6-Dau Be Lake; 7-Cat Ba Lake; 8-Hang Tham Lake; 9-Coastal site I; 10Coastal site II; 11-Coastal site III; 12-Coastal site IV; 13-Coastal site V; 14-Hang Toi Dark Cave; 15Coastal site VI.

159

Table 1: List of the Demosponge species collected from some marine lakes and coastal sites of Ha Long Bay: 1-Hang Luong Lake; 2-Me Cung Lake; 3-Bui Xam Lake; 4-Hang Du I Lake; 5-Hang Du II Lake; 6-Dau Be Lake; 7-Cat Ba Lake; 8-Hang Tham Lake; 9-Coastal site I; 10-Coastal site II; 11-Coastal site III; 12-Coastal site IV; 13-Coastal site V; 14-Hang Toi Dark Cave; 15- Coastal site VI. The species that are new records for Vietnam are marked by *. Species Aaptos cf. pernucleata (Carter, 1870) * Acanthella hispida Pulitzer-Finali, 1980 * Aka mucosa (Bergquist, 1965) * Amorphinopsis excavans Carter, 1887 * Amphimedon sp. Aplysina sp. Biemna megalosigma Hentschel, 1912 * Bubaris sp. Callyspongia sp. Chondrilla australiensis Carter, 1873 Cinachyrella australiensis (Carter, 1886) * Cladocroce sp. Clathria sp. Cliona aurivilli (Lindgren, 1897) Cliona celata Grant, 1826 Cliona orientalis Thiele, 1900 * Cliona sp. Cliothosa hancocki (Topsent, 1888) Desmanthus incrustans (Topsent, 1889) * Dictyonella sp. 1 Dictyonella sp. 2 Dysidea cinerea Keller, 1889 * Dysidea cf. fragilis (Montagu, 1818) Echinodictyum asperum Ridley and Dendy, 1886 Eurypon sp. Gelliodes fibulatus (Carter, 1881) Halichondria sp. Haliclona (Gellius) cymaeformis (Esper, 1794) Haliclona (Haliclona) sp. 1 Haliclona (Haliclona) sp. 2 Haliclona (Haliclona) sp. 3 Haliclona (Haliclona) sp. 4 Haliclona (Haliclona) sp. 5 Haliclona (Haliclona) sp. 6 Haliclona (Reniera) sp. 1 Haliclona (Reniera) sp. 2 Haliclona (Reniera) sp. 3 Haliclona (Gellius) sp. Hyattella intestinalis (Lamarck, 1814) * Ircinia echinata (Keller, 1889) * Ircinia sp. Mycale (Mycale) crassissima (Dendy, 1905) Mycale (Zygomycale) parishi (Bowerbank, 1875) * Mycale (Mycale) philippensis (Dendy, 1896) Mycale (Mycale) sp. Penares cf. sollasi Thiele, 1900 * Petrosia (Petrosia) nigricans Lindgren, 1897 * Pione carpenteri (Hancock, 1867) * Suberites sp. 1 Suberites sp. 2 Protosuberites sp. 1 Protosuberites sp. 2 Spheciospongia solida Ridley and Dendy, 1886 Spheciospongia tentorioides (Dendy, 1905) * Spirastrella cf. cunctatrix Schmidt, 1868 * Spirastrella decumbens Ridley, 1884 * Spongia irregularis (von Lendenfeld, 1885) Stelletta aruensis Hentschel, 1912 * Tedania (Tedania) brevispiculata Thiele, 1903 Terpios cruciata Dendy, 1905 * Tethya seychellensis (Wright, 1881) * Topsentia cavernosa (Topsent, 1897) * Xestospongia cf. testudinaria (Lamarck, 1815) * 1 2 Marine Lakes 3 4 5 6 7 8 9 10 Coastal sites 11 12 13 14 15

160

data but based on field observations, Haliclona (Gellius) cymaeformis, living in symbiotic association with the rodophyte alga Ceratodictyon spongiosum Zanardini, 1878, was the most abundant species, thriving with an impressive number of specimens on all the horizontal substrates, even in very shallow waters where it might be exposed to air at low tide. It was dark green in colour and may vary in shape from thickly encrusting to massive or, more often, bushy (Fig. 2A). A remarkable number of boring sponges i.e. Aka mucosa (Bergquist, 1965), Cliona celata Grant, 1826, C. orientalis Thiele, 1900, Cliothosa hancocki (Topsent, 1888), Spheciospongia tentorioides, and Cliona aurivilli (Lindgren, 1897) the latter according to Calcinai et al. (2006) were found both in the lakes and in the surrounding sea, while the distribution of Pione carpenteri (Hancock, 1867) and Spirastrella decumbens Ridley, 1884 seems to be restricted to the lakes (Table 1). The bay In the costal stations of the bay we have observed that sponges settled on two types of substrates: The calcareous rocky shores of the islands, extending down to 3-4 m in depth and the coral gardens which proliferate on the horizontal surfaces to a depth of about 8 m. On the coast, the suitable substrate for sponge settlement was very scarce, due to the dense belt of bivalve molluscs. Horny sponges such as Dysidea cinerea, Dysidea cf. fragilis (Montagu, 1818), Spongia irregularis (von Lendenfeld, 1885), as well as several species of Haplosclerida (Haliclona spp., Cladocroce sp.) and Tethya seychellensis were the most common species on the rocky shores. In the coral gardens, sponges behave as opportunistic species, dwelling on corals [e.g. Gelliodes fibulatus (Carter, 1881), Amphimedon sp., Ircinia echinata (Keller, 1889)], on pockets of sediment in between corals (Biemna megalosigma Hentschel, 1912), and on coral rubble [Acanthella hispida Pulitzer-Finali, 1980, Spheciospongia tentorioides, Xestospongia cf. testudinaria (Lamarck, 1815)]. These last two species were particularly abundant and seem to contribute to consolidate the coral fragments. Along the coast of the islands one can find some peculiar habitats represented by semi-dark environments, such as a rocky tunnel with a steady current, open at both ends, leading to Xang Luong cove in the Island of Bo Hon and Hang Toi Cave, a cavity with a depth of 1-1.5 m, in the Island of Cong Do (Fig. 1). In these habitats, sponges were abundant and diverse. Cinachyrella australiensis (Carter, 1886), Penares cf. sollasi Thiele, 1900, and Stelletta aruensis Hentschel, 1912 have been recorded in Hang Toi Cave only. In the above mentioned tunnel steady water movement supported numerous colonies of the octocoral Carijoa riisei (Duchassaing and Michelotti, 1860). Several sponge species [Callyspongia sp., Mycale philippensis, Mycale (Mycale) crassissima (Dendy, 1905), Spirastrella cf. cunctatrix Schmidt, 1868, Tedania (Tedania) brevispiculata Thiele, 1903 and some unidentified Haliclona] were epizoic on Carijoa colonies in the Ha Long Bay. They initially use the octocoral skeleton as support and subsequently overgrow it completely. Haliclona (Haliclona) sp. 2 (Fig. 2C) has been observed on a colony already covered by Mycale

philippensis. Carijoa appears to be unharmed by the epizoic sponges because its anthocodia are free to expand and retract (Fig. 2B). The lakes Among the pool of 23 species recorded only in the lakes, strong differences among the different basins were observed. The only species recorded in 50% of the lakes is Haliclona (Gellius) sp.; four other species i.e. Pione carpenteri (Hancock, 1867), Suberites sp. 1, Protosuberites sp. 1, Spirastrella cf. cunctatrix, were present in 25% of the lakes. Each of the other 18 species was recorded only in one lake. The highest number of species (28) was found in the enclosed lake of Bui Xam, which has no detectable connection to the sea. 70% of these species were found both in the lakes and the coastal stations. Three species, Clathria sp., Eurypon sp. and Spirastrella decumbens were found only in this lake. In the enclosed lake of Hang Du I, ten species were identified, 7.5% of which were in common with the coastal stations. However, Aaptos cf. pernucleata (Carter, 1870), Dictyonella sp. 1, Haliclona (Haliclona) sp. 3 and Haliclona (Reniera) sp. 2 were found only in this lake. Suberites sp. 1, a very abundant species as regards the number and size of specimens, was found just in Hang Du I and in Dau Be, a lake connected to the sea by small conduits. Pione carpenteri and Spirastrella decumbens seem to be restricted to the enclosed lakes of Hang Du I and Bui Xam. In three lakes, Hang Luong, Me Cung and Cat Ba, the sponge fauna was completely composed of species that were also present in the bay, while in Hang Du II, Dau Be and Hang Tham the overlapping with the bay fauna ranged between 55 and 75%.

Ecology
Hang Du I lake was studied in detail because of the extreme variability of its environmental conditions directly affecting the sponge fauna. Direct observations of Suberites sp. 1 showed that during the dry season, in winter and early spring, numerous healthy specimens thrived close to the lake surface (Fig. 2D). In late spring and summer, morphological rearrangements and degeneration phenomena due to the combined effect of rainwater stratification with high temperatures were observed in this upper zone (Fig. 2E). Particularly evident was the case of Protosuberites sp. 1 (Fig. 2F, G). As soon as water cooled again, very quick fall and winter growth followed, but the original conditions did not seem to be restored after a single season.
Fig. 2: A. Haliclona (Gellius) cymaeformis: a bushy specimen. B. Sponges associated with colonies of the octocoral Carijoa riisei. Polyps continue their filtering activity; the worm-like organisms are synaptid holothurians. C. Epizoic sponges on Carijoa colonies: Haliclona (Haliclona) sp. 2 (green) and Mycale (Mycale) philippensis (red). Morphological rearrangements of two sponge species of the Hang Du I lake in spring and late summer: D-E. Suberites sp. 1; F-G. Protosuberites sp. 1.

161

162

Biogeography
Twenty-three species, corresponding to 63.8% of the 36 fully identified sponges of our collection, are new records for the coast of Vietnam (Table 1). Most of these species (32 out of 36) are distributed in the Indian Ocean, including the Red Sea, and in the West Pacific Ocean, including Australia. Three of them (Cliona celata, Dysidea fragilis, Tethya seychellensis) are considered cosmopolitan, while a single species, Aaptos pernucleata, is known from the West Indies only.

Discussion
The recorded data suggest that the sponge fauna of the Tonkin Gulf is similar to that of the adjacent tropical areas of Indonesia and Northern Australia. Both the peculiar geomorphology and oceanography of the bay may represent important factors negatively affecting the northern expansion of sponges. The number of species found in the lakes (46) is comparable to that found until now in four anchialine lakes of East Kalimantan which were thought to represent a lagoonal reef of a former barrier reef complex (de Voogd et al. 2006). A species particularly adapted and restricted to the lakes appears to be Suberites sp. 1, very likely cospecific with the Suberites sp. reported from Lake Satonda (Palau) and East Kalimantan Lakes (Indonesia) (de Voogd et al. 2006). The species composition of sponge fauna is very different in the studied lakes without any apparent relation with the observed degree of connection to the open sea. This fact results from the comparison of the number of sponge species, 28 and 10, respectively, recorded in the two enclosed lakes of Bui Xam and Hang Du I. The occurrence of scattered colonies of massive corals which are present in Bui Xam and absent in Hang Du I suggests the presence of an undetected, large connection between the first lake and the open sea. Conversely, the high degree of isolation and the peculiar character of the above mentioned lake are confirmed by the presence of a dense population of the non-stinging jellyfish (Rhizostoma sp.) (Cerrano et al. 2006) and four species of sponges out of a total of 10 which are absent elsewhere. The different degree of isolation from the open sea of these two lakes is demonstrated by the severe water stratification occurring in Hang Du I, while in the Bui Xam lake stratification phenomena are less intense. Due to the stillness of this sheltered basin, a light and cool rainwater layer (salinity < 7 ) as thick as 150 cm (on a maximum lake depth of about 6 m) stratifies on the lake surface thus preventing the normal mixing of the water column. This resulted in an abnormal rising of the bottom temperature, which was as high as 36C in September 2003 (Fig. 3). This thermal crisis produced a surplus of organic debris, coming from vegetable and animal decay, that deposits on the lakes muddy bottom that became anoxic (Cerrano et al. 2006). The spring conditions, recorded in April 2003 and 2004, were each similar and presumably bound to the climatic pattern of the year (Fig. 3). Seasonal variations in temperature and salinity such as those recorded in Hang Du I remarkably affect the sponge fauna. The response to the environmental stress seems to

be related to the sponge position on the bottom, because Clionaid species and Tethya seychellensis, living in sheltered habitats, appear unaffected, whereas Suberites sp. 1 and Pseudosuberites sp. 1, living in exposed locations, show evident regression. Rapid growth rates as those observed after the late summer crisis were already recorded in sponges after negative events (Ayling 1983). These growth rates may be also supported by increased food abundance (Duckworth et al. 2003) resulting from the restoration of the normal conditions in the water column. Summer temperatures in temperate regions normally cause positive growth rates in sponges (Turon et al. 1998, Tanaka 2002) whereas sponge shrinking was associated to colder temperatures (Duckworth and Battershill 2001). However, persistence of high water temperatures in late summer may stress benthic organisms, sometimes triggering mass casualties (Cerrano et al. 2000). Regression and reorganization of adult sponges apparently neither related to environmental stress nor to a seasonal cycle were observed in natural conditions (Pansini and Pronzato 1990, Bell and Smith 2004). Besides salinity variations, another physical factor affecting sponge fauna both in lakes and coastal sites appears to be the high water turbidity. The impact of sedimentation on sponges is well known (Sar and Vacelet 1973, Bell and Smith 2004) as is the defensive reaction set up by sponges in order to avoid clogging of their aquiferous system (Reiswig 1971, Bell 2004, Cerrano et al. 2004). In a shallow water area such as Ha Long Bay, tidal range and competition with bivalve molluscs reduce the space available for sponge settlement on vertical substrates and overhangs, which are protected from sediment deposition. This may cause an overall reduction of the specific richness of porifera but may also select taxa producing fistules (e.g. Biemna megalosigma), adapted to live partially buried by the sediment on horizontal substrates. Symbiosis may be the clue for explaining the great abundance of Haliclona (Gellius) cymaeformis, associated with the rodophyte Ceratodyction spongiosum, in the intertidal and subtidal of the bay. Steindler et al. (2002) suggest that the photosynthetic activity of the algae may fulfill the energetic needs of the sponges when they stop filtering, being partially emerged at low tide, and that symbionts protect them from UV radiation, particularly intense in shallow water. In addition, Pile et al. (2003) showed that the sponge, feeding on nitrogen-rich bacteria and protozoans of the ultraplankton, can meet the nitrogen demand of both partners of the symbiotic association. Therefore, several positive factors could allow Haliclona (Gellius) cymaeformis to thrive in the shallow water environment even in the presence of rather low water transparency. Until the present, marine lake biota is poorly known, but they very likely host many species new to science as the taxonomy of sponges here suggests. As highlighted by Dawson and Hamner (2005) marine lakes offer the possibility to study the founder effect at different stages, in relation to their age and level of isolation. The lakes of North Vietnam are smaller than Palau and Indonesian ones and host a fauna very interesting in relation to speciation processes and to its physiology, being strongly adapted to sudden modifications of the environmental parameters. These aspects may at least partially explain the large variation in qualitative composition

163

Fig. 3: Environmental variations in Hang Du I lake.

of the sponge fauna between different lakes, and between the lakes and the surrounding marine areas. It is important to increase the knowledge of these unique habitats before human activities and climate change would irreparably damage them.

Acknowledgements
We are indebted to Do Co Thung and his team of the Hai Phong Institute of Oceanology for supporting us in the field and to Massimo Sarti of the Universit Politecnica delle Marche for his geological advice.

References
Ayling AL (1983) Growth and regeneration rates in thinly encrusting Demospongiae from temperate waters. Biol Bull 165: 343-352 Bell JJ (2004) Evidence for morphology-induced sediment settlement prevention on the tubular sponge Haliclona urceolus. Mar Biol 146(1): 29-38 Bell JJ, Smith D (2004) Ecology of sponge assemblages (Porifera) in the Wakatobi region, south-east Sulawesi, Indonesia: richness and abundance. J Mar Biol Assoc UK 84: 581-591 Calcinai B, Azzini F, Bavestrello G, Cerrano C, Pansini M, Thung DC (2006) Boring sponges from the Ha Long Bay (Tonkin Gulf, Vietnam). Zool Stud 45(2): 201-212 Cerrano C, Bavestrello G, Bianchi CN, Cattaneo-Vietti R, Bava S, Morganti C, Morri C, Picco P, Sara G, Schiaparelli S, Siccardi A, Sponga F (2000) A catastrophic mass-mortality episode of gorgonians and other organisms in the Ligurian Sea (north-western Mediterranean), summer 1999. Ecol Lett 3: 284-293 Cerrano C, Pansini M, Valisano L, Calcinai B, Bavestrello G, Sar M (2004) Lagoon sponges from Carrie Bow Cay (Belize): ecological benefits of selective sediment incorporation. In: Pansini M, Pronzato R, Bavestrello G, Manconi R (eds). Sponge Science in the new Millenium. Boll Mus Ist Biol Univ Genova 68: 239-252

Cerrano C, Azzini F, Bavestrello G, Calcinai B, Pansini M, Sarti M, Thung DC (2006) Marine lakes of karst islands in Ha Long Bay (Vietnam). Chem Ecol 22(6): 489-500 Dawydoff C (1952) Inventaire des animaux benthique rcolts par moi dans le domaine maritime Indochinois. Porifres. Suppl Bull Biol France Belgique 37: 46-51 Dawson M, Hamner WN (2005) Rapid evolutionary radiation of marine zooplankton in peripheral environments. Proc Natl Acad Sci USA 102 (26): 9235-9240 de Voogd N, de Weerdt WH, van Soest RWM (2006) The sponge fauna of the anchialine lakes of Kakaban and Maratua (East, Kalimantan, Indonesia). In: Custdio MR, Lbo-Hajdu G, Hajdu E, Muricy G (eds). 7th International Sponge Symposium - Book of Abstracts (Armao dos Bzios, Brazil). Museu Nacional, Srie Livros, vol. 16. p. 242 Duckworth AR, Battershill CN (2001) Population dynamics and chemical ecology of New Zealand Demospongiae Latrunculia sp. nov. and Polymastia croceus (Poecilosclerida: Latrunculiidae: Polymastiidae). N Zealand J Mar Freshw Res 35: 935-949 Duckworth AR, Samples GA, Wright AE, Pomponi SA (2003) In vitro culture of the tropical sponge Axinella corrugata (Demospongiae): effect of food cell concentration on growth, clearance rate, and biosynthesis of stevensine. Mar Biotechnol 5(6): 519-527 Hamner WM, Hamner PP (1998) Stratified marine lakes of Palau (Western Caroline Island). Phys Geogr 19: 175-220 Hooper JNA, Kennedy JA, van Soest RWM (2000) Annotated checklist of sponges (Porifera) of the South China Sea region. The Raffles Bull Zool suppl 8: 125-207 Lvi C (1961) ponges intercotidales de Nha Trang (Viet Nam). Arch Zool Exp Gn 100: 127-150 Lindgren NG (1898) Beitrag zur kenntniss der Spongienfauna des Malayischen Archipels und der Chinesischen Meere. Zool Jahrb Abt Syst Geogr Biol Thiere 11: 283-378 Pansini M, Pronzato R (1990) Observations on the dynamics of a Mediterranean sponge community. In: Rtzler K, Macintyre VV, Smith KP (eds). New Perspectives in Sponge Biology. Smithsonian Institution Press, Washington DC. pp. 404-415

164

Pile AJ, Grant A, Hinde R, Borowitzka MA (2003) Heterotrophy on ultraplankton communities is an important source of nitrogen for a spongerhodophyte symbiosis J Exp Biol 206: 4533-4538 Reiswig HM (1971) In situ pumping activities of tropical Demospongiae Mar Biol 9: 38-50 Sar M, Vacelet J (1973) cologie des Dmosponges. In: Grass PP (ed). Trait de Zoologie, Vol. 3, Part. 1. Masson, Paris. pp. 462576 Steindler L, Beer S, Ilan M (2002) Photosymbiosis in intertidal and subtidal tropical sponges. Symbiosis 33: 263-273

Tanaka K (2002) Growth dynamics and mortality of the intertidal encrusting sponge Halichondria okadai (Demospongiae, Halichondrida). Mar Biol 140(2): 383-389 Tang VT (2001) The Eastern Sea Resources and Environment. The Gioi Publishers, Hanoi Turon x, Tarjuelo I, Uriz, MJ (1998) Growth dynamics and mortality of the encrusting sponge Crambe crambe (Poecilosclerida) in contrasting habitats: correlation with population structure and investment in defence. Funct Ecol 12(4): 631-639

Porifera research: Biodiversity, innovation and sustainaBility - 2007

165

Microbial nitrification in Mediterranean sponges: possible involvement of ammonia-oxidizing Betaproteobacteria


Kristina Bayer, Susanne Schmitt, Ute Hentschel(*)
Research Center for Infectious Diseases, University of Wuerzburg. Roentgenring 11, D-97070 Wuerzburg, Germany. Tel.: +49-931-312581. Fax: +49-931-312578. kristina.bayer@mail.uni-wuerzburg.de, susanne.schmitt@mail.uni-wuerzburg.de, ute.hentschel@mail.uni-wuerzburg.de Abstract: The aim of this study was to assess the potential for nitrification in Aplysina aerophoba Schmidt 1862 using a combined physiological and molecular approach. Whole animals were incubated in experimental aquaria and the concentrations of ammonia, nitrate and nitrite were determined in the incubation water using colorimetric assays. Nitrate excretion rates reached values of up to 3.6 mol g-1 fresh weight day-1 (equivalent to 830 nmol g-1 dry weight h-1) and were matched by ammonia excretion rates of up to 0.56 0.09 mol g-1 fresh weight day-1. An accumulation of nitrite was not detected in any of the experiments. Control experiments without sponges showed no variation in nitrogen species in the incubation water. A slight increase in ammonia excretion was observed over 11 days of maintenance in holding tanks that were constantly supplied with fresh, untreated Mediterranean seawater. Other sponges from the same habitat (Dysidea avara Schmidt 1862, Tethya sp., Chondrosia reniformis Nardo 1847) showed high rates of ammonia excretion but nitrate excretion was significantly reduced or absent. Using specific PCR primers, 16S rRNA genes of the betaproteobacterial clade of the Nitrosospira cluster 1 were recovered from A. aerophoba, D. avara and Tethya sp. tissues. In conclusion, this study provides physiological and molecular evidence for the presence of nitrifying bacteria in A. aerophoba while the potential for nitrification in the other sponges remains to be investigated. Keywords: Ammonia-oxidizing Betaproteobacteria, microbial consortia, nitrification, 16S rRNA gene, sponge

Introduction
Sponges (Porifera) are evolutionarily ancient metazoans with a fossil record dating back nearly 600 million years in time (Li et al. 1998). Today, an estimated 13,000 species, classified in three classes (Demospongiae, Calcarea, Hexactinellida) populate virtually all benthic marine and freshwater habitats (Hooper and van Soest 2002). Sponges have a primitive morphology lacking true organs or tissues. Most metabolic functions are carried out by totipotent, amoeboid cells, termed archaeocytes that move freely through the mesohyl matrix. Inhalant and exhalant canals build an aquiferous system through which water is actively pumped by flagellated choanocytes (Brusca and Brusca 1990). As filter-feeders, sponges efficiently take up nutrients like organic particles and microorganisms from the seawater, leaving the expelled water essentially sterile (Reiswig 1974, Pile 1997, Wehrl et al. 2007). Despite the fact that sponges feed on microorganisms, large amounts of extracellular microorganisms populate the mesohyl matrix of many demosponges (for recent reviews, see Hentschel et al. 2003, 2006, Imhoff and Sthr 2003, Hill 2004). Bacterial numbers may constitute as much as

40 - 60 % of the total biomass exceeding concentrations of seawater by two to four orders of magnitude. Molecular diversity analyses showed that the sponge microbiota is phylogenetically complex, yet highly sponge-specific. Members of eight eubacterial phyla [Proteobacteria (Alpha-, Gamma-, Deltaproteobacteria), Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Gemmatimonadetes and Nitrospira], members of the recently discovered candidate phylum Poribacteria (Fieseler et al. 2004), and of one archaeal phylum (Crenarchaeota) are numerically abundant and metabolically active in sponges. As none of these sponge-specific microorganisms have been obtained in pure culture, their function, metabolism, and possibly nutritional interactions with the host sponge are virtually unknown. In this study, we aimed to investigate the process of microbial nitrification in Mediterranean demosponges. Nitrification describes the oxidation of ammonia (NH3) to nitrite (NO2-) by ammonia-oxidizing bacteria (AOB) and archaea (AOA) and subsequently to nitrate (NO3-) by nitrite-oxidizing bacteria (NOB) for energy purposes (Kowalchuk and Stephen 2001). Several lines of evidence suggest that marine sponges are indeed a reservoir for

166

nitrifying microorganisms. Firstly, sponges and many other marine invertebrates release ammonia as a metabolic waste product (Wang and Douglas 1998, Davy et al. 2002) and nitrate excretion has already been documented in Caribbean (Corredor et al. 1988, Pile 1996, Diaz and Ward 1997) and Mediterranean (Jimenez and Ribes 2007) sponges. Secondly, 16S rRNA gene sequences of several clades of ammoniaoxidizing Beta- and Gammaproteobacteria and nitriteoxidizing Nitrospina were recovered from sponge tissues, making microbial nitrification a likely scenario (Hentschel et al. 2002, Diaz et al. 2004). In the present study, a combination of physiological and phylogenetic approaches was employed to explore the potential of microbial nitrification in Aplysina aerophoba Schmidt 1862 and several other Mediterranean demosponges.

DNA extraction and PCR


For the amplification of 16S rRNA genes from ammoniaoxidizing Betaproteobacteria, the primers AOB189f (5GGA GAA AAG CAG GGG ATC G-3) and AOB1224r (5-CGC CAT TGT ATT ACG TGT GA-3) were used, that originally had been designed as the FISH probes NSO190 and NSO1224, respectively (Loy et al. 2003). The PCR reaction mix contained 1 x PCR reaction buffer (Qiagen), 2 mM of each primer, 0.2 mM dNTPs (Sigma) and 1.25 U Taq Polymerase (Qiagen) in a final volume of 50 l. The PCR protocol was as follows: 1 min initial denaturation at 94C followed by 30 cycles of denaturation at 94C for 30 sec, primer annealing at 56C for 30 sec and elongation at 72C for 50 sec. The PCR was terminated with a final elongation step at 72C for 5 min.

Materials and methods Animal collection


Whole, intact colonies of the sponges A. aerophoba, Dysidea avara Schmidt 1862, Tethya sp. and Chondrosia reniformis Nardo 1847 were collected by SCUBA diving offshore Banyuls-sur-Mer (France) (4243N, 1008E) and from Rovinj (Croatia) (4505N, 1338E) at depths from 220 m. The animals were in the range of 30-50 g wet weight (about 15 g for D. avara). Small tissue pieces were removed from freshly collected animals, immediately frozen in liquid nitrogen and stored at -80C until use. Whole, intact animals were maintained in > 1000 L volume, flow-through holding tanks that were constantly supplied with fresh, untreated Mediterranean seawater prior to the experiments.

Cloning, RFLP-Analysis and Sequencing


Purified PCR products (PCR purification kit, Qiagen) were ligated into the pGEMT-easy vector (Promega) and transformed by electroporation into competent E. coli XL 1Blue cells. The enzymes Msp I and Hae III were used for restriction fragment length polymorphism (RFLP) analysis. Plasmid DNA was isolated from selected clones by standard miniprep procedures (Sambrook et al. 1989) and sequencing was performed on an ABI 377XL automated sequencer (Applied Biosystems).

Phylogenetic analysis
Sequences obtained in this study were checked for chimeras with the program Pintail. Sponge sequences together with reference sequences [received from GenBank using BLAST (http://www.ncbi.nlm.nih.gov/BLAST)] were aligned automatically with ClustalX and the alignment was subsequently corrected manually in Align. Neighbor Joining (with Jukes-Cantor correction) and Maximum Parsimony trees were constructed using the ARB software package (Ludwig et al. 2004).

Sponge incubations
Individual specimens were placed into aquaria containing three liters of fresh, untreated Mediterranean seawater. A constant water current was generated by small aquarium pumps (Vita Tech 300, Vitakraft, Germany). Only sponges that were in good physiological condition as judged by their regular filtration activity were chosen for the experiments. The experiments were performed in triplicate while a fourth aquarium without a sponge served as a control. In time intervals, 10 ml aliquots were removed from each aquarium, placed on ice and frozen at -20C until use.

Results and discussion In vivo sponge incubations


For A. aerophoba, nitrate excretion rates of 3.6 0.27 mol g-1 fresh weight day-1 (equivalent to 830 nmol g-1 dry weight h-1) were determined which corresponded to an ammonia excretion of 0.56 0.09 mol g-1 fresh weight day-1 (n = 4 S.E.) (Fig. 1A). The nitrate excretion rate was about six fold higher than the ammonia excretion rate. Ammonia and nitrate did not appear in the incubation water in sponge-free control aquaria. Nitrite was not detected in any of the incubations. In order to measure ammonia uptake rates, 100 or 200 M NH4Cl final concentrations were added to the incubation water. Aplysina aerophoba was capable of ammonia uptake which corresponded to a nitrate excretion rate of 9.2 and 5.0 mol g-1 fresh weight day-1 (Fig. 1B). Aplysina aerophoba was not capable of taking up nitrate which was tested at a concentration of 100 M (data not shown).

Determination of ammonia, nitrate and nitrite concentrations


The ammonia concentration was determined with the Indol-phenol-blue reaction (Parsons et al. 1984). The concentration of nitrite (NO2-) was determined by the Griess reaction (Parsons et al. 1984). Nitrate (NO3-) concentrations were measured indirectly after conversion to nitrite by the nirmutant E. coli strain JBC 606 as described in Pospesel et al. (1998). Ammonia and nitrate standard curves ranging from 0-100 M were performed for each measurement series and fresh standards were prepared on a weekly basis.

167

Fig. 1: A. Ammonia and nitrate excretion by A. aerophoba (n = 4 S.E.). B. Ammonia uptake and nitrate excretion (n = 2). Symbols represent ammonia () and nitrate () concentrations.

Ammonia and nitrate excretion rates of A. aerophoba were determined in correlation to the maintenance time in holding tanks (Fig. 2). After one day of maintenance, the ammonia excretion rate was 0.54 0.07 mol g-1 fresh weight day-1. After six and 11 days of maintenance, the ammonia excretion rates were slightly increased (1.04 0.05 and 1.11 0.05 mol g-1 fresh weight day-1, respectively, Krustal-Wallis Test p=0.051). Nitrate excretion rates were similar over time of maintenance, ranging from 0.74 0.15 (Fig. 2A), to 0.94 0.36 (Fig. 2B) and 0.87 0.43 mol g-1 fresh weight day-1 (Fig. 2C). Interestingly, a correlation between ammonia and nitrate excretion and sponge pumping activity was evident. While ammonia was excreted at almost double rates in non pumping sponges, nitrate was not excreted in sponges whose osculi were closed as judged by visual inspection (data not shown). The observation that the mesohyl of non-pumping sponges becomes anaerobic within 15 min (Hoffmann et al. unpublished) would be consistent with an inhibition of the aerobic process of nitrification. However, possible differences in the diffusion process of ammonia and nitrate in non pumping sponges cannot be excluded.

Fig. 2: Ammonia and nitrate excretion by A. aerophoba over time of maintenance in flow-through holding tanks (n = 3 S.E.). The rates were measured after one day (A), six days (B) and 11 days (C) after collection. Symbols represent ammonia () and nitrate () concentrations.

With respect to microbial loads in the mesohyl tissue, sponges are characterized as high and low microbial abundance sponges (Hentschel et al. 2006). Accordingly, ammonia and nitrate excretion rates were determined in the Mediterranean

168

weight day-1 (Fig. 3C) respectively, nitrate excretion was reduced or not detectable in any of the three sponge species investigated. Furthermore, Tethya sp. and C. reniformis were not able to take up ammonia (data not shown).

Phylogenetic analysis
In total, 200 clones were compared by restriction fragment length polymorphism analysis and four major restriction patterns were detected. After removal of five chimeras, nine, one, and two sequences from A. aerophoba, D. avara and Tethya sp. libraries, respectively, were used for phylogenetic tree construction (Fig. 4). All twelve sequences fell into the marine Nitrosospira cluster 1 of the Betaproteobacteria together with marine seawater and sediment sequences. Except Aplysina aerophoba (F) clone 5, the sequences obtained in this study build a subcluster with a high in-cluster similarity (98.5-99.9%). It is noteworthy that sequences were also recovered from the bacteria-free sponge D. avara as well as Tethya sp. whose mesohyl shows moderate amounts of microorganisms (Thiel et al. 2007). While it cannot be excluded that the cloned AOB sequences represent seawater bacteria rather than true sponge symbionts, the high nitrate excretion rates, at least for A. aerophoba, would argue for a specific and probably symbiotic association. Although primers used in this study also match Nitrosomonas species, no such bacteria could be detected in the clone libraries. Our findings expand those by Diaz (1997) and Diaz et al. (2004) who had reported on the identification of members of the Nitrosomonas eutropha/europae lineage (Betaproteobacteria) in five tropical sponges.

Modelling nitrogen fluxes in the sponge-microbe association


The following scenario, depicted in Fig. 5, is proposed based on this and other studies. The sponge host excretes ammonia as a metabolic waste product, which in turn, is oxidized to nitrite by ammonia-oxidizing bacteria (AOB), such as Nitrosospira (this study), Nitrosococcus (Hentschel et al. 2002) or members of the Nitrosomonas eutropha/ europaea l lineage (Diaz et al. 2004). Nitrite is further oxidized to nitrate by nitrite-oxidizing bacteria (NOB), such as Nitrospina or members of the phylum Nitrospira (Hentschel et al. 2002). The coordinated action of members of these two groups might then be responsible for the conversion of ammonia to nitrate in A. aerophoba and possibly also in other sponges. In addition to eubacteria, the involvement of archaea should be considered in future studies. Recent literature illustrates

Fig. 3: Ammonia and nitrate excretion by D. avara (A), Tethya sp. (B), and C. reniformis (C), (each species, n = 3 S.E.). Symbols represent ammonia () and nitrate () concentrations.

low microbial abundance sponges D. avara and Tethya sp. and the high microbial abundance sponge C. reniformis (Fig. 3). While ammonia was excreted at similar rates of 3.0 0.39 mol g-1 fresh weight day -1 (Fig. 3A), 6.95 0.08 mol g1 fresh weight day-1 (Fig. 3B) and 5.8 0.9 mol g-1 fresh

Fig. 4: Distance 16S rRNA (1053 bp) gene phylogeny showing the relationships between ammonia-oxidizing Betaproteobacteria using the ARB program package. Sponge derived sequences are shown in bold. Neighbor-joining and maximum parsimony (100 pseudoreplicates) bootstrap values are indicated. Arrow to outgroup (Nitrosococcus oceani Nc1 (AJ298727)). Designation of clusters is adapted from Freitag and Prosser (2003). Scale bar indicates 1% sequence divergence.

169

170

Fig. 5: Schematic diagram showing the inferred nitrogen-fluxes resulting from nitrification in the sponge-microbe-association.

that archaea, rather than bacteria, might in fact be involved in nitrification in marine and terrestrial ecosystems (Leininger et al. 2006, Wuchter et al. 2006). In fact, close relatives of the Cenarchaeum symbiosum lineage that are also present in sponges have recently been isolated and were shown to be capable of nitrification (Knneke et al. 2005). Additionally, it needs to be investigated whether nitrate serves as an energy substrate for denitrifying microorganisms under anaerobic conditions. In conclusion, this study contributes to an ongoing effort to link microbial diversity with function in these phylogenetically highly diverse, elusive and so far uncultivated marine sponge-associated microbial communities.

References
Brusca RC, Brusca GJ (1990) Phylum Porifera: the sponges. In: Sinauer AD (ed). Invertebrates. Sinauer Press, Sunderland. pp. 181-210 Corredor JE, Wilkinson CR, Vicente VP, Morell JM, Otero E (1988) Nitrate release by Caribbean reef sponges. Limnol Oceanogr 33(1): 114-120 Davy SK, Trautman DA, Borowitzka MA, Hinde R (2002) Ammonium excretion by a symbiotic sponge supplies the nitrogen requirements of the rhodophyte partner. J Exp Biol 205: 35053511 Diaz MC (1997) Molecular detection and characterization of specific bacterial groups associated to tropical sponges. Proc 8th Int Coral Reef Symp, Balboa 2: 1399-1402 Diaz MC, Ward BB (1997) Sponge-mediated nitrification in tropical benthic communities. Mar Ecol Progr Ser 156: 97-107 Diaz MC, Akob D, Cary CS (2004) Denaturing gradient gel electrophoresis of nitrifying microbes associated with tropical sponges. In: Pansini M, Pronzato R, Bavestrello G, Manconi R (eds). Sponge science in the new millennium. Boll Mus Ist Biol Univ Genova 68: 279-289 Fieseler L, Horn M, Wagner M, Hentschel U (2004) Discovery of a novel candidate phylum Poribacteria in marine sponges. Appl Environ Microbiol 70: 3724-3732 Freitag TE, Prosser JI (2003) Community structure of ammoniaoxidizing bacteria within anoxic marine sediments. Appl Environ Microbiol 69(3): 1359-1371

Acknowledgements
We thank Prof. F. Brmmer (University of Stuttgart), Prof. W.E.G. Mller (University of Mainz), Dr. R. Batel (Institute Rudjer Boskovic, Croatia) and all BiotecMarin colleagues as well as Dr. F. Hoffmann (MPI Bremen, Germany) for sampling support and many interesting discussions. Additionally, we thank C. Gernert for excellent laboratory assistance (University of Wuerzburg). Financial support was provided by bmb+f Center of Competence BiotecMarin (FKZ 03F0414E) and Sonderforschungsbereich SFB 567 - TPC3 grant to U. H.

171

Hentschel U, Fieseler L, Wehrl M, Gernert C, Steinert M, Horn M, Hacker J (2003) Microbial diversity of marine sponges. In: Mller WEG (ed). Molecular biology of sponges. Springer-Verlag, Heidelberg. pp. 59-88 Hentschel U, Hopke J, Horn M, Friedrich AB, Wagner M, Hacker J, Moore BS (2002) Molecular evidence for a uniform microbial community in sponges from different oceans. Appl Environ Microbiol 68: 4431-40 Hentschel U, Usher KM, Taylor MW (2006) Marine sponges as microbial fermenters. FEMS Microbiol Ecol 55(2): 167-177 Hill RT (2004) Microbes from marine sponges: a treasure trove of biodiversity for natural products discovery. In: Bull AT (ed). Microbial diversity and bioprospecting. ASM Press, Washington DC. pp 177-190 Hooper JNA, van Soest RWM (2002) Systema Porifera: a guide to the classification of sponges, vol. 1. Kluwer Academic/Plenum Publishers, New York Imhoff JF, Sthr R (2003) Sponge-associated bacteria: general overview and special aspects of bacteria associated with Halichondria panicea. In: Mller WEG (ed). Molecular biology of sponges. Springer Verlag, Heidelberg. pp. 35-56 Jimnez E, Ribes M (2007) Sponges as a source of dissolved inorganic nitrogen: nitrification mediated by temperate sponges. Limnol Oceanogr 52(3): 948-958 Kowalchuk GA, Stephen JR. (2001) Ammonia-oxidizing bacteria: a model for molecular microbial ecology. Annu Rev Microbiol 55: 485-529 Knneke M, Bernhard AE, de la Torre JR, Walker CB, Waterbury JB, Stahl DA (2005) Isolation of an autotrophic ammonia-oxidizing marine archaeon. Nature 437(7058): 543-546 Leininger S, Urich T, Schloter M, Schwark L, Qi J, Nicol GW, Prosser JI, Schuster SC, Schleper C (2006) Archaea predominate among ammonia-oxidizing prokaryotes in soils. Nature 442(7104): 806-809 Li CW, Chen JY, Hua TE (1998) Precambrian sponges with cellular structures. Science 279: 879-82 Loy A, Horn M, Wagner M (2003) ProbeBase - an online resource for rRNA-targeted oligonucleotide probes. Nucleic Acids Res 31: 514-516 (http://www.microbial-ecology.net/probebase/)

Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar, Buchner A, Lai T, Steppi S, Jobb G, Forster W, Brettske I, Gerber S, Ginhart AW, Gross O, Grumann S, Hermann S, Jost R, Konig A, Liss T, Lussmann R, May M, Nonhoff B, Reichel B, Strehlow R, Stamatakis A, Stuckmann N, Vilbig A, Lenke M, Ludwig T, Bode A, Schleifer KH (2004) ARB: a software environment for sequence data. Nucleic Acids Res 32: 1363-1371 Parsons TR, Maita Y, Lalli CM (1984) Manual of chemical and biological methods for seawater analysis. Pergamon Press, New York Pile AJ (1996) The role of microbial food webs in benthic-pelagic coupling in freshwater and marine ecosystems. PhD thesis. College of William and Mary, Williamsburg Pile AJ (1997) Finding Reiswigs missing carbon: quantification of sponge feeding using dual-beam flow cytometry. Proc 8th Intern Coral Reef Symp, Balboa 2: 1403-1410 Pospesel M, Hentschel U, Felbeck H (1998) Determination of nitrate in the blood of the hydrothermal vent tubeworm Riftia pachyptila using nitrate reduction assay. Pergamon Deep Sea Res I (45): 2189-2200 Reiswig H (1974) Water transport, respiration and energetics of three tropical marine sponges. J Exp Mar Biol Ecol 14: 231-249 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual (second edition). Cold Spring Harbor Press, Cold Spring Harbor, New York Thiel V, Neulinger SC, Staufenberger T, Schmalljohann R, Imhoff JF (2007) Spatial distribution of sponge-associated bacteria in the Mediterranean sponge Tethya aurantium. FEMS Microbiol Ecol 59: 47-63 Wang JT, Douglas AE (1998) Nitrogen recycling or nitrogen conservation in an alga-invertebrate symbiosis? J Exp Biol 201: 2445-2453 Wehrl M, Steinert M, Hentschel U (2007) Bacterial uptake by the marine sponge Aplysina aerophoba. Microb Ecol 53(2): 355-365 Wuchter C, Abbas B, Coolen MJ, Herfort L, van Bleijswijk J, Timmers P, Strous M, Teira E, Herndl GJ, Middelburg JJ, Schouten S, Sinninghe Damste JS (2006) Archaeal nitrification in the ocean. Proc Natl Acad Sci USA 103(33): 12317-12322

Porifera research: Biodiversity, innovation and sustainaBility - 2007

173

Perplexing distribution of 3-alkylpyridines in haplosclerid sponges


Leontine E. Becking(1*), Yoichi Nakao(2), Nicole J. de Voogd(1), Rob W.M. van Soest(3), Nobuhiro Fusetani(2), Shigeki Matsunaga(2)
National Museum of Natural History Naturalis, Dept. Zoology, P.O. Box 9517, 2300 RA Leiden, The Netherlands. becking@naturalis.nnm.nl (2) University of Tokyo, Laboratory of Aquatic Natural Products Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan (3) University of Amsterdam, Zoological Museum, P.O. Box 94766, 1090 GT Amsterdam, The Netherlands
(1)

Abstract: In this study we reviewed the natural product literature for the distribution of 3-alkylpyridines among sponge taxa. In parallel, we traced selected 3-alkylpyridines, amphitoxins, in three haplosclerid genera (Amphimedon, Callyspongia, Haliclona) in order to establish the utility of such compounds as genuine chemotaxonomic markers. We confirmed that this group of compounds had been almost solely extracted from sponges of the order Haplosclerida. Three groups of compounds within the 3-alkylpyridine derivatives were noteworthy, as they appear to be concentrated in restricted taxonomic units within the order Haplosclerida: 1) polymers, 2) cyclic dimers, and 3) bicyclic dimers. There was a concentration of the polymer amphitoxin in the families Niphatidae and Callyspongiidae of the suborder Haplosclerina, and more particularly in the genera Amphimedon and Callyspongia. Our experimental results reconfirmed the presence of amphitoxin in Callyspongia (Euplacella) biru, but we were unable to trace any amphitoxins or 3-alkylpyridines of any kind in Callyspongia (Callyspongia) truncata, Haliclona sp., and Amphimedon aff. queenslandica. Assuming that these classifications are correct, our results diminish the value of both amphitoxins and 3-alkylpyridines as monophyletic markers. Keywords: 3-alkylpyridines, amphitoxin, chemotaxonomy, Order Haplosclerida, secondary metabolites

Introduction
Sponges have proven to be a magnificent source of numerous highly bioactive compounds with pharmaceutical potential (Faulkner 2000, Sipkema et al. 2005). Since the 1960s natural product chemists have been active in extracting these compounds from sponges, which were collected directly and randomly from the seas. For the past thirty decades several authors have been considering sponge compounds as candidates for additional markers that could potentially be useful in verifying or aiding the standing phylogeny of the Porifera. (e.g. Bergquist and Hartman 1969, van Soest and Braekman 1999, Erpenbeck and van Soest 2007). The present classification of sponges is mainly based on external and internal morphological characters and some life history characteristics (Hooper and van Soest 2002). This classification of sponges is not infrequently disputed amongst sponge taxonomists and in fact the phylogenetic tree has been altered several times over the past decades. The field of chemotaxonomy in its turn has, however, yet to prove its utility. The bulk of published data on natural products from marine organisms has been archived in a database called MarinLit (Munro and Blunt 2004) that is updated annually. Andersen et al. (1996) and van Soest and Braekman (1999) reviewed the MarinLit database to discuss the utility of secondary

metabolites of sponges as systematic tools. One conclusion from these studies was that 3-alkylpyridine derivatives might be useful markers for the Order Haplosclerida. Since the publication of the reviews of Andersen et al. (1996) and van Soest and Braekman (1999), numerous additional compounds have been isolated from sponges. We, therefore, once again reviewed the distribution of 3-alkylpyridines in sponge genera. A well-reported problem with allocating taxonomic relevance to a compound based on the results of literature reviews is the strong bias of natural product laboratories to publish only novel compounds, thereby confounding any conclusions on the absence of compounds in certain taxonomic groups. To overcome this problem we selected particular 3-alkylpyridine derivatives, amphitoxins, and traced their distribution in representatives of three genera to establish whether such compounds could be viewed as genuine chemotaxonomic markers.

Material and methods Literature review


MarinLit (Munro and Blunt, 2004), Web of Science and scholar.google.com were reviewed for publications reporting extraction of 3-alkylpyridine derivatives from sponges. Keywords of all known names within this compound

174

group were submitted in the databases, as well as the names of the genera within the Order Haplosclerida.

Results Literature review


We identified 49 records reporting extraction of 3alkylpyridine alkaloids from sponges. The majority of the records were identified to genus-level and belonged to the Order Haplosclerida. There were three records of 3-alkylpyridines extracted from sponges of other orders: Theonella swinhoei (Lithistida, from Kobayashi et al. 1989), Batzella sp. (Poecilosclerida, from Segraves and Crews 2005), and Halichondria sp. (Halichondrida, from Chill et al. 2002). Within the Order Haplosclerida, 3-alkylpyridines have been recorded from the 5 of the 6 main marine haplosclerid families, but have not been recorded from all genera belonging to these families. In the suborder Haplosclerina the families represented were Callyspongiidae (2 genera, 5 species), Chalinidae (2 genera, 3 species), Niphatidae (3 genera, 6 species), and in the suborder Petrosina the families were Petrosiidae (2 genera, 3 species), and Phloedictyidae (1 genus, 1 species). Three groups of compounds appeared to be concentrated in small taxonomic units (see Table 1A-C): 1. Polymers (hali/amphitoxins) 2. Cyclic dimers (cyclostelletamines/haliclamines) 3. Bicyclic dimers (haliclonacyclamines/halicyclamines/ arenosclerins)

Collection, extraction and fractionation


Four tropical reef sponge species were collected at depths ranging from 2-10 m: Callyspongia (Euplacella) biru de Voogd 2004 (Sulawesi, Indonesia), Callyspongia (Callyspongia) truncata (von Lendenfeld, 1887) (Shizuoka, Japan), Amphimedon aff. queenslandica Hooper and van Soest 2006 (Okinawa, Japan), Haliclona sp. (Kagoshima, Japan), (see Fig. 1A-D). Voucher specimens have been deposited at the Zoological Museum, University of Amsterdam, the Netherlands (C. (E.) biru collection number POR.15222), and at the National Museum of Natural History Naturalis, Leiden The Netherlands (C. (C.) truncata, A. aff. queenslandica, Haliclona sp. collection numbers respectively: POR.2971, POR.2972, POR.2973). Two individuals of each species were screened for amphitoxins. Amphitoxins are known to be mainly responsible for the bioactivity of the crude extracts of C. (E.) biru (Dubut 2000 as Callyspongia sp. blue; de Voogd et al. 2005). To date no amphitoxins have been reported from C. (C.) truncata,while a large number of bioactive polyacetylenes have been extracted from this species (Nakao et al. 2002). Each sponge was extracted with MeOH (500 ml x 3) and the extract was partitioned between water and CHCl3. The water-soluble fraction was extracted with n-butanol. The organic phase was partitioned between MeOH/H2O (9:1) and n-hexane. The aqueous MeOH layer was then partitioned between MeOH/H2O (6:4) and CHCl3. Finally the n-butanol fraction and MeOH/H2O (6:4) fraction were combined. All fractions were analyzed by TLC on silica gel with CHCl3/ MeOH/H2O (7:3:0.5) and different mixtures of CHCl3/ MeOH. Tertiary or quaternary bases were visualized by spraying with Dragendorff reagent. The fractions exhibiting orange/red spots were subsequently fractionated by ODS flash chromatography using stepwise elution of aqueous MeOH (0 % to 100 % MeOH), followed by elution with CHCl3/MeOH/H2O (7:3:0.5). The fractions showing spots positive to Draggendorff on TLC were further separated by ODS HPLC (Column: COMOSIL-5C18-ARII 10 x 250 mm; flow rate 2 ml/min.; UV 266 nm) using gradient elution from 35 % MeCN/0.05 % TFA to 80 % MeCN/0.05 % TFA. The peaks positive to Draggendorff were analyzed by 1H NMR spectroscopy to examine if typical proton signals for amphitoxin were present. As C.(E.) biru has previously been shown to contain amphitoxins (Dubut 2000, de Voogd et al. 2005), the above procedure was first performed on this species to obtain a standard reference for amphitoxin. After a standard was obtained, the extracts of the other sponges (after solvent partitioning and ODS flash) were first checked for the presence of a similar compound by comparing Rf values on TLC with that of amphitoxin, before subjected to HPLC.

Extraction and fractionation


Amphitoxin was successfully extracted from the C. (E.) biru specimens, and was present in the 20-100% elution by ODS chromatography of the combined n-butanol and MeOH/ H2O (6:4) fractions. We were unable to trace any amphitoxins or 3-alkylpyridines from the extracts of the C. (C.) truncata, Haliclona sp. or A. aff. queenslandica sp. specimens.

Discussion
We confirmed that 3-alkylpyridines have almost exclusively been isolated from sponges of the order Haplosclerida, after having made the necessary amendments on sponge taxonomy and nomenclature. There are, however, three reports of 3alkylpyridines from sponges of other orders: Theonella swinhoei is a probable case of mislaid labels or undetected haplosclerid overgrowth (cf. van Soest and Braekman 1999), and Batzella sp. and Halichondria sp. remain unresolved for the time being. It must be noted, that these genera share a single diactinal spicule type with Haplosclerida, making it possible that they have been misidentified Haplosclerida. In our view the use of more specific compound structures/ groups, rather than the relatively broad nominator 3alkylpyridine derivatives, ought to be the next step in supporting classifications based on compounds. In previous reviews linear 3-alkylpiperidines appeared to be concentrated in the families Callyspongiidae and Niphatidae, while cyclic 3-alkylpiperidines were concentrated in Chalinidae and Petrosiidae (Andersen et al.

175

Fig. 1A-D: Sponges examined in this study: A. Callyspongia (Euplacella) biru (photo: B. W. Hoeksema), B. Callyspongia (Callyspongia) truncata (photo: S. Hoshino), C. Amphimedon aff. queenslandica (photo: L. E. Becking), D. Haliclona sp. (photo: Y. Nakao).

1996, van Soest and Braekman 1999). Records of extracted compounds published since that review (notably the cyclic 3-alkylpiperidines found in Amphimedon sp. by Matsunaga et al. 2004), however, indicate that a taxonomic distinction based on the presence of cyclic or linear 3-alkylpiperidine derivatives can most likely no longer be upheld. Three groups of compounds within the 3-alkylpyridines were noteworthy in that they have structures with presumed similar biogenetic pathways and appear to be concentrated in small taxonomic units (see Table 1A-C). Based on the common presence of these specific compounds, the close relationship between Callyspongiidae, Chalinidae, Niphatidae, and Petrosiidae is confirmed. Previous works suggested that the closely related polymers halitoxin and amphitoxin might be markers for the family Niphatidae. Our review showed a concentration of these types of compounds not only in Niphatidae, but also in Callyspongiidae, and more particularly in the genera Amphimedon and Callyspongia (Table 1A).

Our laboratory work reconfirmed the presence of amphitoxin in C. (E.) biru, but we were unable to trace any amphitoxins from C. (C.) truncata, nor from the Haliclona sp. and A. aff. queenslandica. In fact, the situation is rather more complex as we did not extract 3-alkylpyridines of any kind from these three specimens. The sponge specimens that did not contain 3-alkylpyridines were collected from Japan, but we do not suspect this is a case of geographic variation of compound distribution as these compounds have been isolated from Japanese sponges before (Fusetani et al. 1989, 1994, Matsunaga et al. 2004). Causes of these results may lie in the frequently reported high natural variation in compound production, which in turn may be due to physical and environmental variation (e.g. Thacker et al. 1998, de Voogd et al. 2004, de Voogd 2007) or to symbionts that may be the true producers of the compounds (e.g. Jadulco et al. 2002, Becerro and Paul 2004). Sponges generally harbor vast amounts of symbionts, among which there are not only the specialists, which may

176

Table 1A-C: Three compound groups that appear to be concentrated in restricted taxonomic units (compound figures reproduced from Andersen et al. 1996): A. polymers, B. cyclic dimers, C. bicyclic dimers.

A. Polymers

Family Callyspongidae Callyspongidae Callyspongidae Niphatidae Niphatidae Niphatidae Niphatidae Niphatidae Niphatidae Niphatidae Chalinidae Chondropsidae

Species Callyspongia (Euplacella) biru Callyspongia (Cladochalina) fibrosa Callyspongia ridleyi Amphimedon compressa Amphimedon erina Amphimedon viridis Amphimedon viridis Amphimedon viridis Amphimedon compressa Amphimedon paraviridis Haliclona (Rhizoniera) sarai Batzella sp.

Compound Amphitoxin Halitoxin Halitoxin Halitoxin Halitoxin Halitoxin Halitoxin Hali/Amphitoxin Amphitoxin Amphitoxin Halitoxin Halitoxin

Location Indonesia Micronesia unknown Caribbean Caribbean Caribbean Brazil Red Sea Bahamas Indonesia Adriatic Sea Madagascar

Identification de Voogd van Soest unknown Hartman Hartman Hartman Hajdu unknown Genoa Museum de Voogd Vacelet Diaz

Reference de Voogd et al. 2005 Davies-Coleman et al. 1993 Scott et al. 2000 Schmitz et al. 1978 Schmitz et al. 1978 Schmitz et al. 1978 Berlinck et al. 1996 Kelman et al. 2001 Albrizio et al. 1995 de Voogd et al 2005 Sepi et al. 1997 Segraves and Crews 2005

B. Cyclic dimers

Family Chalinidae Chalinidae Chalinidae Chalinidae Niphatidae Petrosiidae

Species Haliclona sp. Haliclona (Rhizoniera) viscosa Haliclona sp. Haliclona (Rhizoniera) viscosa Pachychalina sp. Xestospongia sp.

Compound Haliclamines Haliclamines Cyclostellettamines Cyclostellettamines Cyclostellettamines Cyclostellettamines

Location Japan Arctic Japan Arctic Brazil Japan

Identification Watanabe de Weerdt van Soest de Weerdt Hajdu van Soest

Reference Fusetani et al. 1989 Volk et al. 2004 Fusetani et al. 1994 Volk and Kck 2004 de Oliviera et al. 2004 Oku et al. 2004

C. Bicyclic dimers

Family Niphatidae Niphatidae Niphatidae Callyspongiidae Callyspongiidae Chalinidae Chalinidae Petrosiidae Halichondriidae

Species Amphimedon sp. Amphimedon sp. Amphimedon sp. Arenosclera braziliensis Arenosclera braziliensis Haliclona sp. Haliclona sp. Xestospongia sp. Halichondria sp.

Compound Tetradehydrohalicyclamine 22-hydroxyhalicyclamine Tetrahydrohalicyclamine Arenosclerins Haliclonacyclamines Haliclonacyclamines Halicyclamine Halicyclamine Halichondramine

Location Japan Japan Japan Brazil Brazil Australia Indonesia Indonesia Eritrea

Identification van Soest van Soest van Soest Hajdu Hajdu Hooper unknown Diaz van Soest

Reference Matsunaga et al. 2004 Matsunaga et al. 2004 Matsunaga et al. 2004 Torres et al. 2002 Torres et al. 2002 Clark et al. 1998, Charan et al. 1996 Jaspars et al. 1994 Harrison et al. 1996 Chill et al. 2002

177

have co-evolved with the host sponge, but also the generalist microbes which can be found in a wide array of sponges and some of which are even widely present in seawater (Lee et al. 2001, Taylor et al. 2004). When identical or very similar compounds are found in species from different orders or genera, microbial producers are suspected (e.g. bacteria and fungi). If these generalist symbionts play any role in the production of the toxic compounds extracted from the sponges, this could mystify the classifications based on chemotaxonomy. Erpenbeck and van Soest (2007) provide a full review of the various pitfalls in the use of compounds in chemotaxonomy. They illustrate that the major problems are not only related to the great natural variation in compound production and ambiguous origins of compounds, but also the unknown homology of compounds, misidentifications of sponge species and the lack of reporting by natural products chemists of presence/absence of known compounds. To conclude, our results diminish the value of both 3alkylpyridines and amphitoxins as monophyletic markers assuming that the classifications are correct. The classification of haplosclerid families and genera is presently under siege from molecular studies (e.g. McCormack et al. 2002, Nichols 2005). Thus, this study may prove of some value in the ongoing debate on the classification of the poriferan phylum. Any definate conclusions of the utility of 3-alkylpyridines as chemotaxonomic markers would, however, be presumptuous before a more comprehensive systematic study is performed, particularly of genera that have not been examined previously, and the presence in the other orders is unequivocally ruled out.

Acknowledgements
The field- and laboratory-work was financed by NWO-WOTRO and the Japan Prizewinners Programme administered by the Dutch Ministry of Education, Culture and Science. The first author is also grateful for financial support received by the Scientific Committee of the 7th International Sponge Symposium and the Jan Joost ter Pelkwijk Fonds, which made attending the symposium possible. We would like to thank Dr. Hoeksema and Dr. Hoshino for providing photographs and Yamashita-san for all his friendly help in the lab.

References
Albrizio S, Cimiello P, Fattorusso E, Magno S, Pawlik JR (1995) Amphitoxin, a new high molecular weight antifeedent pyridinium salt from the Caribbean sponge Amphimedon compressa. J Nat Prod 58: 647-652 Andersen RJ, van Soest RWM, Kong F (1996) 3-alkylpiperidine alkaloids isolated from marine sponges in the order Haplosclerida. In: Pelletier SW (ed). Alkaloids: Chemical and Biological Perspectives, Vol 10. Pergamon, Oxford, pp 301-356 Becerro MA and Paul VJ (2004) Effects of depth and light on secondary metabolites and cyanobacterial symbionts of the sponge Dysidea granulosa. Mar Ecol Prog Ser 280: 115-128 Bergquist PR, Hartman WD (1969) Free amino acid patterns and the classification of the Demospongiae. Mar Biol 3: 247-268 Berlinck RGS, Ogawa CA, Almeida AMP, Sanchez MAA, Malpezzi ELA, Costa LV, Hajdu E, Freitas de JC (1996) Chemical and Pharmacological characterization of halitoxin from Amphimedon

viridis (Porifera) from the Southeastern Brazilian Coast. Comp Biochem Phys 115C: 155-163 Charan RD, Garson MJ, Brereton IM, Willis AC, Hooper JNA (1996) Haliclonacyclamines A and B, cytotoxic alkaloids from the tropical marine sponge Haliclona sp. Tetrahedron 52: 9111-9120 Chill L, Miroz A, Kashman Y (2000) Haliclonyne, a new highly oxygenated polyacetylene from the marine sponge Haliclona species. J Nat Prod 63: 523-526 Clark RJ, Field KL, Charan RD, Garson MJ, Brereton IM, Willis AC (1998) The Haliclonacyclamines, cytotoxic tertiary alkaloids from the tropical marine sponge Haliclona sp. Tetrahedron 54 (30): 8811-8826 Davies-Coleman MT, Faulkner DJ, Dubowchik GM, Roth GP, Polson C, Fairchild C (1993) A new EGF-active polymeric pyridinium alkaloid from the sponge Callyspongia fibrosa. J Org Chem 58: 5925-5930 de Voogd NJ (2004) Callyspongia (Euplacella) biru spec. nov. (Porifera: Demospongiae: Haplosclerida) from Indonesia. Zool Meded 78: 477-483 de Voogd NJ, Becking LE, Noor A, Hoeksema BW, van Soest RWM (2004) Sponge interactions with spatial competitors in S.W. Sulawesi. In: Pansini M, Pronzato R, Bavestrello G, Manconi R (eds). Sponge science in the new millennium. Boll Mus Ist Biol Univ Genova 68: 253-261 de Voogd NJ, Haftka JJH, Hoeksema BW (2005) Evaluation of the ecological function of amphitoxin in the reef-dwelling sponge Callyspongia (Euplacella) biru (Haplosclerida: Callyspongiidae) at southwest Sulawesi, Indonesia. Contr Zool 74: 51-59 de Voogd NJ (2007) The mariculture potential of the Indonesian reef-dwelling sponge Callyspongia (Euplacella) biru: growth, survival and bioactive compounds. Aquaculture 262:54-64 Dubut D (2000) Isolment et dtermination de structure de toxins dponges du genre Callyspongia. Student Report, Universit Libre de Bruxelles, Belgium Erpenbeck E, van Soest RWM (2007) Status and perspective of sponge chemosystematics. Mar Biotech 9: 2-19 Faulkner DJ (2000) Highlights of marine natural products chemistry. Nat Prod Rep 17: 1-6 Fusetani N, Yasumuro K, Matsunaga S, Hirota H (1989) Haliclamines A and B, cytotoxic macrocyclic alkaloids from a sponge of the genus Haliclona. Tetrahedron Lett 30: 6891 Fusetani N, Asai N, Honda K, Yasumuro K (1994) Cyclostellettamines A-F, pyridine alkaloids which inhibit binding of methyl quinuclidinyl benzylate (QB) to muscarine acetylcholine receptors, from the marine sponge Stelletta maxima. Tetrahedron Lett 35: 3967-3970 Harrison B, Talapatra S, Lobkovsky E, Clardy J, Crews P (1996) The structure and biogenetic origin of (-) Halicyclamine B from a Xestospongia sponge. Tetrahedron Lett 37: 9151-9154 Hooper JNA, van Soest RWM (2002) Systema Porifera: a guide to the classification of sponges. Kluwer Academic/Plenum Publishers, New York Hooper JNA, van Soest RWM (2006) A new species of Amphimedon (Porifera, Demospongiae, Haplosclerida, Niphatidae) from the Capricorn-Bunker Group of Islands, Great Barrier Reef, Australia: target species for the sponge genome project. Zootaxa 1314: 3139 Jadulco R, Brauers G, Angelie R, Ebel R, Wray V, Sudarsono, Proksch P (2002) New metabolites from sponge-derived fungi

178

Curvularia lunata and Cladosporium herbarum. J Nat Prod 65: 730-733 Jaspars M, Pasupathy V, Crews P (1994) A tetracyclic diamine alkaloid, halicyclamine A, from the marine sponge Haliclona sp. J Org Chem 59: 3253-3255 Kelman D, Kashman Y, Rosenberg E, Ilan M, Ifrach I, Loya Y (2001) Antimicrobial activity of the reef sponge Amphimedon viridis from the Red Sea: evidence for selective toxicity. Aquat Microb Ecol 24: 9-16 Kobayashi J, Murayama T, Ohizumi Y, Sasaki T, Ohta T, Nozoe S (1989) Theonelladins A-D, novel antineoplastic pyridine alkaloids from the Okinawa marine sponge Theonella swinhoei. Tetrahedron Lett 30 (22): 2963-2966 Lee YK, Lee JH, Lee HK (2001) Microbial symbiosis in marine sponges. J Microbiol 39(4): 254-264 Matsunaga S, Shinoda K, Fusetani N (1993) Cribrochalinamine oxides A and B, antifungal beta-substituted pyridines with an azomethine N-oxide from the marine sponge Cribrochalina sp. Tetrahedron Lett 34: 5953 Matsunaga S, Miyata Y, van Soest RWM, Fusetani N (2004) Tetradehydrohalicyclamine A and 22-hydroxyhalicyclamine A, new cytotoxic bis-piperidine alkaloids from a marine sponge Amphimedon sp. J Nat Prod 67: 1758-1760 McCormack GP, Erpenbeck D, van Soest RWM (2002) Major discrepancy between phylogenetic hypotheses based on molecular and morphological criteria within the Order Haplosclerida (Phylum Porifera: Class Demospongiae). J Zool Syst Evol Res 40: 237-240 Munro MGH, Blunt JW (2004) MarinLit. A database of the literature on marine natural products for the use of macintosh computers prepared and maintained by the Marine Chemistry Group. Department of Chemistry, University of Canterbury, New Zealand Nakao Y, Uehara T, Matsunaga S, Fusetani N, van Soest RWM (2002) Callyspongic acid, a polyacetylenic acid which inhibits glucosidase, from the marine sponge Callyspongia truncata. J Nat Prod 65: 922-924 Nichols SA (2005) An evaluation of support for order-level monophyly and interrelationships within the class Demospongiae using partial data from the large subunit rDNA and cytochrome oxidase subunit I. Mol Phylogenet Evol 34: 81-96 Oku N, Nagai K, Shindoh N, Tetrada Y, van Soest RWM, Matsunaga S, Fusetani N (2004) Three new cyclostellettamines, which

inhibit histone deacetylase, from a marine sponge of the genus Xestospongia. Bioorg Med Chem Lett 14: 2617-2620 Oliveira JHHL de, Grube A, Kock M, Berlinck RGS, Macedo ML, Ferreira AG, Hajdu E (2004) Ingenamine G and Cyclostelettamines G-I, K and L from the new Brazilian species of marine sponge Pachychalina sp. J Nat Prod 67: 685-1689 Schmitz FJ, Hollenbeak KH, Campbell DC (1978) Marine natural products: halitoxin, toxic complex of several marine sponges of the genus Haliclona. J Org Chem 43: 3316-3822 Scott RH, Whyment AD, Foster A, Gordon KH, Milne BF, Jaspars M (2000) Analysis of the structure and electrophysiological actions of halitoxins: 1,3-alkylpyridinium salts from Callyspongia ridleyi. J Membr Biol 176: 119-131 Segraves NL, Crews P (2005) A Madagascar sponge Batzella sp. as a source of alkylated iminosugars. J Nat Prod 68: 118-121 Sepi K, Guella G, Mancini I, Pietra F, Dalla Serra M, Menestrina G, Tubbs K, Macek P, Turk T (1997) Characterization of anticholinesterase-active 3-alkylpyridinium polymers from the marine sponge Reniera sarai in aqueous solutions. J Nat Prod 60: 991-996 Sipkema D, Franssen MCR, Osinga R, Tramper J, Wijffels RH (2005) Marine Sponges as Pharmacy. Mar Biotech 7: 142-162 Taylor MW, Schupp PJ, Dahllof I, Kjelleberg S, Steinberg PD (2004) Host specificity in marine sponge-associated bacteria, and potential implications for marine microbial diversity. Environ Microbiol 6: 121-130 Thacker RW, Becerro MA, Lumbang WA, Paul VJ (1998) Allelopathic interactions between sponges on a tropical reef. Ecology 79: 1740-1750 Torres YR, Berlinck RGS, Nascimento GGF, Fortier SC, Pessoa C, de Moraes MO (2002) Antibacterial activity against resistant bacteria and cytotoxicity of four alkaloid toxins isolated from the marine sponge Arenosclera brasiliensis. Toxicon 40: 885-891 van Soest RWM, Braekman J-C (1999) Chemosystematics of Porifera: a review. Memoir Queensl Mus 44: 569-589 Volk CA, Lippert H, Lichte E, Kck M (2004) Two new haliclamines from the Arctic sponge Haliclona viscosa. Eur J Org Chem 14: 3154-3156 Volk CA, Kck M (2004) Viscosaline: new 3-alkyl pyridinium alkaloid from the arctic sponge Haliclona viscosa. Org Biomol Chem 2: 1827-1830 Web of Science: http://portal.isiknowledge.com

Porifera research: Biodiversity, innovation and sustainaBility - 2007

179

Lake Baikal endemic sponge Lubomirskia baikalensis: structure and organization of the gene family of silicatein and its role in morphogenesis
Sergey I. Belikov(1,2), Oksana V. Kaluzhnaya(1,2,3), Heinz C. Schrder(3,2), Isabel M. Mller(3), Werner E.G. Mller(2,3*)
Limnological Institute of the Siberian Branch of Russian Academy of Sciences, Ulan-Batorskaya 3, RUS-664033 Irkutsk, Russia (2) Limnological Institute of the Siberian Branch of Russian Academy of Sciences, Joint Russian-German Laboratory for Biology of Sponges, Ulan-Batorskaya 3, RUS-664033 Irkutsk, Russia (3) Institut fr Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universitt, Duesbergweg 6, D-55099 Mainz, Germany. tel.: +49-6131-392-5910; fax.: +49-6131-392-5243. wmueller@uni-mainz.de
(1)

Abstract: Lake Baikal is known for its abundant endemic fauna and flora. The photosymbiotic sponges are the most numerous sessile animals in the Baikals littoral zone. These endemic sponges are grouped to the family Lubomirskiidae and, based on molecular data, they are separated from the cosmopolitan family Spongillidae. The endemic sponges are monophyletic and originate from a common ancestor with the freshwater sponges Ephydatia fluviatilis / Spongilla lacustris. They are grouped to the class of Demospongiae having spicules that are composed of hydrated, amorphous, noncrystalline silica. With the Baikalian sponge Lubomirskia baikalensis it could be shown that silica is deposited around an organic filament. A major step forward to elucidate the formation of the siliceous spicules on molecular level was the finding that the axial organic filament of siliceous spicules is an enzyme, silicatein, which mediates the apposition of amorphous silica and hence the formation of spicules. The formation of siliceous spicules is certainly genetically controlled; this process initiates the morphogenesis phase and involves primarily silicatein. In the present study the existence of silicatein- genes in the fresh-water Lake Baikal sponge L. baikalensis is demonstrated. The intron-exon structure of the full-size silicatein- gene was determined. A comprehensive phylogenetic analysis with new sequences sheds light upon the evolution of the cathepsin L and silicatein families which occurred at the base of the metazoan phyla. It is concluded, that in parallel with the emergence of these enzymes at first the number of introns increased, especially in the coding region of the mature enzyme. Later in evolution the number of introns decreased again. We postulate that modification of the catalytic triad, especially of its first amino acid, is a suitable target for a chemical modulation of enzyme function of the silicateins/cathepsins L. Keywords: cathepsin, intron/exon structure, Lubomirskia baikalensis, silicatein, Suberites domuncula

Introduction Sponges in the Neo-Proterozoic Eon


Sponges were designated as living fossils (Mller 1998) because they represent the evolutionary oldest, still extant taxon which testifies the developmental level of animals living in the Neo-Proterozoic Eon (1000 to 542 million years ago [MYA]). This is important to note since two major snowball earth events (Walker 2003) occurred during this period, the Sturtian glaciation (710 to 680 MYA) and the Varanger-Marinoan ice ages (605 to 585 MYA), which very likely resulted in a continuous ice cover of the earth that supposedly caused extinction of most organisms on Earth at that time (Hoffman et al. 1998). The primordial earth surface comprised initially insoluble silicates, carbonates, and also phosphates. During the cycle of silicate weathering and carbonate precipitation, prior or

simultaneously with the glaciations, a dissolution of surface rocks composed of insoluble silicates [CaSiO3] resulted in the formation of soluble calcium carbonate [CaCO3] and soluble silica [SiO2], under consumption of atmospheric CO2 (Walker 2003). These soluble minerals leached subsequently out to the oceans, rivers and lakes and there again led to a reprecipitation of the dissolved minerals to new compositions as part of the sedimentary rocks. Such processes are dependent upon temperature, pH and atmospheric carbon dioxide; passively, the minerals are transformed diagenetically to secondary structures. In contrast to passive re-precipitation, biogenic deposition of minerals by metazoans is first seen in sponges. The oldest sponge fossils (Hexactinellida) have been described from Mongolia and were assessed to have lived coeval with the diverse Ediacara fauna of Namibia more than 540 MYA (Brasier et al. 1997). Hence, the Hexactinellida are the oldest

180

group of sponges as documented there and later in fossil records of the Sansha section in Hunan (Early Cambrian; China; Steiner et al. 1993), where more or less completely preserved sponge fossils, e.g. Solactiniella plumata (Mehl et al. 1998), have been found. The oil-shales of the Messel pit, near Darmstadt (Germany), are very rich in fossil freshwater sponges; among them is Spongilla gutenbergiana from the Middle Eocene (Lutetian), approximately 50 MYA (Mller et al. 1982). Based on sequencing data of informative genes, which code for structural and functional proteins, it had been calculated that the sponges diverged from the common metazoan ancestor approximately 650 MYA (Schcke et al. 1994). This calculation is in close accordance with fossil records and implies that the sponges evolved between the two glaciations, Sturtian and Varanger-Marinoan. The existence of a large genetic repertoire in the Porifera, the basis for the establishment of complex metabolic and morphogenetic pathways, may have contributed to the rapid evolution of sponges occurred between the snowball periods 710 to 680 MYA and 605 to 585 MYA. At present it cannot be ruled out that other animal phyla evolved simultaneously with the Porifera, but became extinct during the last ice age.

Sponges in the Quaternary Eon at Lake Baikal


An exceptional freshwater sponge fauna has evolved, as endemic taxa, in the Lake Baikal in the last few million years. Interestingly enough these species branched off from a common ancestor with cosmopolitan freshwater sponges during ice periods. Fossil sponge spicules have been described from the Pliocene, 3.1 to 2.9 MYA (Weinberg et al. 2003). Paleoclimatic records over the period of 5 million years revealed two cold episodes, each approximately 300,000 years long, at the time intervals 2.8-2.5 MYA and 1.8-1.5 MYA (Kashiwaya et al. 2000). Therefore, it can be assumed/ postulated that the radiation of the freshwater sponges started in Lake Baikal more than 3 MYA. This view is also consistent with the finding of Weinberg et al. (2003) who described fossil sponge spicules from the Academician Ridge. At present, this ridge forms a submerged elevation of 400 m, while the maximal depth of the lake is 1,473 m. During the Upper Pliocene-Eopleistocene period (3.5 MYA) the ridge might have formed a land barrier between the Northern Basin and the Central Basin of the lake (Ovchinnikova 2005). During those ice periods the average temperature in the lake dropped from 20C (Eocene; 70-35 MYA) to <10C (Upper Pliocene; 3.5 MYA). Todays average water temperature is 35C, but changes considerably; in January the temperature is 0 to 4C while during the short summer period from June to August/September it rises to 12C (Scientific Council 1993). Nevertheless the metabolic activities, the pumping rates (Savarese et al. 1997) and the feeding efficiencies (Pile et al. 1997) of the Baikalian sponges are identical to those found in species in the marine tropic environment. Baikalian species are generally larger in size than other freshwater species, they grow up to 1.5 m high. The three most abundant species in the lake are Lubomirskia baikalensis Dybowski 1880 (Fig. 1AB), followed by Baikalospongia bacillifera Dybowski, 1880 and Baikalospongia intermedia Dybowski, 1880 (Pile et al.

1997). The habitus of the Baikalian sponges is highly variable; even though the basic Bauplan remains constant (Belikov et al. 2005, Mller et al. 2006b). In depths between 2 and 4 m L. baikalensis is an irregular crust. However, below 4 m an arborescent growth pattern is seen; from the 2 cm thick crusts, arborescent branches protrude with a modular arrangement of the tissue units. At larger depths fusion of the branches starts (> 7 m). Under certain environmental conditions the shape of the branches changes from cylindrical to flattened. Related species, e.g. Baikalospongia intermedia profundalis Rezvoj, 1936 or Baikalospongia fungiformis Makuschok, 1927 are deep-water sponges (they occur between 40-140 m [B. fungiformis] and up to 540 m [B.media profundalis]); a fact which is of prime ecological importance (Efremova 2001). One most remarkable chemical component of Lake Baikal is the relatively high level of dissolved silicic acid. On average the lake water contains 30-100 mol L-1 of silicic acid (Grachev 2002). In the marine coastal areas the mean silicon concentration at the surface is less than 3 mol L-1 (Maldonado et al. 1999); while it gradually increases with depth (Trguer et al. 1995). The high content of silicic acid in Lake Baikal is caused by the heavy influx of silicon from the rivers (Bukharov and Fialkov 2001). The turnover rate of silicic acid in the lake is elevated; more than 70% of the mineral is cycled during the year through biological uptakebiomineralization-sedimentation-redissolving as well as by upwelling and diffusion back to the lake.

Monophyly of sponges in Lake Baikal


Variability of populations, adaptive changes in populations, geographic variation and speciation are all processes that are described by the term microevolution, while macroevolution refers to processes that occur above the species level (Mayr 2001). The microevolutionary events that took place during the last several hundreds to a few million years can be studied exemplarily in the populations of endemic animal species in Lake Baikal and in some surrounding lakes. According to geological records that go back to 24 million years Lake Baikal is the oldest and deepest (1,637 m) of the earths ancient lakes; its water mass contributes to one fifth of the worlds unfrozen freshwater. More than 1,500 endemic species inhabit Lake Baikal (Timoshkin 1997) with the highly diverse sponges being the dominant animals in the littoral zone (Kozhov 1972); it is assumed that there are up to 18-20 species, all belonging to the taxon Spongillidae/Spongillina (Efremova 2004). Also other ancient lakes have a similar rich endemic sponge fauna, e.g. Malawispongiidae Manconi and Pronzato, 2002, Metschnikowiidae Czerniavsky, 1880 or Potamolepidae Brien, 1967 (see: Manconi and Pronzato 2000).

Molecular phylogeny: cytochrome oxidase-tubulinsilicatein


Nucleotide sequence data obtained from protein-coding sponge genes, such as the mitochondrial cytochrome oxidase subunit I (COI) gene and the exon/intron sequences framing intron-2 of the tubulin gene evidenced the evolution of the different sponge species in Lake Baikal from a common

181

Fig. 1: Lubomirskia baikalensis. A. The first illustration of this species by Dybowski (1880). B. Bush-like growth form of L. baikalensis. Specimens from a depth of 10 m, showing the transition from the crust growth form to the arborescent pattern organization. The photograph was taken in March 2006 from animals growing under the ice cover of 1 m. They appear in bright green, due to abundance of the dinoflagellates, highly related to Gymnodinium sanguineum.

ancestor which was also the origin for the ubiquitous freshwater sponge Spongilla lacustris Linnaeus, 1758 (Spongillidae Gray, 1867; Schrder et al. 2003). The degree of nucleotide substitutions within the 18S rDNA region of the different Baikalian sponges proved not sufficiently high to draw unequivocal conclusions (Itskovich et al. 1999). Even the degree of sequence identity in the COI gene of the different Baikalian sponges required additional support from analyses of intron-2 of the tubulin genes (Schrder et al. 2003). A phylogenetic tree (Fig. 2B) could be constructed on the ground of the different lengths of the introns (Fig. 2A) and their building blocks. The sequence of intron-2 from tubulin from the marine demosponge Suberites domuncula (Olivi, 1792) (Hadromerida Topsent, 1894: Suberitidae Schmidt, 1870) served as outgroup. The cosmopolitan freshwater sponge S. lacustris formed the next similar branch from wich the Baikalian sponges, all belonging to the family Lubomirskiidae Rezvoj, 1936, branch off; first, the member of the genus Swartschewskia Makuschok, 1927, S. papyracea Dybowski, 1880, followed by the three species among the Baikalospongia Annandale, 1914, B. recta Efremova, 2001, B. intermedia Dybowski, 1880 and B. bacillifera Dybowski, 1880 (Fig. 2B) and finally the species Lubomirskia baikalensis (Pallas, 1776). This branching pattern was supported also by the COI sequence data, even though their statistical significance was low. Paleontological data also indicate that Swartschewskia-species appeared before Baikalospongiaand Lubomirskia-species in Lake Baikal (Weinberg et al. 2003). Analysis of the protein sequence of the gene encoding silicatein, the major protein in the axial filament of spicules from Demospongiae, further supported this phylogeny. The organic filament in the central canal of the spicules, composed

of a cathepsin L-related enzyme, was termed silicatein by the group of Morse (Shimizu et al. 1998). They cloned two of the proposed three isoforms of silicateins, the - and -form, from the demosponge Tethya aurantium Pallas, 1766 (Cha et al. 1999). Later silcateins were also cloned from other sponges, among them S. domuncula and L. baikalensis (Krasko et al. 2000, 2002, Schrder et al. 2004, Kaluzhnaya et al. 2005b, Wiens et al. 2006, Mller et al. 2006a). For the studies of the relationship between the endemic Baikalian sponges and the cosmopolitan freshwater sponges, S. lacustris and Ephydatia fluviatilis Lamarck, 1816, the silicatein cDNAs from these two species were isolated. Phylogenetic analysis was performed with the silicateins from the demosponges L. baikalensis, T. aurantium and S. domuncula as well as with the cathepsins L from Metazoa and the papain cysteine peptidase from the plant species Arabidopsis thaliana. The phylogenetic tree showed that the cathepsin L sequences form the basic branch from which the silicatein sequences diverge (see: Wiens et al. 2006) indicating that the silicateins have a common ancestor with the cathepsin L sequences from the marine hexactinellids such as Aphrocallistes vastus Schulze, 1899 and demosponges, here from S. domuncula. Among the silicateins those from the cosmopolitan species S. lacustris and E. fluviatilis form the basal branch for the polypeptides of the Baikalian sponges L. baikalensis (Fig. 3; [Mller et al. 2006c]). From the results obtained by three different molecular biological approaches (mitochondrial genes [Schrder et al. 2003]; introns of tubulin [Fig. 2; Schrder et al. 2003]; silicatein genes [Fig. 3 and review: Mller et al. 2006c]) it may be concluded that the endemic sponges in Lake Baikal evolved monophyletically from the cosmopolitan freshwater sponges. Studies are in progress to investigate the silicatein

182

Fig. 2: Phylogenetic relationship of the sequences of intron 2 in tubulin genes of freshwater sponges and the marine sponge S. domuncula. A. Intron/exon organization of the human tubulin beta 2 gene (accession number X02344); coding region of the tubulin gene interspersed with (at least) three introns. The intron numbers, their sizes (in nucleotides) as well as their positions within the human tubulin are indicated; the numbers refer to the corresponding cDNA (accession number NM_006088; length 1338 nts). B. Phylogenetic tree (slanted cladogram), constructed after alignment of the sequences for intron-2 from the tubulin genes of the freshwater sponges [FW-sponge] S. lacustris (SLAC$TUI2), L. baikalensis (LBAI$TUI2), B. intermedia (BINT$TUI2), B. recta (BREC$TUI2), B. bacillifera (BBAC$TUI2), S. papyracea (SPAP$TUI2) and the corresponding sequence from the marine demosponge S. domuncula (SDOM$TUI2), which was used as outgroup. The analysis was performed by neighbour-joining. The numbers at the nodes indicate the level of confidence (in percent) for the branches after 1000 bootstrap replicates. Modified after Schrder et al. (2003a) and Mller et al. (2006c).

surfaces of the branches and have diameters of 3-4 mm. The spicules of L. baikalensis are slightly curved amphioxea which are covered by many spines (Fig. 4A and B); they measure 150-210 m in length and 8-15 m in diameter (Dybowski 1880, Masuda et al. 1997). The spicules of Lubomirskia are other than in the related genus Baikalospongia embedded into a horny sheet (Annandale 1914). This organic support allows a regular construction of the bundles within the sponge tissue. X-ray analysis revealed a highly ordered arrangement of the 150 m to 220 m long spicules within the body into longitudinal bundles. These structures are to some extent interrupted by transversal lines which demarcate 1 cm strong growth annuli (Kaluzhnaya et al. 2005a). Longitudinal cross-sections through the skeleton of L. baikalensis show the pronounced apical-basal organization of the branches into approximately 1 cm long modules (Wiens et al. 2006) determined by the arrangement of spicule bundles. The ascending, longitudinal bundles of one module originate from the annulus, a concavely curved demarcation zone between modules which is formed by a dense fusiform arrangement of the bundles. Annuli are composed of a ramified network of longitudinal and traverse bundles which appear very bright in cross-sections (Fig. 5A and B). Within the branches, the spicules are embedded in an organic matrix which fixes them in the ordered skeleton (Fig. 5C). At the uppermost tips of the branches the spicules protrude freely into the environment (Fig. 5D).

Enzymatic activities of L. baikalensis axial filaments


According to earlier studies (Cha et al. 1999, Krasko et al. 2000) the axial filament is composed of silicatein which causes a polymerization of silica from monomeric forms, like from tetraethylorthosilicate (TEOS). The filaments from L. baikalensis were incubated with TEOS (Belikov et al. 2005, Mller et al. 2006d); after a reaction period of 180 min the filaments were washed and analyzed by SEM. The images revealed that the smooth surfaces from the rhomboid to cylindrical axial filaments (Fig. 4C-a) found immediately after isolation from the spicules, changed after incubation with TEOS and were decorated with silica lumps (Fig. 4Cb). In a second approach to demonstrate that the axial filaments catalyze the formation of biosilica from monomeric TEOS, the filament samples were stained with Rhodamine 123 and inspected by fluorescence microscopy. At time zero, only the filaments are stained (Fig. 4D), while after 30 min (Fig. 4E) or after 180 min the filaments are surrounded with silica depositions which are intensively stained (Fig. 4F). It is known (see: Sumerel and Morse 2003, Mller et al. 2003) that the silicatein proteins share high sequence similarity with the cathepsins L. Therefore, we investigated by an in situ detection assay for proteinases whether silicatein displays in addition to its ability to synthesize polymeric biosilica also proteolytic activity (see: Mller et al. 2006d). At the beginning of the incubation period with a synthetic substrate no staining is seen, while after an incubation for 30 min a distinct color reaction proceeds which is first seen at the surfaces of the filaments and later more diffusely also

sequences from other endemic sponge species of Lake Baikal, especially those living in the deep water, below 30 m.

Morphological characteristics of L. baikalensis


The Lubomirskiidae are one major family of endemic sponges in Lake Baikal (Masuda et al. 1997). The species L. baikalensis is found on every hard bottom down to at least 15 m (Efremova 2001); as reported by Savarese et al. (1997) it is one of the most abundant species in the littoral-zone. The initial description of this species came from Pallas (1776), with the precise description by Dybowski (1880) (Fig. 1A). In some locations L. baikalensis grows to more than 120 cm high specimens with dichotomous branches between 1 and 3 cm in diameter (Fig. 1B). The oscules are located at the lateral

183

Fig. 3: Phylogenetic relationship of the silicateins, the enzyme which catalyzes the polymerization process of biosilica in the sponge spicules. Four deduced silicatein sequences of the isoform silicatein- [-1, -2, -3 and -4] from L. baikalensis (SILICAa1_LUBAI, accession number AJ872183; SILICAa2_LUBAI, AJ968945; SILICAa3_LUBAI, AJ968946; SILICAa4_LUBAI, AJ968947) and the two cathepsin L sequences (CATL1_LUBAI, AJ96849; CATL2_LUBAI, AJ968951) were aligned with silicatein- from S. domuncula (SILICAa_SUBDO; CAC03737.1) from Tethya aurantia [T. aurantium] (SILICAa_TETYA, AAD23951) and with the -isoenzymes from S. domuncula (SILICAb_SUBDO, AJ547635.1) and T. aurantia (SILICAb_TETYA, AF098670), as well as with the cathepsin L sequences from sponges S. domuncula (CATL_SUBDO, AJ784224), G. cydonium (CATL_GEOCY, Y10527) and Aphrocallistes vastus (CATL_APHRVAS, AJ968951); and the related papain-like cysteine peptidase XBCP3 from Arabidopsis thaliana (PAPAIN_ARATH, AAK71314) [outgroup]. Additionally the deduced silicateins from the cosmopolitan freshwater sponges E. fluviatilis (SILCA1_EPHYDAT and SILCA2_EPHYDAT) and S. lacustris (SILCA_SPONGILLA) are included in this analysis. The numbers at the nodes are an indication of the level of confidence for the branches as determined by bootstrap analysis [1000 bootstrap replicates]. The slanted phylogenetic tree was constructed after the alignment of these sequences and had been rooted with the plant enzyme (papain from A. thaliana) as an outgroup.

as patches around them. Prolonged incubation for 120 min results in a strong increase of the color reaction. In parallel experiments it was established that the axial filaments of L. baikalensis show no proteolytic activity if the substrate for elastase was applied.

Intron/exon structures of sponge silicatein and cathepsin L genes


The cDNAs from the sponge silicateins and cathepsins L were used to construct the primers to clone the complete genes. Interestingly the intron/exon structures of the genes for these two groups of proteins are very similar among the sponges and characteristically different from other metazoan proteins. The two genes from L. baikalensis for silicatein and cathepsin L are shown here (Fig. 6A); the other new genes are in the data base.

nts (accession number AJ872183). Six introns which are delimited by characteristic donor splice sites and acceptor splice sites are located between nt274 to nt344 (intron-1; phase 0), nt498 to nt820 (intron-2; 0), nt936 to nt1313 (intron-3; 1), nt1424 to nt1501 (intron-4; 0), nt1655 to nt1728 (intron-5; 0) and nt1848 to nt1930 (intron-6; 2).

The cathepsin L gene from L. baikalensis


The 2,149 nts long sequence, which encompasses the ORF of LBCATL (accession number AJ872184) comprises the following introns: between nt82 to nt298 (intron-1; phase 0), nt443 to nt619 (intron-2; 0), nt773 to nt988 (intron-3; 0), nt1104 to nt1187 (intron-4; 1), nt1298 to nt1553 (intron-5; 0), nt1710 to nt1830 (intron-6; 0) and nt1950 to nt2043 (intron-7; 2).

The silicatein- gene from L. baikalensis


The full-length sequence comprising the ORF (nt43 to nt2027) of the corresponding cDNA, LBSILICA, has 2,040

The silicatein- gene from S. domuncula


The ORF is located from nt63 to nt2271 within the SDSILICAa gene. The six introns are found between nt303 to nt510 (intron1; phase 0), nt667 to nt832 (intron-2; 0), nt948 to nt1085 (intron-3;

184

Fig. 4: Spicules and axial filament/silicatein from L. baikalensis. A. SEM micrograph of amphioxea. B. Broken spicules show the axial canal (ac); SEM analysis. C. Axial filaments which are composed of silicatein (SEM). One axial filament is shown immediately after isolation from a spicule (C-a), or after incubation with TEOS for 180 min (C-b). The time-dependent formation of biosilica from TEOS and mediated by silicatein is shown after staining with Rhodamine 123 (D - F). The silica production was analyzed with the dye Rhodamine 123 at time zero (D), or after the incubation period of 15 min (E) or 180 min with (F) with TEOS. The reaction products are visualized by immunofluorescence microscopy; they are deposited around the axial filaments.

1), nt1196 to nt1368 (intron-4; 0), nt1522 to nt1896 (intron-5; 0) and nt2016 to nt2171 (intron-6; 2).

The silicatein- gene from S. domuncula


The 1,740 long gene includes the complete ORF which is located from nt63 to nt1725 within the SDSILICAb gene. The five introns are located as follows: between nt390 to nt469 (intron-1; phase 0), nt683 to nt737 (intron-2; 0), nt853 to nt907 (intron-3; 1), nt1033 to nt1289 (intron-4; 0) and nt1562 to nt1628 (intron-5; 2).

The cathepsin L gene from S. domuncula


The intron/exon borders of cathepsin L exist in the mature enzyme from S. domuncula between aa130 and aa131 (phase 0), within aa165 (phase 1), between aa201 and aa202 (phase 0), aa252 and aa253 (phase 0) and within aa292 (phase 2).

Conservation of the intron/exon borders of sponge silicatein and cathepsin L genes


The comparison of the intron/exon borders of sponge silicateins with those of cathepsins L from sponges and other Metazoa and with the plant cysteine protease from A. thaliana shows a characteristic distribution/conservation within the

mature deduced enzymes. While in the sponge enzymes, both in the silicateins- and -, five introns are found, the human, insect and nematode cathepsin L genes have less introns. In Fig. 6 the introns within the mature enzyme region in all sequences are indicated consecutively by small letters; e.g. intron-a corresponds to intron-2 in the LBSILICA gene, or to intron-3 in LBCATL gene. It is obvious that intron-a in all sponge silicatein and cathepsin genes is of phase 0, intronb of phase 1, intron-c of phase 0, intron-d of phase 0 and intron-e of phase 2 and they are all located at the same sites within the deduced coding segments (Fig. 6A). In the human cathepsin L gene intron-b is missing, while all other introns exist at the same site within the gene. In D. melanogaster none of the introns exists within the gene region encoding the mature cathepsin L. The only existing unusual intron (Le Boulay et al. 1998) in this region of the cathepsin L gene does not match with any other intron discussed here (Fig. 6A and B). Interestingly enough intron-a, intron-c and intron-d exist at the same sites in sponges as the corresponding introns in the human sequence. This comparison underlines once more that the intron/exon borders and even their phases are highly conserved among the Metazoa. The number of introns is lower in the protostomians D. melanogaster and C. elegans in comparison to sponges and human (Fig. 6B). Moreover

185

Fig. 5: Microscopical analysis of the skeleton of L. baikalensis. SEM images through a longitudinal section of a branch. A. The lower magnification shows the two modules (mo) which are separated from each other by an annulus (an). (B to D) At higher magnification it is seen that the longitudinal bundles (lo) are fortified by traverse bundles (tr). The top of one branch is marked (to). Size bars are given. Modified according to Wiens et al. (2006).

the presence of the comparatively high number of the introns in the plant sequence underscores the phylogenetically conserved relationship of the cysteine proteases.

Consequence: Joint Russian-German Laboratory


Furthermore, the lesson of history has been learnt and transformed to a sustainable collaboration between scientists of two nations by the foundation of the Joint RussianGerman Laboratory for Biology of Sponges in Irkutsk. Through the interlinking of the expertise of the two groups headed by S.I. Belikov, O.V. Kaluzhnaya [Irkutsk] and W.E.G. Mller, H.C. Schrder [Mainz] under the umbrella of academician M.A. Grachev a tradition, which had been started by German scientists e.g. Gmelin, Pallas and von Humboldt, is continued and will contribute to a sustainable social and commercial progress in an under-populated area, which is so extremely rich in natural resources. Focusing on the scientific biotechnological issue, biosilica will perhaps even dominate the present day high economical trade factor, e.g. oil/gas or metals. The treasure of Lake Baikal is larger, it is a unique place where (i) evolution in action can be studied, (ii) a unique and conserved climate weather situation exists, which will provide us with early warning markers for the potential present day global warming process, (iii) solid methane, a powerful greenhouse gas that is also a valuable fuel to generate mechanical and electrical energy. (iv) More biotechnological innovations will emerge, e.g. those coming from other biosilica forming organisms, e.g. diatoms (Popovskaya et al. 2002). Needless to say, that the Joint

General conclusion
In summary, sponge silicatein, here with the example L. baikalensis silicatein, catalyzes biosilica formation from monomeric silicon alkoxides. The in situ analyses showed an impressive deposition of biosilica. Even though the catalytic triad of the enzyme is changed from cysteine to serine (in the first amino acid of the triad) the molecule exhibits also proteolytic activity towards a substrate which is specific for cathepsin L. This biochemical result is supported by cDNA analysis and the elucidation of the intron/exon structure of the genes, showing the high sequence similarity between these two groups of enzymes. The data also show that in parallel with the emergence of the silicateins the number of introns increased in Porifera, the oldest phylum which branched off from the common metazoan ancestor, the Urmetazoa. This study will also contribute to the development of new strategies to chemically modify the active sites of the silicateins/cathepsins in the direction to change their enzymic properties.

186

Fig. 6: A. Comparison of the intron/exon borders (}{In-1 to 5) of the silicatein genes from the sponges L. baikalensis (silicatein- from L. baikalensis (SILCAa_LUBAI, truncated from aa 1-110; accession number AJ877018) and S. domuncula (silicatein- (SILCAa_SUBDO, aa1-114; CAC03737.1 and silicatein- SILCAb_SUBDO, aa 1-162), with the related cathepsin L genes from L. baikalensis (CATL_LUBAI, aa 1-108; AJ877019) and S. domuncula (CATL_SUBDO, aa 1-108) and also from human (CATL_HUMAN, aa 1-114; HGNC:2537MIM:116880), D. melanogaster (CATL_DROME, aa 1-124; CG6692-FBgn0013770) and C. elegans (CATL_CAEEL, aa 1-120; 2B354K02E7.10). From the protein sequences shown, the propeptides have been truncated. The characteristic sites of the catalytic triad and, the serine cluster and the cleavage site of the signal peptide are marked. The sites of the introns are marked and numbered consecutively with small letters (In-a to In-e). B. For the slanted phylogenetic tree the above sequences were aligned with the cysteine protease from A. thaliana (CyP_ARATH; BAB08269.1). The sponge cathepsins (CATL) and the silicateins (SILCA) are in gray. If present, the introns with the respective numbers within the range of the mature polypeptides are given.

187

Laboratory is open for the scientific community and surely will soon attract also the commercially directed institutions. At present the focus of the Laboratory centers around the topic sponge biosilica. The proof on concept of biosilica for the application in nano-biotechnology applicable in biomedicine has been documented. The rich presence of silicatein genes in Lake Baikal endemic sponges, with L. baikalensis as a model, has been recognized, their genes analyzed and the product of this key molecule [silicatein] has been secured. Biosilica can be synthesized by recombinant technologies, and the material will have pronounced impact in biomedicine, since it is biocompatible, biodegradable and mechanically stabile. To close with von Humboldt (1844): analyze the richness of Siberia to a global view and exploit those for the technological development and hence for the prosperity of the population.

Acknowledgements
This work was supported by grants from the European Commission, the Deutsche Forschungsgemeinschaft, the Bundesministerium fr Bildung und Forschung Germany [project: Center of Excellence BIOTECmarin], WTZ Germany - Russia (German-Russian cooperation through the BMBF [Founding of the Joint RussianGerman Laboratory for Biology of Sponges, Irkutsk]) and the International Human Frontier Science Program, as well as by a grant from the Presidium of the Russian Academy of Science (no. 25.5) and from RFBR (no. 03-04-4985).

References
Annandale N (1913) Notes on some sponges from Lake Baikal in the collection of the Imperial Academy of Science [St. Petersburg]. Ann Mus Zool Acad Sci St. Petersburg 18:18-101 Belikov SI, Kaluzhnaya OV, Schrder HC, Krasko A, Mller IM, Mller WEG (2005) Metabolism of spicules from the Baikalian sponge Lubomirskia baicalensis. Cell Biol Intern 29: 943-951 Brasier M, Green O, Shields G (1997) Ediacarian sponge spicule clusters from southwest Mongolia and the origins of the Cambrian fauna. Geology 25: 303-306 Bukharov AA, Fialkov VA (2001) Baikal in numbers. Irkutsk: Baikal Museum Cha JN, Shimizu K, Zhou Y, Christianssen SC, Chmelka BF, Stucky GD, Morse DE (1999) Silicatein filaments and subunits from a marine sponge direct the polymerization of silica and silicones in vitro. Proc Natl Acad Sci USA 96: 361-365 Dybowski W (1880) Studien ber die Spongien des russischen Reiches mit besonderer Bercksichtigung der Spongien-Fauna des Baikal-Sees. Mm Acad Sci St Petersburg sr 7, 27:1-71 Efremova SM (2001) Porifera. Index of animal species inhabiting Lake Baikal and its catchment area. Novosibirsk: Nauka, - Vol. 1: Lake Baikal, Book 1; pp. 182-192 Efremova SM (2004) New genus and new species of sponges from family Lubomirskiidae Rezvoy, 1936. Index of Animal species inhabiting Lake Baikal and its catchment area. - Novosibirsk: Nauka, - Vol. 1: Lake Baikal, Book 2; p.p.1261-1278 Grachev MA (2002) On the current state of Baikal ecosystem. Novosibirsk: Publishing House SD RAS Hoffman PF, Kaufman AJ, Halverson GP, Schrag DP (1998) A Neoproterozoic snowball earth. Science 281: 1342-1346

Itskovich VB, Belikov SI, Efremova SM, Masuda Y (1999) Phylogenetic relationships between Lubomirskiidae, Spongillidae and some marine sponges according partial sequences of 18S rDNA. Memoir Queensl Mus 44: 275-280 Kaluzhnaya OV, Belikov SI, Schrder HC, Rothenberger M, Zapf S, Kaandorp JA, Borejko A, Mller IM, Mller WEG (2005a) Dynamics of skeletal formation in the Lake Baikal sponge Lubomirskia baicalensis. Part I: Biological and biochemical studies. Naturwissenschaften 92 128133 Kaluzhnaya OV, Belikov SI, Schrder HC, Rothenberger M, Zapf S, Kaandorp JA, Borejko A, Mller IM, Mller WEG (2005b) Dynamics of skeletal formation in the Lake Baikal sponge Lubomirskia baicalensis. Part II: Molecular biological studies. Naturwissenschaften 92: 134-138 Kashiwaya K, Tanaka A, Sakai H, Kawai T (2000) Paleoclimatic signals printed in Lake Baikal sediments. In: Minoura K (ed). Lake Baikal. Elsevier, Amsterdam. pp 53-70 Kozhov MM (1972) Essays on Baikalology. East-Siberian Publishers, Irkutsk (In Russian) Krasko A, Batel R, Schrder HC, Mller IM, Mller WEG (2000) Expression of silicatein and collagen genes in the marine sponge Suberites domuncula is controlled by silicate and myotrophin. Europ J Biochem 267: 4878-4887 Krasko A, Schrder HC, Batel R, Grebenjuk VA, Steffen R, Mller IM, Mller WEG (2002) Iron induces proliferation and morphogenesis in primmorphs from the marine sponge Suberites domuncula. DNA Cell Biol 21: 67-80 Le Boulay C, Sellos D, Van Wormhoudt A (1998) Cathepsin L gene organization in crustaceans. Gene 218: 77-84 Maldonado M, Carmona MC, Uriz MJ, Cruzado A (1999) Decline in Mesozoic reef-building sponges explained by silicon limitations. Nature 401: 785-788 Manconi R, Pronzato R (2000) Suborder Spongillina subord. nov.: freshwater sponges. In: Hooper JNA, van Soest RWM (eds). Systema Porifera: a guide to the classification of sponges. Kluwer Academic/Plenum Publishers, New York. pp 921-1019 Masuda Y, Itskovich VB, Veinberg EV, Efremova SM (1997) Studies on the taxonomy and distribution of freshwater sponges in the Lake Baikal. In: Miyzaki N (ed). Animal community, environment and phylogeny in Lake Baikal. Otsuchi Marine Center/Ocean Research Institute, Tokyo. pp 21-41 Mayr E (2001) What evolution is. Basic Books, Tokyo Mehl D, Mller I, Mller WEG (1998) Molecular biological and palaeontological evidence that Eumetazoa, including Porifera (sponges), are of monophyletic origin. In: Watanabe Y, Fusetani N (eds). Sponge Science - Multidisciplinary Perspectives. SpringerVerlag, Tokyo. pp 133-156 Mller WEG, Zahn RK, Maidhof A (1982) Spongilla gutenbergiana n.sp., ein Swasserschwamm aus dem Mittel-Eozn von Messel. Senckenbergiana lethaea 63: 465-472 Mller WEG (1998) Origin of Metazoa: sponges as living fossils. Naturwissenschaften 85: 11-25 Mller WEG, Krasko A, Le Pennec G, Steffen R, Ammar MSA, Wiens M, Mller IM, Schrder HC (2003) Molecular mechanism of spicule formation in the demosponge Suberites domuncula: silicatein - collagen - myotrophin. Progr Molec Subcell Biol 33: 195-221 Mller WEG, Belikov S I, Tremel W, Perry CC, Gieskes WWC, Boreiko A, Schrder HC (2006a) Siliceous spicules in marine

188

demosponges (example Suberites domuncula). Micron 37: 107120 Mller WEG, Kaluzhnaya OV, Belikov SI, Rothenberger M, Schrder HC. Reiber A, Kaandorp JA, Manz B, Mietchen D, Volke F (2006b) Magnetic resonance imaging of the siliceous skeleton of the demosponge Lubomirskia baicalensis. J Struct Biol 153: 3141 Mller WEG, Schrder HC, Wrede P, Kaluzhnaya OV, Belikov SI (2006c) Speciation of sponges in Baikal-Tuva region (an outline). J Zool Syst Evol Res 44: 105-117 Mller WEG, Gamulin V, Kaluzhnaya OV, Schrder HC, Grebenjuk VA, Borejko A, Mller IM, Krasko A, Belikov SI (2006d) Silicateins, the major biosilica forming enzymes present in demosponges: protein analysis and phylogenetic relationship. Gene 395:62-71. Ovchinnikova NS (ed). (2005) Atlas of Lake Baikal: past-presentfuture. Omskaya kartographicheskaya Fabr, Omsk Pallas PS (1771-1776) Reise durch die verschiedenen Provinzen des russischen Reiches. Theil 3-Buch 2. Kayserliche Academie der Wissenschaften, St. Petersburg Pile AJ, Patterson MR, Savarese M, Chernykh VI, Fialkov VA (1997) Trophic effects of sponge feeding within Lake Baikals littoral zone. 2. Sponge abundance, diet, feeding efficiency, and carbon flux. Limnol Oceanogr 42: 178-184 Popovskaya GI, Genkal SI, Likhoshway YV (2002) Diatoms of the plankton of Lake Baikal. Nauka, Novosibirsk Savarese M, Patterson MR, Chernykh VI, Fialkov VA (1997) Trophic effects of sponge feeding within Lake Baikals littoral zone. 1. In situ pumping rate. Limnol Oceanogr 42: 171-178 Schcke H, Mller IM, Mller WEG (1994) Tyrosine kinase from the marine sponge Geodia cydonium: the oldest member belonging to the receptor tyrosine kinase class II family. In: Mller WEG (ed). Use of aquatic invertebrates as tools for monitoring of environmental hazards. Gustav Fischer Verlag, Stuttgart, New York. pp 201-211 Schrder HC, Efremova SM, Itskovich V B, Belikov S, Masuda Y, Krasko A, Mller IM, Mller WEG (2003) Molecular phylogeny of

the freshwater sponges in Lake Baikal. J Zool Syst Evol Research 41: 80-86 Schrder HC, Perovi-Ottstadt S, Wiens M, Batel R, Mller IM, Mller WEG (2004) Differentiation capacity of the epithelial cells in the sponge Suberites domuncula. Cell Tissue Res 316: 271-280 Schulze FE (1904) Hexactinellida. Wissenschaftliche Ergebnisse der Deutschen Tiefsee-Expedition; vol 4. Fischer-Verlag, Jena Scientific Council (1993) Atlas of Baikal. Federal Agency of Geodesy and Maps of Russia, Moscow Shimizu K, Cha J, Stucky GD, Morse DE (1998) Silicatein alpha: cathepsin L-like protein in sponge biosilica. Proc Natl Acad Sci USA 95: 6234-6238 Steiner M, Mehl D, Reitner J, Erdtmann BD (1993) Oldest entirely preserved sponges and other fossils from the Lowermost Cambrian and a new facies reconstruction of the Yangtze Platform (China). Berl Geowiss Abhand (E) 9: 293-329 Sumerel JL, Morse D (2003) Biotechnological advances in biosilicification. Progr Molec Subcell Biol 33: 225-247 Timoshkin OA (1997) Biodiversity of Baikal fauna: state-of-theart (preliminary analysis). In: Wada E, Timoshkin OA, Fujita N, Tanida K (eds). New scope on boreal ecosystems in east Siberia. DIWPA series vol. 2; Siberian Branch of the Russian Academy of Science, Novosibirsk. pp 35-76 Trguer P, Nelson DM, van Bennekom AJ, DeMaster DJ, Leynaert A, Quguiner B (1995) The silica balance in the world ocean: a reestimate. Science 268: 375-379 Walker G (2003) Snowball Earth. Bloomsbury, London Weinberg E, Weinberg I, Efremova S, Tanichev A, Masuda Y (2003) Late Pliocene spongial fauna in Lake Baikal (from material from the deep drilling core BDP-96-1). In: Kashiwaya K (ed). Long continental records from Lake Baikal. Springer Verlag, Tokyo. pp 283-293 Wiens M, Belikov SI, Kaluzhnaya OV, Krasko A, Schrder HC, Perovic-Ottstadt S, Mller WEG (2006) Molecular control of serial module formation along the apical-basal axis in the sponge Lubomirskia baicalensis: silicateins, mannose-binding lectin and mago nashi. Dev Genes Evol 216: 229-242

Porifera research: Biodiversity, innovation and sustainaBility - 2007

189

Sponges from a submarine canyon of the Argentine Sea


Marco Bertolino(1), Laura Schejter(2), Barbara Calcinai(3*), Carlo Cerrano(1), Claudia Bremec(2)
Dipartimento per lo studio del Territorio e delle sue Risorse, C.so Europa, 26, 16132, Genova, Italy. marco.bertolino75@libero.it, cerrano@dipteris.unige.it (2) Laboratorio de Bentos, Instituto Nacional de Investigacin y Desarrollo Pesquero, Paseo Victoria Ocampo 1, (B7602HSA) Mar del Plata, Argentina schejter@inidep.edu.ar, cbremec@inidep.edu.ar (3) Dipartimento di Scienze del Mare, Via Brecce Bianche, 60131, Ancona, Italy. b.calcinai@univpm.it
(1)

Abstract: During a research cruise to assess the abundance and distribution of Patagonian scallop Zygochlamys patagonica (King and Broderip, 1832), a submarine canyon was discovered using a multibeam SIMRAD EM1002 sonar. The canyon is positioned at 4335S and 5933W, close to the southern commercial scallop beds in the Argentine Sea. The existence of submarine canyons on the continental shelf of Argentina was already known, but their edaphic and biotic conditions remain unstudied. A sample of the benthic community was collected at the head of the canyon at 360 m depth. In total nine species of demosponges were identified; two of them represent new records for the Argentine Sea and two are new to science. Keywords: Argentina, new records, new species, Porifera, submarine canyon

Introduction
The existence of submarine canyons on the continental shelf of Argentina was known (Parker et al. 1997), but their exact location, number, edaphic and biotic conditions remain unstudied. During a research to assess the distribution and abundance of the Patagonian scallop Zygochlamys patagonica (King and Broderip, 1832), a submarine canyon was discovered. The Patagonian scallop is distributed in the Magellanic Biogeographical Province and its exploitation results in the destruction of associated species. Porifera are particularly affected (Schejter et al. 2006). The Argentinian sponge fauna is relatively well known; with the earliest studies commencing in the 19th century (Ridley and Dendy 1886, 1887, Sollas 1886, 1888, Schulze 1887). More recent contributions include Boury-Esnault (1973), Sar (1978), Mothes de Moraes and Pauls (1979), and particularly Cuartas (1986, 1991, 1992a, 1992b, 1992c, 1995). Despite our knowledge of the Argentinian sponge fauna, a recent paper on the Porifera associated with commercial Patagonian scallop beds reported four new records for the area (Schejter et al. 2006). Here we describe 9 species of demosponges recorded in a canyon close to scallop beds. In particular Pseudosuberites cf. antarcticus Carter, 1876 and Guitarra dendyi (Kirkpatrick, 1907) were previously known only for the Antarctic area, representing new records for the Argentine Sea, while two (Stelodoryx argentinae sp. nov. and Tedania (Tedaniopsis) sarai sp. nov.) are new to science.

Materials and methods


The canyon was discovered using a multibeam SIMRAD EM1002 sonar, during a research cruise (R/V Oca Balda, INIDEP, April 2005) for assessment of the Patagonian scallop (Zygochlamys patagonica). The canyon is positioned at 4335S and 5933W, close to the southern commercial scallop beds, in the Argentine Sea (Fig. 1). Sampling was carried out on board the R/V Oca Balda (INIDEP, National Fisheries Research and Development Institute) during April 2005, at 360 m depth. The samples were labelled OB-4-05 with the indication of the ship (Oca Balda) and the date of collection, and with progressive numbers (e.g. OB-4-05 caon 14). Specimens were frozen upon collection and fixed in 5% formaldehyde in sea water and then preserved in alcohol 70%, or dried, in the laboratory. For the study of spicules, small fragments of sponge tissue were heat-dissolved in nitric acid, rinsed in water, and dehydrated in ethanol; then spicules were mounted on microscope slides. Spicule dimensions are given as range of lengths and of widths and as average (in brackets); they were obtained measuring 20 to 30 spicules per category. Sections were cut by hand, perpendicularly and tangentially to the sponge surface, using a razor blade. For SEM analyses spicule dissociations were transferred onto stubs and sputter-coated with gold. SEM studies were carried out on a Philips XL 20 scanning electron microscope. Photographs of the specimens were taken using a Nikon Coolpix 4500.

190

Class Demospongiae Sollas, 1885 Order Spirophorida Bergquist and Hogg, 1969 Family Tetillidae Sollas, 1886 Genus Craniella Schmidt, 1870 Craniella leptoderma (Sollas, 1886) Examined material: OB-4-05: caon 14, caon 15 Sponge round or elongate, egg-shaped (Fig. 2A, B). Surface hispid, conulose. Several small oscules are located on the tip of the sponge conules (Fig. 2A). In caon 15 the surface is more regular (without conules) and more hispid (Fig. 2B). The consistency is hard. The colour is dirty white, or brown-pinkish in alcohol. Skeleton: Spicule tracts radiate from the centre of the sponge towards the surface (Fig. 2C). Spicules: Megascleres are large oxeas, fusiform; they are frequently broken. Small oxeas (Fig. 2D), straight or slightly curved, frequently centrotylote. They measure 540 (913.6) 1500 x 5 (25) 30 m. Anatriaenes 1, with thin clads (Fig. 2E). The rhabdomes are frequently broken; the clads measure 100 (150) 200 x 15 (18) 20 m. Anatriaenes 2, with thick and short clads 70 (105) 120 x 20 (32.5) 40 m (Fig. 2F). Rhabdomes are frequently broken. Protriaenes 1, with rhabdomes that reach more than 8 cm in length, while the clads are 40 (140.5) 200 m long (Fig. 2G). Protriaenes 2 are filiform, up to 1 cm long (Fig. 2H) and with a clad longer than the others; the long clads are 26 (32.5) 44 m long and the short ones 10 m long. Microscleres are spinispires (Fig. 2I); they measure 5 (7.8) 10 m. Distribution: Antarctic shores, South Georgia, Straits of Magellan, Malvinas, Kerguelen and Heard Islands (Sar et al. 1992), South Shetland Islands (Ros et al. 2004), Chile, Atlantic coast of South America (mouth of the Rio de la Plata) (Desqueyroux-Fandez 1989). Remarks: This species has a highly variable habitus; the body is round, elongate egg-shaped (Koltun 1964) or massive, spherical (Desqueyroux-Fandez 1989); the surface varies from even and smooth, to more or less bristly (Koltun 1964), to strongly hispid (Desqueyroux-Fandez 1989). Order Hadromerida Topsent, 1894 Family Suberitidae Schmidt, 1870 Genus Pseudosuberites Topsent, 1896 Pseudosuberites cf. antarcticus Carter, 1876 Examined material: OB-4-05: caon 6, caon 10 Massive sponge (Fig. 3A) with a cavernous structure covered by remains of a thin membrane easily detachable from the sponge body. The consistency is soft. The colour is beige-grey in alcohol. The sample caon 6 hosted numerous samples of the bivalve Hiatella solida (Sowerby, 1834). Skeleton: The ectosomal skeleton is made of tylostyles tangentially arranged (Fig. 3B); the choanosomal skeleton is made of well defined spicule tracts running towards the surface (Fig. 3C). Spicules: Tylostyles and subtylostyles (Fig. 3D-H), often slightly curved. They have well formed heads (Fig. 3H),

Fig. 1: Location of the canyon and of the sampling station in the Atlantic Ocean, Argentine Sea, Argentina.

The present collection is preserved at the Laboratorio de Bentos, Instituto Nacional de Investigacin y Desarrollo Pesquero (INIDEP). The type materials of the new species here described are deposited at the Museo Civico di Storia Naturale G. Doria of Genoa (MSNG).

Results
In total nine species were identified. Craniella leptoderma (Sollas, 1886), Myxilla (Myxilla) mollis Ridley and Dendy, 1886, Tedania (Tedaniopsis) charcoti Topsent, 1907, Tedania (Tedaniopsis) massa Ridley and Dendy, 1886 and Tedania (Trachytedania) mucosa Thiele, 1905 are already known for the area. Pseudosuberites cf. antarcticus Carter, 1876 and Guitarra dendyi (Kirkpatrick, 1907) were previously known only for the Antarctic area and so they represent new records for the Argentine Sea. Stelodoryx argentinae sp. nov. and Tedania (Tedaniopsis) sarai sp. nov. are new for science. All species dealt with are described below.

191

Fig. 2: A, B. Specimens of Craniella leptoderma (Sollas, 1886); C. Radial skeleton; D. Small oxeas; the arrow points the central tyle; E. Anatriaene 1; F. Anatriaene 2; G. Protriaene 1; H. Protriaene 2; I. Spinispires.

sometimes sub-terminal (Fig. 3G) and acerate tips (Fig. 3E). They measure 350 (1063.6) 1350 x 5 (15.6) 25 m. Distribution: Antarctic shores, Heard and Kerguelen Islands (Koltun 1964). Remarks: This specimen is easily recognisable as Pseudosuberites antarcticus Carter, 1876 in spicule characteristics (comparable size and shape), but it differs from the holotype as well as from other records of the species, in the habitus. Pseudosuberites antarcticus was previously reported as erect and ramified (Ridley and Dendy 1887, Topsent 1902). This represents a new record for the Argentine Sea that enlarges the known distribution of this species northwards.

Order Poecilosclerida Topsent, 1894 Suborder Myxillina Hajdu, van Soest and Hooper, 1994 Family Myxillidae Dendy, 1922 Genus Myxilla Schmidt, 1862 Myxilla (Myxilla) mollis Ridley and Dendy, 1886 Examined material: OB-4-05: caon 3 Massive sponge with an irregular surface and an irregular system of cavities (Fig. 4A). No dermal membrane was present. The sponge is compressible and elastic. The colour is beige in alcohol. Skeleton: The ectosomal skeleton is absent. The choanosome is a loose reticulation of smooth styles and anisotylotes (Fig.

192

Fig. 3: A. Specimen of Pseudosuberites cf. antarcticus Carter, 1876; the arrow points the thin membrane; B. Ectosomal skeleton; C. Choanosomal skeleton; D, F, H. Heads of tylostyles; E. Tip of a tylostyle; G. Subtylostyle.

4B). Microscleres are abundantly scattered all over the choanosome. Spicules: Megascleres: Smooth, slightly curved styles (Fig. 4C). Rare subtylostyles. They measure 325 (407.5) 437.5 x 12 (14) 15 m. Anisotylotes are straight or slightly sinuous, with swollen and microspined extremities (Fig. 4D). They measure 225 (250) 275 x 6 m. Microscleres: Spatuliferous anchorate isochelas 1, with three teeth, slightly curved (Fig. 4E). They measure 24 (35.4) 40 m. Spatuliferous anchorate

isochelas 2 of similar shape (Fig. 4F). They measure about 20 m. Sigmas 1 are C- or S- shaped (Fig. 4G, H). They measure 40 (51) 60 m. Sigmas 2 are C- shaped (Fig. 4F). They measure 15 (25) 30 m. Distribution: West and East coast of South America; Malvinas Islands (Desqueyroux-Fandez and van Soest 1996); Antarctic shores; Strait of Magellan, Kerguelen Island (Sar et al. 1992).

193

Fig. 4: A. Specimen of Myxilla (Myxilla) mollis Ridley and Dendy, 1886; B. Choanosomal skeleton; C. Smooth style; D. Anisotylote; E. Spatuliferous anchorate isochela 1; F. Spatuliferous anchorate isochela 2; G. Sigma 1; H. Sigma 1 and 2 (arrow).

Remarks: This specimen has entirely smooth styles as originally described by Ridley and Dendy (1886) and two categories of sigmas and isochelas as reported by Desqueyroux-Fandez and van Soest (1996). Genus Stelodoryx Topsent, 1904 Stelodoryx argentinae sp. nov. Examined material: OB-4-05: caon 4 Holotype MSNG 54057 Comparative material: holotype of Myxilla cribrigera Ridley and Dendy 1886, Natural History Museum, London (Challenger coll. BMNH: 87.5.2.138.) The species consists of a single specimen about 6.5 cm long, massive, with an irregular surface (Fig. 5A). Some oscules, slightly elevated (0.5-1 mm), 1-2 mm in diameter are visible. The consistency is soft and elastic when alive, fragile and friable in the dried state. The colour is black in alcohol and dried. The sponge includes a large amount of sand. Skeleton: The ectosomal skeleton consists of brushes of anisostrongyles with spined ends (Fig. 5B). A thin tangential, dermal membrane is present (Fig. 5B). The choanosomal skeleton (Fig. 5C) is a paucispicular reticulum of main styles, thin styles and anchorate chelae. Spicules: Megascleres are smooth styles (Fig. 5D), straight or slightly curved with acerate or conical tips; they measure 287.5 (351) 412.5 x 10 (13) 15 m. Straight, thin styles are 188.7 (220.5) 260 x 2.6 m (Fig. 5E). Straight anisostrongyles (Fig. 5F), with finely spined extremities (Fig, 5G, H); the

diameter of the spicule decreases from an extremity to the other. They measure 209 (240) 262.5 x 5 (7.8) 10 m. Microscleres are polydentate, spatuliferous isochelae (Fig. 5I) with five teeth. They measure 40.8 (52.4) 65 m. A few unguiferous anchorate isochelae are present (Fig. 5J). Etymology: The name of this species refers to the sea of origin. Remarks: Among the 11 known species of Stelodoryx, S. cribrigera (Ridley and Dendy, 1886), reported also for the Malvinas Islands is very close to the specimen described here. The study of the holotype of S. cribrigera has shown some differences: the species of Ridley and Dendy is characterized by tylotornotes with slight swelling on the tips. Our species has anisostrongyles with different tips, never swollen. Moreover the species of Ridley and Dendy has larger styles (650 x 25 m) and lacks thin styles. In the skeleton preparation of the holotype an ectosomal layer of isochelas is evident (also reported by Desqueyroux-Fandez and van Soest (1996) from additional material), but it is not present in our species. Family Tedaniidae Ridley and Dendy, 1886 Genus Tedania Gray, 1867 Subgenus Tedaniopsis Dendy, 1924 Tedania (Tedaniopsis) charcoti Topsent, 1907 Examined material: OB-4-05: caon 1 Massive sponge with conulose surface, uneven and with numerous oscules evident. The sponge is soft and brownish in alcohol (Fig. 6A).

194

Fig. 5: A. Stelodoryx argentinae sp. nov.: Holotype; B. Ectosomal skeleton; the arrow points the thin dermal membrane; C. Choanosomal skeleton; D. Smooth style; E. Thin style; F. Straight anisostrongyle; G, H. tips of an anisostrongyle; I. Spatuliferous anchorate isochela 1; J. Unguiferous anchorate isochela 2.

Skeleton: Ectosomal skeleton consists of tangentially disposed anisotornotes which form a loose reticulum. The choanosome is an irregular and confused reticulation of longitudinal tracts of styles, and free onychaetes (Fig. 6B).

Spicules: Megascleres are smooth styles, slightly curved (Fig. 6C). They measure 412.5 (432) 462.5 x 10 m. Smooth, mucronate, straight anisotornotes (Fig. 6D). They measure 262.5 (292) 312.5 x 5 m. Microscleres (Fig. 6E): Onychaetes

195

Fig. 6: A. Specimen of Tedania (Tedaniopsis) charcoti Topsent, 1907; B. Choanosomal skeleton; C. Smooth style; D. Anisotornotes; E. Onychaetes 1 and onychaetes 2.

1 measure 200 (278.6) 462.5 x 2 m. Onychaetes 2 measure 50 (56.6) 60 m. Distribution: Antarctic shores, South Georgia, South Sandwich, South Orkney, Strait of Magellan; Kerguelen and Malvinas Islands (Sar et al. 1992). Argentine Sea (Mar del Plata), Chile (Desqueyroux and Moyano 1987, Cuartas 1992b). South Shetland Islands (Ros et al. 2004). Remarks: This is a very common species frequently collected in the area. In Desqueyroux-Fandez and van Soest (1996), this species is well described. Our material fits very well with the description provided by these authors. Tedania (Tedaniopsis) massa Ridley and Dendy, 1886 Examined material: OB-4-05: caon 11, caon 13 The specimen consists of two small fragments of a massive, lobose sponge (Fig. 7A). The surface is irregular and minutely hispid. The consistency is soft. The colour is beige.

Skeleton: In the ectosome tornotes project towards the surface in divergent brushes (Fig. 7B). The choanosome is a loose reticulum of styles and fibres of onychaetes. These run to the surface, anastomosing and connecting to secondary fibres. Spicules: Megascleres are smooth styles slightly to strongly curved (Fig. 7C). They measure 400 (440) 500 x 15 (16) 20 m. Mucronate anisotornotes are 287.5 (373) 500 x 10 m (Fig. 7D). Microscleres (Fig. 7E): Onychaetes 1 measure 437.5 (480) 660 m. Onychaetes 2 measure 45 (67.8) 85 m. Distribution: South Atlantic Ocean: from Uruguay to the Strait of Magellan, Argentina (Mar del Plata) (Mothes and Pauls 1979, Cuartas 1992b, 1992c). Antarctic shores, South Georgia, Malvinas Islands (Sar et al. 1992). Tedania (Tedaniopsis) sarai sp. nov. Examined material: OB-4-05: caon 8; Holotype MSNG 54058

196

Fig. 7: A. Fragments of Tedania (Tedaniopsis) massa Ridley and Dendy, 1886; B. Ectosomal skeleton made of tornotes in divergent brushes (arrow) and loose reticulum of styles of the choanosome; C. Smooth style; D. Mucronate anisotornotes; E. Onychaetes 1 and onychaetes 2.

Comparative material: holotype of Tedania armata Sar, 1978, Museo Civico di Storia Naturale G. Doria of Genoa (Ant. 3). Specimen of Tedania lanceta Koltun, 1964, identified by V. M. Koltun; pictures of spicules of the specimen labelled NN 6767 and NN 6751 as the type material is missing. This species is massive, cavernous with a smooth, uneven surface (Fig. 8A). The consistency is hard. The colour is brown in alcohol, dirty grey in the dried state, clearer in the interior. Skeleton: In the ectosome anisotornotes make tangential surface tracts. The choanosome is a reticulation of smooth styles (Fig. 8B). Spicules: Megascleres: Curved, often slightly flexuous styles (Fig. 8C), with frequent lanceolate or abruptly pointed tips (Fig. 8D). The heads of the styles are elongated, blunt (Fig. 8E). The thickness of the shaft is often reduced below the elongated extremity of the style. Measurements 387.6 (430.9) 469 x 10.4 (12.3) 13 m. Straight or slightly curved, anisotornotes, with lanceolate, slightly inflated extremities (Fig. 8F, G). They measure 275.4 (319.3) 375 x 5.2 (6.8) 7.8 m. Microscleres: onychaetes 1 (Fig. 8H) with short spines (Fig. 8I); they measure 387.6 (434.4) 489.6 x 2.6 m; onychaetes 2 with different extremities (Fig. 8J) and longer spines (Fig. 8K); they measure 44 (63.7) 122 x 1 m. Etymology: Named after the late Prof. Michele Sar for his contribution to the knowledge of Porifera in general,

including an important contribution towards Argentine sponge taxonomy. Remarks: This species is very close to T. (Tedaniopsis) lanceta Koltun, 1964 and to T. armata Sar, 1978. Koltuns species differs in having stouter styles (400-480 m long and 16-22 m wide) that are not flexuous and have lanceolate tips. In the description of Koltun (1964) the anisotornotes are thicker (360-400 m long and 14-16 m wide), and moreover onychates 1 are shorter (270-320 m long). The holotype is missing so any direct comparison is not possible. Thanks to Dr. Alexander Ereskovsky (St. Petersburg State University, Russia) we had the possibility to compare the spicule complement of this species with some pictures of spicules of T. lanceta Koltun, 1964, determined by Koltun. This comparison confirmed the previous observed differences. Moreover the shape of the spicules of this specimen seems different from our species: the extremities of the anisotornotes are often asymmetrical and bent. Tedania armata Sar, 1978 was synonymised with T. charcoti by Desqueyroux-Fandez and van Soest (1996), but the authors gave no reason for this decision. We suggest the synonymy between T. armata and T. charcoti should be reconsidered on the basis of the examined material and on our experience dealing with antarctic and subantarctic sponges. Tedania charcoti has in fact true styles while in T. armata these principal spicules are subtylostyles with long and acuminate tips (Fig. 9), and also the tornotes are different from those of T. charcoti that have mucronate tips (see e.g.

197

Fig. 8: Tedania (Tedaniopsis) sarai sp. nov.: Holotype; B. Choanosomal skeleton; C. Style; D. Magnification of an abruptly pointed tip of the style; E. Magnification of the blunt head of the style; F. Lanceolate anisotornotes; G. Magnification of the lanceolate tip of the anisotornotes; H. Onychaetes 1; I. Magnification of onychaetes 1; J. Onychaetes 2; K. Magnification of onychaetes 2.

Desqueyroux-Fandez and van Soest (1996), Figs. 107-110, page 57). Comparison with the holotype of T. armata (Fig. 9A) illustrates the primary differences between this species and T. sarai sp. nov.: in T. armata the principal spicules are subtylostyles with rounded heads (Fig. 9B, C, D), while the tips are similar to those of T. sarai sp. nov. (Fig. 9B). In T. armata numerous styles modified to oxeas (Fig. 9D) and

expanded just before the distal tip, rendering the spicule lancelike (lanceolated, Fig. 9B) are common. The dimensions of the styles are also different: in T. armata these spicules are shorter and thinner (300-350 x 6-8 m, Sar (1978); 308-372 x 8 m, Desqueyroux-Fandez and van Soest (1996)). Tornotes have rounded, and mucronate tips in T. armata (Fig. 9E-H), while in our species they have lanceolate tips. The dimensions of

198

Fig. 9: A. Tedania armata. Sar, 1978: Holotype (Ant 3); B. Subtylostyle; C. Heads of subtylostyles; D. Style modified in oxea; E, F. Tornotes with rounded tips; G. Magnification of a tip of a tornote; H. Anisotornote with mucronate and rounded tips; I. Onychaete.

Fig. 10: A. Specimen of Tedania (Trachytedania) mucosa Thiele, 1905; B. Loose reticulation of tracts of styles and abundant onychaetes of the choanosomal skeleton; C. Smooth style; D. Mucronate tornote; E. Onychaetes 1 and onychaetes 2.

199

the tornotes in T. armata are also smaller. Onychaetes 1 in T. sarai sp. nov. are longer: 150-180 m (Fig. 9I). Subgenus Trachytedania Ridley, 1881 Tedania (Trachytedania) mucosa Thiele, 1905 Examined material: OB-4-05: caon 5 The sponge is massive, irregularly elongate. Surface uneven with scattered oscules (Fig. 10A). The consistency is hard, the colour light brown. Skeleton: The ectosome is a perpendicular palisade of densely arranged mucronate tornotes. The choanosomal skeleton is a loose reticulation of tracts of styles and abundant onychaetes (Fig. 10B). Spicules: Megascleres are smooth, slightly curved styles (Fig. 10C). They measure 250 (266.5) 287.5 x 12 (13) 15 m. Mucronate tornotes (Fig. 9D) measure 200 (215) 245 x 6 m. Microscleres (Fig. 10E): Onychaetes 1 measure 135 (186.5) 210 m; onychaetes 2 measure 40.8 (54) 71 m. Distribution: Malvinas Islands, Chilean coast (Desqueyroux 1972). Argentina, Mar del Plata (Cuartas 1992b, DesqueyrouxFandez and van Soest 1996); Strait of Magellan (Sar et al. 1992). Remarks: Two kinds of onychaetes are present as reported by Desqueyroux-Fandez and van Soest (1996). Suborder Mycalina Hajdu, van Soest and Hooper, 1994 Family Guitarridae Dendy, 1924 Genus Guitarra Carter, 1874 Guitarra dendyi (Kirkpatrick, 1907) Examined material: OB-4-05: caon 2 Massive, cushion-shaped sponge with uneven surface (Fig. 11A). The consistency is soft and in alcohol the colour is brick red. Skeleton: In the ectosome the skeleton is made of exotyles arranged in bouquets with apices pointing toward the surface of the body and scattered sigmas. In the choanosome strongyles are irregularly arranged. Spicules: Megascleres are rare exotyles with a spherical, wrinkled apex and a rounded base (Fig. 11B). They measure 210 (300) 400 x 10 (15) 20 m; head diameter measures 50 (65) 80 m. Straight anisostrongyles (Fig. 11C). They measure 375 (454.5) 512.5 x 6 (8) 9 m. Microscleres: placochelae (Fig. 11C-F); they measure 75 (84.5) 90 m. C-shaped sigma. They measure 10 (12) 15 m. Distribution: Antarctic shores and South Shetland Islands (Ros et al. 2004). Remarks: This is the first record of Guitarra dendyi for the Argentine Sea. The distribution of this species was limited to the Antarctic shores (Wilhem II Coast, Banzare Coast, Victoria Land) (Ros et al. 2004) and to the South Shetland

Islands, therefore its geographical range is considerably extended northwards.

Discussion
Among the nine species collected, only one (Tedania charcoti) was previously found associated with Patagonian scallop beds (Schejter et al. 2006). The present findings, including two species new for science, suggest the importance to continue the study of these deep areas, still widely unexplored. Our results extend the geographical range northwards for Pseudosuberites cf. antarcticus and Guitarra dendyi, for which species these are the first records outside the Antarctic sea. The lack of data on deep-water faunas around Antarctica has been recently highlighted by Brandt et al. (2007). These Authors evidenced how the abyssal Antarctic fauna has strong links with others oceans, mainly Atlantic, but only when taxa are good dispersers. For example, isopods, ostracods and nematodes include many species known exclusively in Antarctica differently from the foraminifera. In this way our results suggest once again the importance to study these environments to better evaluate also the relative importance of dispersal by larvae or by floating propagules (as suggested by Burton 1932) to account for a possible relationship between sponge distribution and oceanic current systems. Although not as deep as the community described by Brandt et al. (2007), there are just few studies of the Argentinian Sea waters of subantarctic origin in the continental shelf, the shelf break and submarine canyons. The Argentinian side of the Magellanic Biogeographic Province is influenced by the Malvinas Current, a relatively fresh and cold branch of the Circumpolar Current, strongly flowing northward along the continental shelf of Argentina (Garzoli 1993, Piola and Rivas 1997, Vivier and Provost 1999). However, based on evidence from faunal analysis done in the study area and also considering the shelters and dead shells found by Bremec et al. (2006), it is possible that important fluxes from the shelves to deeper oceanic waters were performed throughout submarine canyons. Fishing effort can produce several ecological consequences, for instance changes in species richness and biodiversity, loss of erect and fragile epifauna, widely damaging epibiotic community (Turner et al. 1999, Coleman and Williams 2002, Thrush and Dayton 2002). Sponges are frequently collected in the invertebrate by-catch of the Patagonian scallop fishery in the neighbouring shelf of the study area and represented approximately 510% of total community biomass (Bremec et al. 1998, 2000, Bremec and Lasta 2002, Schejter et al. 2006). Porifera biomass at Patagonian scallop beds in the Argentine Sea decreased between 1995 and 1998 in exploited areas (Bremec et al. 2000). Between 1998 and 2001, the sponge contribution in these areas represented an average of 0.3 kg / 100 m2 (wet weight) (Schejter 2004). It will be interesting to know if a long term consequence of the fishing activity will

200

Fig. 11: A. Specimen of Guitarra dendyi (Kirkpatrick, 1907); B. Light microscopy observation of an exotyle; C. Anisostrongyle and placochela (arrow); D, E, F. Placochelae.

be, owing to sponge fragmentation caused by trawling, the increase in the distribution of some sponge species in respect to other species on intermediately deep Argentinian bottoms.

Ereskovsky (St. Petersburg State University, Russia) for sending us V.M. Koltuns material for comparison.

References
Boury-Esnault N (1973) Campagne de la Calypso au large des ctes atlantiques de lAmrique du Sud (1961-1962). 29. Spongiaires. Rs Sci Camp Calypso 10: 263-295 Brandt A, Gooday AJ, Brando SN, Brix S, Brkeland W, Cedhagen T, Choudhury M, Cornelius N, Danis B, De Mesel I, Diaz RJ,

Acknowledgments
We thank A. Madirolas and G. Alvarez Colombo (Hydroacoustics Lab., I.N.I.D.E.P. Argentina) for the information and images of the Canyon and Maurizio Pansini (Dip. Te. Ris, Genoa University) for his helpful suggestions. The authors are indebted to Dr. Alexander

201

Gillan DC, Ebbe B, Howe JA, Janussen D, Kaiser S, Linse K, Malyutina M, Pawlowski J, Raupach M, Vanreusel A (2007) First insights into the biodiversity and biogeography of the Southern Ocean deep sea. Nature 447: 307-311 Bremec CS, Lasta ML (2002) Epibenthic assemblage associated with scallop (Zygochlamys patagonica) beds in the Argentinian shelf. Bull Mar Sci 70(1): 89-105 Bremec C, Lasta M, Lucifora L, Valero J (1998) Anlisis de la captura incidental asociada a la pesquera de vieira patagnica (Zygochlamys patagonica (King and Broderip 1832)). Inf Tcn INIDEP 22: 1-18 Bremec C, Brey T, Lasta M, Valero J, Lucifora L (2000) Zygochlamys patagonica beds on the Argentinian shelf: Part I: Energy flow through the scallop bed community. Arch Fish Mar Res 48(3): 295-303 Bremec C, Schejter L, Madirolas A, Tripode M (2006) Comunidades de aguas profundas: macrofauna bentnica de un can submarino localizado en la plataforma patagnica (4335S, 5933W). VI Jornadas Nacionales de Ciencias del Mar, Puerto Madryn, Argentina. Book of abstracts. p. 128 Burton M (1932) Sponges. Discov Rep 6: 237-392 Carter HJ (1876) Descriptions and figures of deep-sea sponge and their spicules from the Atlantic ocean, dredged up on board HMS Porcupina Chiefly in 1869 (concluded). Ann Mag Nat Hist, ser. 4, 20: 38-42 Coleman FC, Williams SL (2002) Overexploiting marine ecosystem engineers: potencial consequences for biodiversity. Trends Ecol Evol 17: 40-44 Cuartas EI (1986) Porferos de la Provincia Biogeogrfica Argentina. Physis A 44 (106): 37-41 Cuartas EI (1991) Demospongiae (Porifera) de Mar del Plata (Argentina) con la descripcin de Cliona lisa sp. n. y Plicatellopsis reptans sp. n. Neritica 6(1-2): 43-63 Cuartas EI (1992a) Porferos intermareales de San Antonio Oeste, provincia de Ro Negro, Argentina (Porifera: Demospongiae). Neotropica 38(100): 111-118 Cuartas EI (1992b) Porferos de la Provincia Biogeogrfica Argentina. III. Poecilosclerida (Demospongiae) del litoral marplatense. Physis A 47 (113): 73-88 Cuartas EI (1992c) Algunas Demospongiae (Porifera) de Mar del Plata, Argentina, con descripcin de Axociella marplatensis sp. n. Iheringia Sr Zool 73: 3-12 Cuartas EI (1995) Redescripcin de Clathria burtoni nomen novum de C. prolifera Burton, 1940. (Porifera: Demospongiae). Ann Mus Civ St Nat G. Doria XC: 571-576 Desqueyroux R (1972) Demospongiae (Porifera) de la costa de Chile. Gayana Zool 20: 1-71 Desqueyroux-Fandez R (1989) Demospongiae (Porfera) del litoral chileno antrtico. Ser Cient Inach 39: 97-158 Desqueyroux-Fandez R, Moyano HI (1987) Zoogeografa de Demospongias chilenas. Bol Soc Biol Concepcin (Chile) 58: 3966 Desqueyroux-Fandez R, van Soest RWM (1996) A review of Iophonidae, Myxillidae and Tedaniidae occurring in the south east Pacific (Porifera: Poecilosclerida). Rev Suisse Zool 103: 3-79 Garzoli SL (1993) Geostrophic velocity and transport variability in the Brazil-Malvinas confluence. Deep Sea Res 40: 1379-1403

Koltun VM (1964) Sponges of the Antarctic. I. Tetraxonida and Cornacuspongida. In: Pavlovskii EP, Andriyashev AP, Ushakov PV (eds). Biol Rep Soviet Antarct Exped (1955-1958), Akademya Nauk SSSR [English translation, 1966, Israel Program for Scientific Translations]. Vol. 2 (10). S. Monson, Jerusalem. pp. 6133, 443-448 Mothes-de-Moraes B, Pauls SM (1979) Algunas esponjas monaxonidas (Porifera: Demospongiae) do litoral sul do Brasil, Uruguay e Argentina. Iheringia Sr Zool 54: 57-66 Parker G, Paterlini MC, Violante RA (1997) El fondo marino. In: Boschi E (ed). El mar argentino y sus recursos pesqueros, I. antecedentes histricos de las exploraciones en el mar y las caractersticas ambientales. pp. 65-87 Piola AR, Rivas AL (1991) Corrientes en la plataforma continental. Mar Arg Rec Pesq 1: 119-132 Ridley S, Dendy A (1886) Preliminary report on the Monaxonida collected by H.M.S. Challenger. Part I and II. Ann Mag Nat Hist 18(5): 325-351, 470-493 Ridley S, Dendy A (1887) Report on the monaxonida collected by H.M.S. Challenger during the year 1873-1876. Rep Sci Res Voy H.M.S. Challenger, Zool 20: 1-275 Ros P, Cristobo FJ, Urgorri V (2004) Poecilosclerida (Porifera, Demospongiae) collected by the Spanish Antarctic expedition BENTART-94. Cah Biol Mar 45: 97-119 Sar M (1978) Demospongie di acque superficiali della Terra del Fuoco (Spedizioni A.M.F.Mares-G.R.S.T.S. e S.A.I). Boll Mus I Biol Univ Genova 46: 7-117 Sar M, Balduzzi A, Barbieri M, Bavestrello G, Burlando B (1992) Biogeographic traits and checklist of Antarctic demosponges. Polar Biol 12: 559-585 Schejter L (2004) Estructura comunitaria en un banco de vieira patagnica Zygochlamys patagonica (Mollusca: Bivalvia: Pectinidae) sujeto a arrastres pesqueros, Mar Argentino. MSc Thesis. Universidad Internacional de Andaluca, Baeza Schejter L, Calcinai B, Cerrano C, Bertolino M, Pansini M, Giberto D, Bremec C (2006) Porifera from the Argentina Sea: diversity in Patagonian scallop beds. Ital J Zool 73 (4): 373-385 Schulze FE (1887) Report on the Hexactinellida collected by H.M.S. Challenger during the years 1873-76. Rep Sci Res Voy H.M.S. Challenger, Zool 21: 1-513 Sollas WJ (1886) Preliminary account of the Tetractinellid sponges dredged by H.M.S. Challenger 1872-76. Part I. The Choristida. Scient Proc R Dubl Soc 5: 177-199 Sollas WJ (1888) Report on the Tetractinellida collected by H.M.S. Challenger during the years 1873-76. Rep Sci Res Voy H.M.S. Challenger, Zool 25: 1-458 Thrush SF, Dayton PK (2002) Disturbance to marine benthic habitats by trawling and dredging: implications for marine biodiversity. Ann Rev Ecol Syst 33: 449-473 Topsent E (1902) Spongiaires. Expd. antarct. belge. Rs Voy S Y Belgica, 1897-1899: 1-54 Turner SJ, Thrush ST, Hewitt JE, Cumming UJ, Funnell G (1999) Fishing impacts and the degradation or loss of habitat structure. Fish Manag Ecol 6: 401-420 Vivier F, Provost C (1999) Direct velocity measurements in the Malvinas Current. J Geophys Res 104: 21083-21103

Porifera research: Biodiversity, innovation and sustainaBility - 2007

203

Excavating rates and boring pattern of Cliona albimarginata (Porifera: Clionaidae) in different substrata
Barbara Calcinai(1), Francesca Azzini(1), Giorgio Bavestrello(1), Laura Gaggero(2), Carlo Cerrano(2*)
Dipartimento di Scienze del Mare, Universit Politecnica delle Marche, Via Brecce Bianche I-60131 Ancona, Italy. b.calcinai@univpm.it, cianfry77@hotmail.com, g.bavestrello@univpm.it (2) Dipartimento per lo studio del Territorio e delle sue Risorse, Universit di Genova, Corso Europa 26 I-16100 Genova, Italy. gaggero@dipteris.unige.it, cerrano@dipteris.unige.it
(1)

Abstract: Eroding sponges create a series of connected chambers and galleries into calcareous substrata where they live. While it is well known that only calcium carbonate is etched by sponge activity, no comparative data are available regarding the different forms of carbonate. In this work we investigate the erosion rates and erosion pattern of the tropical boring sponge Cliona albimarginata in different biogenic and non-biogenic calcareous rocks. In particular, we tested portions of the shell of the large bivalve Hippopus sp. and of the branches of the stony coral Acropora sp. together with different kinds of carbonatic stones such as the Carrara marble, the Majolica of the Conero Promontory, the Finale medium-grained calcarenite, the Prun fine-to medium-grained limestone and the homogeneously fine-grained Vicenza limestone. The dissolution rates of the sponge on the different kinds of carbonate are highly variable and these differences are discussed in terms of crystal shape and aggregation, the rock fabric and the presence of other minerals. Keywords: boring pattern, Cliona albimarginata, excavating rates, Indonesia, Porifera

Introduction
Excavating sponges are able to live in carbonatic substrata, perforated by mechanical and chemical activity of specialised etching cells (Rtzler and Rieger 1973, Pomponi 1980). The contact of sponge canal system with water is ensured by portions of the sponge, called papillae, protruding from its substratum surface. This growing form known as -stage is, in some species, substituted by a complete removal of the substratum such that the sponge becomes a free-living organism (-stage). In other cases the epilithic portion continues to develop until the papillae are connected by a more or less thick crust of sponge (-stage). The activity of boring sponges has been documented especially in tropical waters, and several authors have shown their ecological role in coral reefs such as their influence on the balance of calcium carbonate stored in the reef (Hutchings 1986), the production of fine sediments (Ftterer 1974), their influence on reef morphology (Goreau and Hartman 1963), and their influence on coral asexual reproduction (Tunnicliffe 1981). Sponges are able to excavate into both inorganic and biogenic calcareous materials. In some biogenic structures like mollusc shells, organic components, such as conchiolin, are digested by acid phosphatase released by the etching cell (Pomponi 1980) while the periostracum layer may prevent sponge erosion (Mao Che et al. 1996, Kaehler and McQuaid 1999). In the same way living tissue protects corals from

sponge erosion as very few species are able to attack living tissue (Tunnicliffe 1979, 1981, MacKenna 1997, Schnberg and Wilkinson 2001, Lpez-Victoria et al. 2003, LpezVictoria and Zea 2005). Very few comparative data are available regarding the differences in boring sponge activity in the substrate with different textures. Neumann (1966) suggested that mineralogy of the carbonates does not affect the process while the density of the substrate improves penetration (Highsmith 1982, Rose and Risk 1985, Schnberg 2002). Other physical and biological factors may also affect erosion, such as nutrient availability, temperature, symbiosis with zooxanthellae (Hill 1996), etc. In this study we investigated the influence of the substrata with similar carbonatic composition but different microtexture and mineralogy composition on the erosion pattern and rate of the coral reef species Cliona albimarginata Calcinai et al. 2005. In particular we tested: i) metamorphic rocks with medium and regular grain and isorientation of calcitic grains; ii) sedimentary rocks with fine and homogeneous grain; iii) sedimentary rocks with fine and irregular grains; iv) sedimentary rocks with medium and irregular grain and a detrital component.

Materials and methods


Our experiment was carried out in the Bunaken Marine Park (North Sulawesi, Indonesia). Carbonatic blocks of

204

different mineral composition, texture and porosity were fixed, using wires and nails, on a single large specimen of C. albimarginata (about 15 m2) living 5 m deep on the edge of the coral reef (Fig. 1A, B). In this way, avoiding sponge grafts commonly used in this kind of experiment (Neumann 1966, Rtzler 1975, Schnberg and Wilkinson 2001), we limited the stress to the sponge due to the handling procedure. As both light and current enhance boring activity in sponges with zooxanthellae (Hill 1996) we chose a single specimen with the same light exposure and uniform current. The following materials were tested: 15 blocks of coral coming from branches of dead colonies of Acropora sp; 15 blocks obtained from the umbo portion of large shells of Hippopus sp.; 15 blocks of Carrara marble; 4 blocks of Finale fine-medium grained calcarenite; 5 blocks of Conero fine grained mudstone (Majolica); 4 blocks of homogeneously fine grained Vicenza limestone; 4 blocks of Prun fine-to medium-grained limestone. As control, for each kind of material 5 blocks were placed out of the sponge to evaluate the excavation due to microborers. Before the test, the blocks were dried and weighted. Each block surface was calculated using program Image-J v1.37. After 200 days the blocks were removed from the sponge, cleaned in hydrogen peroxide (120 vol.), dried and weighed again. Encrusting organisms present on the blocks were manually removed. Sponge erosion rates (Kg/m2/y) were calculated as the difference in weight of the blocks, before and after the experiment, as microerosion due to microboring organisms was negligible. To study the penetration of sponges together with rock microtexture some blocks were consolidated by epoxy resin, then processed to 30 m thick section. The mineralogical and petrographic analysis of the biogenic or inorganic substrata was carried out by stereoscopic and transmitted light microscopy. The mineral composition of substrata was analysed by Xray diffraction (XRPD) using a Philips PW1140-X-CHANGE

diffractometer (CuK radiation; current 30 mA, voltage 40 kV, scan speed, 0.5 2/min; scan interval, 3-70 2) and interfaced with PC-APD software for data acquisition and processing.

Results
The boring rates of Cliona albimarginata into the different kinds of carbonates were highly variable (Fig. 2): 29.5 2.2 Kg/m2 /y for the Carrara marble; 24.2 2.3 Kg/m2/y for the Finale calcarenite; 24.0 2.6 Kg/m2/y for Hippopus sp. (umbo); 15.6 4.7 Kg/m2/y for the Acropora (branch); 12.6 2.9 Kg/m2/y for the Conero majolica; 11.01 1.0 Kg/m2/ y for the Vicenza limestone and 2.9 1.2 Kg/m2/y for the Prun limestone. The dissolution rate in all the blocks placed outside the sponge, considered as control, was negligible. Plotting the erosion rate vs specific gravity of the different materials it was possible to observe that for four materials (Vicenza limestone, Acropora, Hippopus, and Marble) the rate of erosion is directly related to the density of the different rocks while for the Finale calcarenite, the Majolica and the Prun limestone no obvious relationships could be observed (Fig. 3). The maximum vertical penetration was also variable in different substrata, with the highest penetration in the biogenic substrata and in the marble, while the limestones were harder to penetrate and the Prun limestone was attacked only on the surface (Fig. 4). The boring pattern produced by C. albimarginata was different in the tested substrata (Fig. 5). In the marble the sponge produced vertically elongated excavations, oval in section and tidily organised (Fig. 5A). In the compact Hippopus umbo, the sponge produced similar boring patterns in every direction (Fig. 5B). In the porous Acropora the sponge largely penetrated the pre-existing canals, producing a fine spongious pattern of erosion (Fig. 5C). In the Majolica the boring activity produced circular tunnels running in an

Fig. 1: Images of the field experiment. A. General view of the experimental set with Carrara marble. Scale bar: 5 cm B. A detail of experimental set with Acropora sp. Scale bar: 2 cm.

205

Fig. 2: Dissolution rates of Cliona albimarginata in the tested substrata.

Fig. 3: Dissolution rate vs. specific gravity of the tested substrata.

indefinite directions (Fig. 5D). The Prun limestone was only slightly etched with superficial erosion marks (Fig. 5E). The Finale calcarenite was heavily affected by the sponge action that had detached the large biogenic elements (Fig. 5F). Also at the level of rock microtexture, the activity of etching cells produced different results in the tested materials. Hippopus shell has crystals organized in layers that are deformed as kinks. The erosion pattern developed in the laminated layers of the shell produced rounded excavations

0.3 -1 mm that fused into each other to form lobated cavities (Fig. 6A, B). Acropora is made of large rare aragonitic crystals (more than 0.2 mm in diameter). From this compact structure, sheaves with a centrifuge growth pattern of calcite fibres (about 0.002-4 mm long) had originated. The growth of coral was visible because of the parallel arrangement of fibrous radiating structure. The erosion pattern was affected by the isoorientation of the crystals. In fact the sponge produced

206

Fig. 4: The maximum vertical penetration of Cliona albimarginata in the tested substrata.

elongated excavations (0.15-1.5 mm) that followed the orientation of the fibrous crystals (Fig. 6C, D). In the Carrara marble (Fig. 6E, F) the average crystal size is 0.2 mm and the grains are regularly arranged in a mosaic texture. The sponge produced excavations that were regularly spaced following the regular orientation of the crystals. Under a transmitted polarised light, the boundary of the cavity was clearly thinned, and the sites of more pervasive etching exhibited micro-lobes along the external portion of the calcite grain. In fact, excavations can merge but the regular pattern of corrosion is maintained. The Prun mudstone displays an inhomogeneous texture characterized by fine-grained calcite with patches of coarser sparry calcite and scarce detrital fraction made by chlorite. The rock showed submillimetric fractures filled by clay minerals and hydroxides and clusters of forams occurring as biointraclasts. The erosion pattern was evident only where the sponge met an embedded shell that the sponge reached through a fracture filled by coarse grained spatic calcite (Fig. 6G, H). The Vicenza limestone is a biodetritic rock with the grain size varying irregularly between 0.004 and 0.1 mm. It contains forams, bivalve shells and sea urchin fragments, together with iron oxides and hydroxides. The primary porosity was distributed irregularly. The erosion pattern of the sponge was very irregular (Fig. 6I, J). The Finale calcarenite is composed of fine-mediumgrained detrital limestone, rich in detrital fossiliferous content and terrigenous minerals (quartz, lithic fragments) in sparry cement. As a consequence the texture within recrystallized areas showed irregular granulometry. Calcite grains present in the cement were between 0.01 and 0.2 mm of diameter. The sponge avoided the cement. The erosion pattern showed merging lobate excavations that led to irregular and wide galleries (Fig. 6K, L).

The Conero majolica mudstone is fine-grained (< 0.01 mm) and pervasively recrystallized. It includes nannofossils and scarce impurities of detrital quartz. The calcite crystals are fine but coarser than those found in the Prun mudstone. The erosion pattern produced lobate cavities with stochastic directions (Fig. 6M, N).

Discussion
Even if Cliona albimarginata exclusively perforates corals in the field, it is able to bore a wide variety of both mineral and biogenic substrates. The sponge had excavated all the substrata used with different intensity. The microtexture of rock substrata affects the microscopic pattern of erosion. Observation by optical techniques (transmitted light microscopy) reveals that the erosion pattern of sponge erosion may be affected by the mineral setting (i.e. rock fabric) of the substrate. In fact, the sponge produces excavations that follow the preferred orientation of fibrous calcite crystals (Acropora), or the parallel lamination within the Hippopus sp. shell. Conversely, the anisotropic granoblastic and mosaic texture of Carrara marble turns out to be etched with preference along the flow schistosity. Within a fine-grained, virtually isotropic material such as limestones (Conero Majolica or in Vicenza limestone), the general behaviour of the sponge is to etch the rock with irregular, randomly oriented, erosion patterns. The boring pattern is also affected at macroscopic scale by the characteristic of the substrata such as crystal preferred orientation (e.g. marble), pre-existing cavities (e.g. Acropora) or the presence of silicatic fragments (e.g. Prun limestone). For example in the compositionally homogeneous, massive, flow oriented, medium grained marble, the sponge produces vertically elongated excavations, oval in section and regularly organised. This suggests that dissolution is driven by crystal organisation. In the Acropora skeleton the sponge widely uses

207

Fig. 5: Macroscopic boring pattern of Cliona albimarginata in the tested substrata. A. Vertical, tidy organised excavations in the compact marble. B. A boring pattern, similar in both directions is produced in Hippopus sp. umbo. C. A fine spongious pattern produced by C. albimarginata that uses in Acropora the pre-existing canals of the coral. D. Tunnels, circular in section and running in indefinite direction in the Majolica. E. Superficial erosion marks are produced by C. albimarginata in the Prun stone. F. C. albimarginata detaches the large biogenic elements in the Finale stone, that is strongly eroded. Scale bars: 1 cm.

the pre-existing canals producing a fine spongious pattern around the principal tunnels (Fig. 5C). The regular pattern is lost in the Finale stone because the sponge detaches the large biogenic elements. In this way the substratum appears to be widely destroyed. In the Prun stone, chambers or tunnels are not detectable because the rock is only slightly etched with superficial erosion due to the presence of silicatic fragments in the stone that are not excavated by the sponge. Some authors (Hoeksema 1983, Bromley and DAlessandro 1984) report that the macroscopic pattern of excavation, produced by a sponge species, may be different because of its substrate dimension or its age. Other authors (Rtzler

1974, Schnberg 2000) have demonstrated that this character might be useful to differentiate among various species. Our data demonstrate how the previous cited characteristics of the substrata may affect the macroscopic erosion pattern produced by the boring sponge. In this way the use of the erosion pattern as a taxonomic tool requires some caution. Also the erosion rates are affected by the characteristic of the substrata. C. albimarginata is a highly destructive species. It may erode 300 - 400 kg per year of corals supplying a corresponding amount of fine sediments to the bottom. These values are similar to those of a few other excavating sponges (Schnberg 2002). Neumann (1966) studied the

208

Fig. 6: Polarized light microphotographs. A. Longitudinal section of perforated Hippopus sp. The texture is microcrystalline; the finegrained crystals are optically discontinuous and define a layered fabric of the shell, having kinked or sheaf textured appearance inside the laminae. The erosion proceed from the outer (large cavities) to inner layers (finer cavities) of the shell, following the shape of calcite grains. B. Longitudinal section of perforated Hippopus sp. The stratified structure is evidenced by the contrasting orientation of crystals. Inside the cavities a detrital fraction is preserved, most likely deriving from the mechanical action of the sponge. C. Acropora sp. exhibits an inner fabric characterized by fibrous calcite (about 0.1 mm long) elongated towards the top of the organism. The erosion pattern is sinuous and cavities are oblate and generally parallel to the fibre length. In the cavities, a fine grained detrital fraction, most likely deriving from the mechanical erosion, is preserved. D. Open cavity in Acropora sp.: the overall shape is elongated with sinuous with lobate boundaries. E. Carrara marble has homogeneous granoblastic mosaic texture. The cavities are rounded with apparently lobate boundaries like bites. F. Close up of the rock-hole area in Carrara marble: the calcite grain is thinned and corroded with lobate geometry. A detrital fraction is preserved in the cavity. G. The Prun fine-grained limestone is a mudstone (bioclasts < 10%). H. The erosion on the Prun limestone was restricted to coarse grained bioclasts (bivalve shell fragment), made of sparry (i.e. coarser than matrix) calcite. I. The Vicenza limestone is characterized by abundant bio- and intraclasts cemented by sparry calcite. The excavations are regularly dispersed, rounded to sub-rounded and flattened when two rounded holes merge. J. Detail of an excavation in the Vicenza limestone: the boundaries are finely lobated, with selective etching at the expence of sparry calcite. K. Microtexture of the Finale calcarenite. The rock is mostly formed of sparry cement including the terrigenous fraction. The excavation is developed in a patch of sparry calcite. L. Detail of an excavation etched in an organic fragment, made of sparry calcite. M. The Conero Majolica is made of nannofossils in a very fine-grained micrite matrix (mudstone). N. Erosion pattern in the Conero Majolica. Subrounded excavations tend to merge. Top, centre: a relic mudstone septum is almost isolated within a cavity. Scale bars A-D, G-N: 1 mm; E-F: 0.5 mm.

209

210

boring activity of Cliona lampa on substrata with a different mineralogy (calcite or aragonite) concluding that the density of the substrata is important to determine the value of the erosion rates. This is due to the fact that the sponge in a porous material first occupies the available spaces before excavating resulting in reduced erosion rates. Schnberg (2002) came to a similar conclusion for C. orientalis. Our data suggest a more complex scenario where density is only one of the factors affecting erosion rate. Here we have shown that there is a group of substrata where the erosion rate is directly related to substratum density. Nevertheless, other characteristics of the substratum are involved in this phenomenon. High values of erosion rates are obtained for the Finale calcarenite in spite of its low density due to the removal of entire large clasts of the biogenic fraction during the erosion activity. On the contrary, the presence of silicatic fragments in the substrate, that are not excavated by the sponge reduces the erosion rate in the Prun stone. Also the size of the crystals affects the erosion rate. In fact the low erosion rate showed by the Majolica in spite of its high density (Fig. 3) is quite likely related to the randomly oriented very small grains.

Acknowledgements
This manuscript is dedicated to Prof. L. Cortesogno who greatly stimulated this research. The project was partially funded by the Executive program of scientific co-operation between Italy and Indonesia (project 2004-07 STE-16).

References
Bromley RG, DAlessandro A (1984) The ichnogenus Entobia from the Miocene, Pliocene and Pleistocene of Southern Italy. Riv Ital Paleo Strat 90: 227-296 Calcinai B, Bavestrello G, Cerrano C (2005) Excavating sponge species from the Indo-Pacific Ocean. Zool Stud 44(1): 5-18 Ftterer DK (1974) Significance of the boring sponge Cliona for the origin of fine grained material of carbonate sediments. J Sediment Petrol 44: 79-84 Goreau TF Hartman WD (1963) Boring sponges as controlling factors in the formation and maintenance of coral reefs. Publ Am Assoc Advan Sci 75: 25-54 Highsmith RC (1982) Reproduction by fragmentation in corals. Mar Ecol Prog Ser 7: 207226 Hill MS (1996) Symbiotic zooxanthellae enhance boring and growth rates of the tropical sponge Anthosigmella varians forma varians. Mar Biol 125: 649-654 Hoeksema BW (1983) Excavation patterns and spiculae dimensions of the boring sponge Cliona celata from the SW Netherlands. Senckenberg Marit 15: 55-85

Hutchings PA (1986) Biological destruction of coral reefs. Coral Reefs 4: 239-252 Kaehler S, McQuaid CD (1999) Lethal and sub-lethal effects of phototrophic endoliths attacking the shell of the intertidal mussel Perna perna. Mar Biol 135: 497-503 Lpez-Victoria M, Zea S, Weil E (2003) New aspects on the biology of the encrusting excavating sponges complex Cliona caribbea - Cliona langae - Cliona aprica. In: Pansini M, Pronzato R, Bavestrello G, Manconi R (eds). Sponge science in the new millennium. Boll Mus Ist Biol Univ Genova 68: 425-432 Lpez-Victoria M, Zea S (2005) Current trends of space occupation by encrusting excavating sponges on Colombian coral reefs. Mar Ecol 26 (1): 33-41 MacKenna SA (1997) Interactions between the boring sponge, Cliona lampa and two hermatypic corals from Bermuda. Proc 8th Int Coral Reef Symp, Balboa 2: 1369-1374 Mao Che L, Le Campion-Alsumard T, Boury-Esnault N, Payri C, Golubic S, BZac C (1996) Biodegradation of shells of the black pearl oyster, Pinctada margaritifera var. cumingii, by microborers and sponges of French Polynesia. Mar Biol 126: 509-519 Neumann AC (1966) Observations on coastal erosion in Bermuda and measurements of the boring rate of the sponge Cliona lampa. Limnol Oceanogr 11: 92-108 Pomponi SA (1980) Cytological mechanisms of calcium carbonate excavation by boring sponges. Int Rev Cytol 65: 301-319 Rose CS, Risk MJ (1985) Increase in Cliona delitrix infestation of Montastrea cavernosa heads on an organically polluted portion of the Grand Cayman. PSZN Mar Ecol 6: 345-363 Rtzler K (1974) The burrowing sponges of Bermuda. Smithsonian Contrib Zool 165: 1-32 Rtzler K (1975) The role of burrowing sponge in bioerosion. Oecologia 19: 203-216 Rtzler K, Rieger G (1973) Sponge burrowing: fine structure of Cliona lampa penetrating calcareous substrata. Mar Biol 21: 144162 Schnberg CHL (2000) Bioeroding sponges common to the Central Australian Great Barrier Reef: description of three new species, two new records, and additions to two previously described species. Senckenberg Marit 30: 161-221 Schnberg CHL (2002) Substrate effects on the bioeroding Demosponge Cliona orientalis. 1. Bioerosion rates. PSZN Mar Ecol 23: 313326 Schnberg CHL, Wilkinson CR (2001) Induced colonization of corals by a clionid bioeroding sponge. Coral Reefs 20: 6976 Tunnicliffe V (1979) The role of boring sponges in coral fracture. In: Lvi C, Boury-Esnault N (eds). Biologie des spongiaires. Coll Int CNRS 291: 309-315 Tunnicliffe V (1981) Breakage and propagation of the stony coral Acropora cervicornis. Proc Nat Acad Sci USA 78(4): 2427-2431

Porifera research: Biodiversity, innovation and sustainaBility - 2007

211

The possible role of Echinometra lucunter (Echinodermata: Echinoidea) in the local distribution of Darwinella sp. (Porifera: Dendroceratida) in Arraial do Cabo, Rio de Janeiro State, Brazil
Emiliano Nicolas Calderon(1*), Carla Zilberberg(2), Paulo Csar de Paiva(1)
Universidade Federal do Rio de Janeiro, Instituto de Biologia, Departamento de Zoologia, Laboratrio de Polychaeta. CCS. Bloco A, Ilha do Fundo, s/n, 21940-590, Rio de Janeiro, RJ, Brazil. encalderon@yahoo.com.br, ppoliqueta@hotmail.com (2) Departamento de Biologia Molecular e Gentica, Instituto de Biologia Roberto Alcntara Gomes, Universidade do Estado do Rio de Janeiro. Rua So Francisco Xavier 524 PHLC, sala 205. 20550-013. Rio de Janeiro (RJ), Brazil. carlazilber@ yahoo.com.br
(1)

Abstract: Sea urchins are known to control algal populations, leading to an increase in substrate accessibility for many organisms, thus having a fundamental role in the maintenance of diversity in those habitats. Darwinella sp. is a common sponge at several sites in Arraial do Cabo, Rio de Janeiro State, Brazil, but factors determining its distributional patterns are still unknown. The goal of this study was to establish the relationship among Echinometra lucunter density, algal and Darwinella sp. cover. The study was conducted at Saco do Cherne (SC) and Porcos Island (PI), Arraial do Cabo. At both sites, the density of E. lucunter and the percent cover of Darwinella sp. and algae were quantified. Additionally, Darwinella sp. percent cover was compared between portions of high and low E. lucunters density. Spearman Rank correlation analyses were performed between E. lucunter density and algae cover, E. lucunter density and Darwinella sp. cover, and between Darwinella sp. and algae cover. Results show that areas with high urchin density support a significantly higher cover of Darwinella sp. compared to areas with low density of urchins. Correlation analyses demonstrate that, in general, algae percent cover was negatively related to the density of E. lucunter. The same pattern was found between algae and Darwinella sp. percent covers. On the other hand, the relationship between E. lucunter density and Darwinella sp. cover was weakly positive. The results from this study suggest that algae is possibly competing with Darwinella sp. for space, but the presence of E. lucunter is probably mediating this interaction by decreasing the percent cover of algae. However, the weak relationship observed between E. lucunters density and Darwinella sp. cover suggests that other factors besides competition with algae are affecting the percent cover and distribution of the sponge Darwinella sp. at the studied sites. Keywords: Algae, herbivory, sea urchin, sponge, competition

Introduction
Rocky marine substrates are characterized by the presence of clonal sessile invertebrates, such as sponges, corals and ascidians (Jackson 1977, Buss 1979, McKinney 1992). In these habitats, competition for space and predation act as major biotic determinants of sessile invertebrates distribution (Connell 1961, 1973, Sutherland 1976, Jackson 1977, Buss 1980). Competition for space is particularly strong in taxa that grow horizontally along the substrate, since the fitness of these organisms is often related to their size (Jackson 1977, Buss 1990). In shallow hard substratum communities, competition with algae seems to negatively affect sessile invertebrate distribution by settlement, growth inhibition or overgrowth (Sammarco 1980, Sammarco 1982, Jernakoff 1985, Jompa and McCook 2002). Algae, for example, can inhibit invertebrate larvae

settlement by chemical or physical means (Sammarco 1980, Jernakoff 1985, Jompa and McCook 2002). Additionally, algae have greater advantage over invertebrates by their extremely fast growth rates at shallower depths (Lobban and Harison 1994), which allow them to overgrow most benthic invertebrates (McCook et al. 2001). This negative effect has been demonstrated, for example, by Jompa and McCook (2002), where the presence and consequent overgrowth of the red algae Lobophora variegata (Lamouroux) Womersley ex. Oliveira 1977 caused significant colony tissue mortality on the coral Porites cylindrica Dana, 1846. Herbivores, such as sea urchins and many fishes, are known to control algal populations leading to an increase in substrate accessibility for many organisms (Paine 1966, Jackson and Winston 1982, Ferreira et al. 1998, Tuya et al. 2004). Therefore, the presence of herbivores has a fundamental role

212

in the maintenance of diversity in marine hard substratum communities (Paine and Vadas 1969, Carpenter 1986, Jompa and McCook 2002). One remarkable example of a herbivore impact in a marine community was the mass mortality of the sea urchin Diadema antillarum Philippi, 1845 around the Caribbean region in 1983/1984 (Carpenter 1990). The decrease in 95-99% of D. antillarum population in Caribbean reefs had a large negative impact on scleractinian coral cover, changing entire reef systems from coral to algal dominated communities (Hughes 1994). Only with the slow increase in D. antillarium densities algal cover began to decrease and coral cover consequently increase (Edmunds and Carpenter 2001). Therefore, it is clear that the presence of sea urchins have a positive effect on marine hard substratum communities by the reduction of algal cover and, consequently, a greater diversity of invertebrate fauna. Sponges are often competitively superior to most benthic invertebrates (Jackson and Winston 1982, Bell and Barnes 2003). However, algae might be competitively superior to sponges as demonstrated in a study of competition between a coralline algae (Corallina vancouveriensis Yendo, 1901) and the sponge Halichondria panicea (Pallas, 1766) in temperate seas (Palumbi 1985). If sponges are inferior competitors to algae, their distribution in rock bottom communities may be negatively affected by algal competition, particularly in the absence of an effective herbivore (Bell and Barnes 2003). The negative association between sponges and macroalgae in temperate rocky subtidal communities has been suggested to be caused by competition between these two groups of organisms (Witman and Sebens 1990, Bell 2002). Conversely, it has been argued that the observed macroalgae and sponge distribution in those habitats might be, instead, the result of abiotic factors, such as depth and substratum inclination (Preciado and Maldonado 2005). What remains unknown is whether in habitats with similar abiotic factors competition with algae has a negative effect on sponge distribution. Wulff (2005) has argued that in the absence of abiotic factors, that could prevent a species from occurring in a particular habitat, biotic factors, such as competition, can have a large effect on sponge distribution. Consequently, if sponges are outcompeted by algae, the presence of an efficient herbivore is important to mediate these interactions and avoid the competitive exclusion of sponges in a particular habitat (Palumbi 1985). Darwinella sp. is a demosponge that fifteen years ago was rarely seen around the Arraial do Cabo region (Rio de Janeiro State, Brazil) (Muricy et al. 1991). Currently though, Darwinella sp. can be commonly seen at some localities around Arraial do Cabo (ENC, personal observation). Although frequent, Darwinella sp. distribution in a scale of meters to kilometers seems patchy at shallower depths (0-8 m; ENC, personal observation), however, the factors affecting the distribution of this species remains unknown. In Arraial do Cabo, the sea urchin Echinometra lucunter (Linnaeus, 1758) is the most conspicuous benthic herbivore in shallow waters (Castro et al. 1995), although herbivore fishes in the area are also common (Ferreira et al. 1998). It has been shown that 98% of E. lucunters diet is constituted by algae, while the remaining 2% is composed of invertebrates that are probably ingested accidentally (Oliveira 1991). Therefore, one possible

biotic factor determining the distribution of Darwinella sp. around Arraial do Cabo might be the relative abundance of algae, which should be regulated by E. lucunter. The goal of the present study was to evaluate the relationship among E. lucunter density, algal and Darwinella sp. cover in Arraial do Cabo, Rio de Janeiro State, Brazil.

Materials and methods Study area


The study was conducted at Porcos Island (PI) and Saco do Cherne (SC), in Arraial do Cabo, RJ, Brazil (Fig. 1). PI is characterized by low wave action, sheltered from the northeastern winds that are predominant in the area. The substratum morphology is characterized by rocky walls with variable inclination interspaced by small portions of vertical walls. SC is a small inlet, characterized by high wave action, exposed to the predominant northeastern winds. Substratum morphology is characterized by a single rocky wall predominantly vertical. On both sites PI and SC, the interface between the rocky wall and the bottom happens abruptly in approximately 90o angle.

Sampling
Eight 5 m portions of the rocky substrate were haphazardly chosen at PI and four at SC, where every portion was located at the interface between the bottom and the vertical wall (4-6 m depth), where the urchin E. lucunter was most abundant. Within each portion, five to six vertical rectangles (60 x 40

Fig. 1: Map showing the study area in Arraial do Cabo, Rio de Janeiro state, Brazil. The two studied localities were at Porcos Island (PI) and Saco do Cherne (SC).

213

Data analyses
Percent covers were arcsin transformed prior to all statistical analyses (Sokal and Rohlf 1995). Nonparametric tests were performed when data did not conform to assumptions of normality and homoscedasticity (Sokal and Rohlf 1995). A Mann-Whitney test compared the percent cover of Darwinella sp. between areas of high and low densities of E. lucunter. To choose areas with significant differences in sea urchin densities (i.e., high and low densities), at PI, out of the eight sampled portions of the rocky substratum, two with the highest and two with the lowest average densities of E. lucunter were chosen to be used in the comparison of Darwinellas percent cover (Fig. 3). A one way ANOVA followed by a Tukey post hoc test, established whether there were significant differences in sea urchins density within and between high and low density portions. No significant differences in urchin densities were found within low (F = 2.87; df = 3, 19; P > 0.5) or high (F = 0.000; df = 3, 19; P > 0.5) urchin density portions, while portions of high density were significantly different form portions of low urchin density (F = 14.20; df = 3, 19; P < 0.005). Therefore, the two portions of low density, as well as the two of high density were pooled for the comparison of the percent cover of Darwinella sp. To establish the relationship among E. lucunter densities, algal and Darwinella sp. cover, three pair-wise Spearman Rank correlation analyses were performed: 1) E. lucunter density and Darwinellas cover; 2) E. lucunter density and algal cover; 3) algal cover and Darwinellas cover. All three correlations were performed separately for each locality and afterwards by pooling data from both PI and SC. In all tests, the portions along the rocky bottom were pooled at each locality and the photographed rectangles were used as replicates.

Fig. 2: Schematic representation of the sampling area at PI showing the disposition of the photographed rectangles (Rt) on the substratum.

cm) were randomly chosen and photographed (Fig. 2) using a digital camera (SONY Cyber-Shot DSC-P41). To estimate the percentage cover of Darwinella sp., algae, and other sessile invertebrates within each portion of the rocky substrate, all digital images were analyzed with the CPCe V.3.3 software (National Coral Reef Institute/New Southeastern University) using a 60 point grid system. Preliminary tests showed no difference in percent cover when estimated with a 60 or 100 point system. Density of E. lucunter was quantified by counting all sea urchins that had some portion of its body within each sampled rectangle. In the present study algae was defined by the combination of algal turfs (represented primarily by coralline filamentous algae: Steneck and Dethier 1994) and a few other macroalgae, such as Codium spp. Stackh, 1797, Sargassum furcatum Ktzing 1843 and Dictyota spp. Lamouroux 1809, that occur in lower densities but in conjunction with the predominant algal turfs (Yoneshigue and Valentin 1988).

Results
The density of Echinometra lucunter varied greatly among portions of the rocky bottom at each locality (Fig. 3). At PI, sea urchin density varied between 3.33 1.56 and 65.28 8.95 individuals/m2, while density at SC varied between 6.25 2.57 and 32.64 4.15 individuals/m2 (mean + SD; Fig. 3). Darwinella sp.s percent cover varied between 0% and 17.69 7.30% at PI and 2.68 3.16% and 6.47 2.66% at SC

Fig. 3: Density of Echinometra lucunter within sampled portions of the rocky substratum at Porcos Island (PI) and Saco do Cherne (SC). The portions of High (H) and Low (L) urchin density are shown above bars (see Material and methods for details). Densities are number of individuals/m2. Mean + SD are shown.

214

Fig. 4: Density of Echinometra lucunter (individuals/m2), Darwinella sp. (% cover), algae (% cover) and other invertebrates (% cover) at Porcos Island (PI; N=46) and Saco do Cherne (SC; N=24). Mean + SE are shown.

with averages of 3.92% and 7.53% (PI and SC, respectively; Fig. 4). Algal cover was extremely high at both localities, reaching up to 90.33% at PI and 74.64% at SC (Fig. 4). All other invertebrates together reached maximum densities of 26.66% and 78.33% at PI and SC, correspondingly. Darwinella sp. cover was significantly different between portions of high (62.5 + 7.43 individuals/m2) and low (9.85 + 3.24 individuals/m2; mean + SE; Fig. 3) density of sea urchins (U = 121.00; N =12; P < 0.0001; Fig. 5). The percent cover of Darwinella sp. was more than ten-fold higher in areas of high (11.92 + 2.73%; mean + SE) compared to areas with low sea urchin densities (0.08 + 0.08%; mean + SE; Fig. 5). In general, algal cover was negatively related to the density of E. lucunter (R = -0.807; N = 70; P < 0.0001; Fig. 6A). A similar relationship was found between the percent cover of algae and Darwinella sp. (R = -0.649; N = 70; P < 0.0001; Fig. 6B). Conversely, the relationship between sea urchin density and Darwinella sp. cover, although significant, was weakly positive (R = 0.470; N = 70; P < 0.001; Fig. 6C). The same pattern was found when analyses were performed solely for PI (Fig. 7A, 7B, 7C). At SC, most relationships were weak (Fig. 7D, 7E, 7F), with the strongest relationship being between E. lucunters density and algal cover (R = 0.430; N = 24; P < 0.05). From the total number of Darwinellas quantified in the present study (N = 52), 75% of them were in total or partial contact with algal turfs. Less than 20% were in partial contact with other invertebrates, particularly the bryozoan Schizoporella errata (Walters, 1878). In most partial contacts between Darwinella sp. and other invertebrates, only 10% of the sponge surface was actually in contact with the invertebrate.

Fig. 5: Darwinella sp.s percent cover in areas of low and high urchin densities (see Material and methods for details). The mean density of Echinometra lucunter is also shown (individuals/m2). Mean + SE are shown (N=12).

Discussion
The present study demonstrates that areas with high density of the sea urchin Echinometra lucunter have a significantly higher cover of the sponge Darwinella sp. than areas with low sea urchin density. This observation, in addition with the negative correlations found between E. lucunters density and algal cover, and also between Darwinella sp. and algal cover suggest that Darwinella sp. and algae are

Fig. 6: Sperman Rank correlation analyses including both localities (PI and SC; N=70). A. Echinometra lucunter density and Darwinella sp. percent cover; B. Echinometra lucunter density and algae percent cover. C. Algae and Darwinella sp. percent cover. All percent covers were arcsin transformed.

215

Fig. 7: Sperman Rank correlation analyses performed by localities. PI = A, B, C (N=46); SC = D, E, F (N=24); A. Echinometra lucunter density and Darwinella sp. percent cover; B. Echinometra lucunter density and Darwinella sp. percent cover; C. Echinometra lucunter density and algae percent cover; D. Echinometra lucunter density and algae percent cover; E. Algae and Darwinella sp. percent cover; F. Algae and Darwinella sp. percent cover.

possibly competing for space and that the sea urchin might be controlling algal population. This result is corroborated by the frequent observation of contacts between Darwinella sp. and algae, however, manipulative experiments would be required to confirm this hypothesis. On the other hand, the weak relationship found between E. lucunters density and Darwinella sp. cover also suggests that there might be other factors besides competition with algae that is affecting the density and distribution of Darwinella sp. in Arraial do Cabo. Species of the genus Echinometra usually possess strong homing behavior and are often distributed in an aggregated pattern (McClanahan and Murtiga 2001). In the present study, the large variation in sea urchin density found among portions of the rocky substratum at both localities suggests that E. lucunter is distributed in an aggregated manner (in a scale of less than 0.5 meters), as observed in other species of this genus (McClanahan and Kurtis 1991). Therefore, it is expected that areas of high sea urchin density will support a low algal cover

and vice versa, since this urchins diet is mainly composed of algae (Oliveira 1991). The negative correlation between sea urchin density and algal cover, in the present study, supports Oliveiras (1991) findings that algae is E. lucunters main diet. However, it has been argued that in the absence of algae, E. lucunter can also feed on benthic invertebrates, such as sponges and cnidarians (McClintock et al. 1982, Oliveira 1991, McClanahan and Murtiga 2001). During the course of this study it was observed that Darwinella sp. that was found close to sea urchin aggregations presented irregular shapes that visually appeared to be caused by sea urchin removal (e.g., predation or abrasion). Thus, the weak relationship found between urchin density and Darwinella sp. cover could be due to the sea urchins grazing activity (predation) or by the accidental removal of sponge tissue (abrasion) when sea urchins move along the substratum. Although few studies have focused on sponge/algae competitive interactions (but see Palumbi 1985), studies on the negative effect of algae on the distribution of

216

other invertebrates are abundant (Quinn 1982, Jernakoff 1985, McCook et al. 2001). Algae are often the dominant competitive organism owing to their relative fast growth rates and the presence of secondary metabolites (Duffy and Hay 1990), although, sponges are also known to produce large quantities of chemical compounds (Thacker et al. 1998). The negative correlation between algae and Darwinella sp. cover in the present study suggests that, if competition is occurring, algae are competitively superior to sponges. If that is the case then, it would be interesting to determine what factors make algae competitively superior to sponges, and how general is this phenomenon. In general, the relationships among sea urchin density, Darwinella sp. and algal cover were similar at both localities, although they were weaker at SC compared to PI. The weak relationships at SC might be due to a lower sample size compared to PI, or it can possibly be due to site differences, such as fauna and flora composition or abiotic factors. At SC there was a higher abundance of benthic invertebrates, other than Darwinella sp., when compared to PI (Fig. 4). The high cover of other invertebrates at SC could have a negative effect on the distribution of Darwinella sp., through competition, which could have led to the observed weak relationship between algae and Darwinella sp. and also between Darwinella sp. and E. lucunter. The algal composition was also different between localities, with PI being dominated primarily by algal turfs, while SC had a larger abundance of macroalgae such as Codium sp. and Dictyota spp. (ENC, personal observation). Besides their greater palatability, macroalgae have a relatively higher biomass than algal turfs (Duffy and Hay 1990). Therefore, sea urchins at SC might have smaller grazing patches than at PI, since it has been demonstrated that patch size and grazing patterns are dependent on food availability (Russo 1977, McClanahan and Murtiga 2001). Many authors in the past have suggested that sponge distribution was intimately related to competition (reviewed by Wulff 2006). However, recent studies have pointed out the importance of abiotic factors as determinants of sponge distribution (Preciado and Maldonado 2005). What has recently been argued is that abiotic factors could actually exclude species from a locality (Alcolado 1994, Wulff 2005), but if a species is not inhibited by these factors, competitive interactions may greatly affect species distribution (Wulff 2005). In the present study, abiotic factors such as substratum inclination and depth were similar; however, water flow regimes were different with SC being an exposed site and PI a sheltered one (Fig. 1). This difference in water flow regimes might affect the distribution of Darwinella sp. and its relationship with algae and other invertebrates. To conclude, the results of this study point to a possible competition for space between algae and Darwinella sp., which is probably mediated by the sea urchin E. lucunter. Nevertheless, manipulative studies are needed in order to confirm the negative effect of algal cover on the cover of Darwinella sp, and the positive impact of the sea urchin on Darwinella sp. distribution.

Acknowledgements:
We thank Isaac Zilberberg for providing logistic support in Arraial do Cabo (boat and lodge); Mariana Melo and Fernanda Oliveira for help during the field work. ENC thanks Renato Ventura for insightful conversations that greatly improved the contents of this manuscript. This manuscript was submitted as partial fulfillment of the PhD degree to ENC at Universidade Federal do Rio de Janeiro, Brazil. This study was supported by a fellowship from CAPES (Coordenao de Aperfeioamento de Pessoal de Nvel Superior) to ENC.

References
Alcolado PM (1994) General trends in coral reef sponge communities in Cuba. In: van Soest RWM, van Kempen TMG, Braekman JC (eds). Sponges in time and space: biology, chemistry, paleontology. Balkema, Rotterdam. pp. 251-256 Bell JJ (2002) The sponge community in a semi-submerged temperate sea cave: density, diversity and richness. Mar Ecol 23: 297-311 Bell JJ, Barnes DKA (2003) The importance of competitor identity, morphology and ranking methodology to outcomes in interference competition between sponges. Mar Biol 143: 415-426 Buss LW (1979) Habitat selection, directional growth and spatial refuges: why colonial animals have more hiding places. In: Coulson JC, London DF (eds). Biology and systematics of colonial organisms. Academic Press, New York. pp. 459-497 Buss LW (1980) Competitive intransitivity and size-frequency distributions of interacting populations. Proc Natl Acad Sci USA 77(9): 5355-5359 Buss LW (1990) Competition within and between encrusting clonal invertebrates. Trends Ecol Evol 5(11): 352-356 Carpenter RC (1986) Partitioning herbivory and effects on coral reef algal communities. Ecol Monog 56(4): 345-363 Carpenter RC (1990) Mass mortality of Diadema antillarum. I. Long-term effects on sea urchin population-dynamics and coral reef algal communities. Mar Biol 104: 67-77 Castro CB, Echeverria CA, Pires DO, Mascarenhas MJ, Freitas SG (1995) Distribuio de Cnidaria e Echinodermata no infralitoral de costes rochosos de Arraial do Cabo, Rio de Janeiro, Brasil. Rev Bras Biol 55(3): 471-480 Connell JH (1961) The influence of interspecific competition and other factors on the distribution of the barnacle Chthamalus stellatus. Ecology 42: 710-723 Connell JH (1973) Population ecology of reef-building corals. In: Jones OA, Endean R (eds). Biology and geology of coral reefs. Academic Press, New York. pp. 205-245 Duffy JE, Hay ME (1990) Seaweed adaptations to herbivory: chemical, structural, and morphological defenses are often adjusted to spatial or temporal patterns of attack. BioScience 40(5): 368375 Edmunds P, Carpenter RC (2001) Recovery of Diadema antillarum reduces macroalgal cover and increase abundance of juvenile corals on a Caribbean reef. Proc Natl Acad Sci 98: 5067-5071 Ferreira CEL, Gonalves JEA, Coutinho R, Peret AC (1998) Herbivory by the Dusky Damselfish Stegaster fuscus (Cuvier, 1830) in a tropical rocky shore: effects on the benthic community. J Exp Mar Biol Ecol 229: 241-264 Hughes TP (1994) Catastrophes, phase shifts, and large-scale degradation of a Caribbean coral reef. Science 265: 15471551

217

Jackson J, Winston JE (1982) Ecology of cryptic coral reef communities. I. Distribution and abundance of major groups of encrusting organisms. J Exp Mar Biol Ecol 57: 135-147 Jackson J (1977) Competition on marine hard substrata: the adaptive significance of solitary and colonial strategies. Am Nat 111(980): 743-767 Jernakoff P (1985) The effect of overgrowth by algae on the survival of the intertidal barnacle Tesseropora rosea Krauss. J Exp Mar Biol Ecol 94: 89-97 Jompa J, McCook LJ (2002) Effects of competition and herbivory on interactions between a hard coral and brown alga. J Exp Mar Biol Ecol 271: 25-39 Lobban CS, Harison PJ (1994) Seaweed ecology and physiology. Cambridge University Press, Cambridge McClanahan TR, Kurtis JD (1991) Population regulation of the rock-boring sea urchin Echinometra mathaei (de Blainville). J Exp Mar Biol Ecol 147: 121-146 McClanaham TR, Muthiga N (2001) The ecology of Echinometra. In: Lawrence JM (ed). Edible sea urchins: biology and ecology. Developments in Aquaculture and Fisheries Science, vol. 32. Elsevier Science, Amsterdam. pp. 225-243 McClintock JB, Klinger TS, Lawrence JM (1982) Feeding preferences of echinoids for plant and animal food models. Bull Mar Sci 32: 365-369 McCook LJ, Jompa J, Diaz-Pulido G (2001) Competition between corals and algae on coral reefs: a review of evidence and mechanisms. Coral Reefs 19: 400-417 McKinney FK (1992) Competitive interactions between related clades: evolutionary implications of overgrowth interactions between encrusting cyclostome and cheilostome bryozoans. Mar Biol 114: 645-652 Muricy G, Hajdu E, Custdio MR, Klautau M, Russo C, Peixinho S (1991) Sponge distribution at Arraial do Cabo, SE Brazil. In: Magoon OT, Converse H, Tippie V, Tobie LT, Clark D (eds). Proc VIIth Symp Coast Ocean Manag, Long Beach. ASCE Publ. pp. 1183-1195 Oliveira MC (1991) Survival of seaweeds ingested by three species of tropical sea urchins from Brazil. Hydrobiologia 222: 13-17 Paine RT (1966) Food web complexity and species diversity. Am Nat 100: 65-75 Paine RT, Vadas R (1969) The effects of grazing by sea urchins, Strongylocentrotus spp., on benthic algal populations. Limnol Oceanogr 14: 710-719

Palumbi SR (1985) Spatial variation in an alga-sponge commensalismand the evolution of ecological interactions. Am Nat 126: 267274 Preciado I, Maldonado M (2005) Reassessing the spatial relationship between sponges and macroalgae in sublittoral rocky bottoms: a descriptive approach. Helgol Mar Res 59: 141-150 Quinn JF (1982) Competitive hierarchies in marine benthic communities. Oecologia 54: 129-135 Russo AR (1977) Water flow and the distribution and abundance of echinoids (genus Echinometra) on a Hawaiian Reef. Aust J Mar Freshw Res 28: 693-702 Sammarco PW (1980) Diadema and its relationship to coral spat mortality: grazing, competition, and biological disturbance. J Exp Mar Biol Ecol 45: 245-272 Sammarco PW (1982) Echinoid grazing as a structuring force in coral communities: whole reef manipulations. Exp Mar Biol Ecol 61: 31-35 Sokal RR, Rohlf FJ (1995) Biometry, the principles and practices of statistics in biological research (3th ed). WH Freeman and Company, San Francisco Steneck RS, Dethier MN (1994) A functional group approach to the structure of algal-dominated communities. Oikos 69: 476-498 Sutherland J (1976) Species diversity gradients: synthesis of the roles of predation, competition, and temporal heterogeneity. Am Nat 110(973): 351-369 Thacker RW, Becerro MA, Lumbang WA, Paul VJ (1998) Allelopathic interactions between sponges on a tropical reef. Ecology 79(5): 1740-1750 Tuya F, Boyra A, Sanchez-Jerez, Barbera C, Haroun R (2004) Can one species determine the structure of the benthic community on a temperate rocky reef? The case of the long-spined sea-urchin Diadema antillarum (Echinodermata: Echinoidea) in the eastern Atlantic. Hydrobiologia 519: 211-214 Witman JD, Sebens KP (1990) Distribution and ecology of sponges at a subtidal rock ledge in the Central Gulf of Maine. In: Rutzler K (ed). New perspectives in sponge biology. Smithsonian Institution Press, Washington DC. pp. 391-396 Wulff JL (2005) Trade-offs in resistance to competitors and predators, and their effects on the diversity of tropical marine sponges. J Anim Ecol 74: 313-321 Wulff JL (2006) Ecological interactions of marine sponges. Can J Zool 84: 146-166 Yoneshigue Y, Valentin JL (1988) Comunidades algais fotfilas de infralitoral de Cabo Frio, Rio de Janeiro, Brasil. Gayana Bot 45(14): 61-75

Porifera research: Biodiversity, innovation and sustainaBility - 2007

219

Sponges (Porifera, Demospongiae) from Bransfield strait, off Joinville Island, collected by Brazilian Antarctic Program - PROANTAR
Maurcio Campos(1,2*), Beatriz Mothes(1), Cla Lerner(1), Joo Lus Carraro(1,3), Inga Ludmila VeitenheimerMendes(2)
Museu de Cincias Naturais, Fundao Zoobotnica do Rio Grande do Sul. Rua Salvador Frana 1427, 90690-000. Porto Alegre-RS, Brazil. mrcpoa@hotmail.com, bmothes@fzb.rs.gov.br, cblerner@fzb.rs.gov.br, jlc_rs@hotmail.com (2) Programa de Ps-Graduao em Biologia Animal, Universidade Federal do Rio Grande do Sul. Av. Bento Gonalves 9500, 91501-970. Porto Alegre-RS, Brazil. inga.mendes@ufrgs.br (3) Programa de Ps-Graduao em Ecologia, Universidade Federal do Rio Grande do Sul. Av. Bento Gonalves 9500, 91501-970. Porto Alegre-RS, Brazil
(1)

Abstract: This is the first taxonomic study of the sponges of Joinville Island (Bransfield Strait, Antarctica). In total, 10 species were identified, viz. Iophon terranovae, Artemisina apollinis, Myxilla (Ectyomyxilla) mariana, Mycale (Oxymycale) acerata, Isodictya erinacea, Haliclona (Gellius) rudis, Haliclona (Rhizoniera) dancoi, Haliclonissa verrucosa, Microxina benedeni and Microxina phakelloides. Iophon terranovae and M. (E.) mariana are recorded for the first time for this Antarctic region; I. terranovae, M. (E.) mariana, H. verrucosa and M. phakelloides had their bathymetric ranges extended. Keywords: Antarctica, Demospongiae, distribution, taxonomy, PROANTAR

Introduction
The taxonomy of Antarctic sponges was studied by many authors, who described over 300 species collected through several oceanographic expeditions undertaken in the past 110 years. Highlights are the works of Topsent (1901, 1908, 1913, 1917), von Lendenfeld (1907), Kirkpatrick (1908), Hentschel (1914), Burton (1929, 1932, 1934, 1938) and Koltun (1964). More recently, new records were made by Desqueyroux-Fandez (1989), Barthel et al. (1990, 1997), Pansini et al. (1994), Gutt and Koltun (1995), Mothes and Lerner (1995), Calcinai and Pansini (2000), and Ros et al. (2004). Additionally, important contributions were made on the taxonomy of sponges from the subantartic region, which belongs to the Antarctic Faunistic Complex (Sar et al. 1992), by Ridley (1881), Ridley and Dendy (1887), Sollas (1888), Thiele (1905), Burton (1940), Sar (1978), Boury-Esnault and van Beveren (1982) and Desqueyroux and Moyano (1987). In spite of the many studies conducted, some areas are still unsatisfactorily sampled, such as the South Atlantic Ocean islands, the South Shetland Islands and neighboring areas (Ros et al. 2004). The conduction of new faunistic surveys in the Antarctic continent will be of great importance in order to correlate abundance with environmental factors, to improve understanding of yearly changes and also to extend geographic and bathymetric distributions (Desqueyroux-

Fandez 1989), besides describing new species. The present study aims to increase the knowledge of the sponge fauna from Antarctica, and also to provide a complete illustration of all the identified species.

Materials and methods


The sponges studied here were collected with a beamtrawl during the IVth expedition of the Brazilian Antarctic Program, near Joinville Island (6253S-5627W / 6301S5449W; Fig. 1), between 82 and 274 m depth. The specimens are deposited in the Porifera collection of Museu de Cincias Naturais, Fundao Zoobotnica do Rio Grande do Sul, Brazil. Taxonomic identification was based on dissociated spicules mounts and thick sections of skeletal architecture, following the techniques of Mothes-de-Moraes (1978) and Mothes et al. (2004); preparations for SEM study were done according to Mothes and Silva (2002). Abbreviations used are BMNH (The Natural History Museum, London, England); MCNPOR (Porifera Collection, Museu de Cincias Naturais, Fundao Zoobotnica do Rio Grande do Sul, Brazil); MSNG (Museo Civico di Storia Naturale Giacomo Doria, Genova, Italy); ZMB (Zoologische Museum fr Naturkunde an der Universitt Humboldt zu Berlin, Berlin, Germany).

220

Spicules: Megascleres: styles I: 350-450-550 / 12-18-21 m (Figs. 2C-D); styles II: 330-410-520 / 2.5-4.8-9.0 m (Fig. 2E); anisochelae: 48-55-62 m (Fig. 2F); bipocilli: 7.5-10-12 m (Fig. 2G). Remarks: Slightly malformed styles I, albeit seen in comparative material, were not observed in the specimen from Joinville Island. Distribution: Antarctica (Victoria Land, Bransfield Strait, Joinville I.). Bathymetry: 82-135 m. Family Microcionidae Artemisina apollinis (Ridley and Dendy, 1886) (Figs. 3A-J) Amphilectus apollinis Ridley and Dendy, 1886: 350. Artemisina apollinis; Koltun, 1976: 188; DesqueyrouxFandez, 1989: 125, figs. 22a-e; Ros et al., 2004: 103, figs. 5A-H. Further synonymy see Desqueyroux-Fandez (1989). Material: MCNPOR 1999, Est. 4866: 6253 S - 5627 W, 194 m, 03.II.1986.
Fig. 1: Map showing the studied area; marked points indicate the specific collecting places.

Comparative material: BMNH 1908.2.5.166d - Artemisina apollinis (Ridley and Dendy, 1886). Description: Massive specimen (Fig. 3A), dimensions 7.5 x 4.5 x 3.2 cm; surface hispid to the touch with ramified conules, oscules 0.3-0.4 cm diameter. Preserved material fragile, with brittle consistency, colour light brown. Skeleton: (Fig. 3B) Ectosome without specialization. Choanosome formed by multispicular directionless tracts, whereas closer to the surface such tracts are perpendicularly arranged, producing the surface hispidation. Isochelae dispersed throughout the skeleton. Spicules: Megascleres: styles I: 550-663-730 / 30-34-38 m (Figs. 3C-D); styles II: 320-412-530 / 5.0-6.0-9.0 m (Figs. 3E-F); isochelae: 12-15-16 m (Fig. 3G); toxas I: 340-520740 m (Fig. 3H-I); toxas II: 118-157-195 m (Fig. 3J). Remarks: Spicule sizes reported for the species are varied, but nevertheless measurements obtained for the sample from Joinville Island are slightly different. The comparative material analysed has thinner styles I and smaller toxas, not separable in different size classes (remeasured, in m: styles I: 503-582-665 / 13-15-17; styles II: 295-348-485 / 4.0-5.07.0; isochelas: 14-16-18; toxas: 80-186-370). Samples from Kerguelen (Koltun 1976, Boury-Esnault and van Beveren 1982) have smaller styles I, and toxas separable in distinct size classes only in some descriptions (Topsent 1908, BouryEsnault and van Beveren 1982, Desqueyroux-Fandez 1989).

Results
Order Poecilosclerida Suborder Microcionina Family Acarnidae Iophon terranovae Calcinai and Pansini, 2000 (Figs. 2A-G) Iophon terranovae Calcinai and Pansini, 2000: 371, figs. 2AJ, figs. 3A-F Material: MCNPOR 1951, Est. 4865: 6255 S - 5516 W, 82 m, 03.II.1986. Comparative material: MSNG 31 - Iophon terranovae Calcinai and Pansini, 2000. Description: Cylindrical specimen, incomplete (Fig. 2A), dimensions 6.7 x 3.2 x 3.4 cm; surface smooth, oscules not observed. Preserved material extremely friable consistency, colour dark brown. Skeleton: (Fig. 2B) Ectosome with styles I, perpendicular to the surface and protruding beyond it. Choanosome a confused reticulation, styles I and II arranged in multispicular tracts or dispersed. Anisochelae and bipocilli occur throughout the skeleton.

221

Fig. 2: Iophon terranovae Calcinai and PansinI. 2000. A. Specimen. B. Skeleton. C. Styles I. D. Extremities of style I. E. Styles II. F. Anisochela. G. Bipocilli.

Distribution: Subantarctic region (Kerguelen I.), South Atlantic (South Georgia), Antarctica (Wilhelm II Land, Graham Land, Victoria Land, South Shetland Is., Bransfield Strait, Joinville I.). Bathymetry: 7-380 m. Suborder Myxillina Family Myxillidae Myxilla (Ectyomyxilla) mariana Ridley and Dendy, 1886 (Figs. 4A-J) Myxilla mariana Ridley and Dendy, 1886: 472. Ectyomyxilla mariana; Koltun, 1964: 76. Further synonymy see Koltun (1964). Material: MCNPOR 1962, Est. 4865: 6255 S - 5516 W, 82 m, 03.II.1986. Comparative material: BMNH 1887.5.2.108 - Myxilla mariana Ridley and Dendy, 1886. Description: (Fig. 4A) Fragment, 7.7 x 4.5 x 3.8 cm in dimensions; surface rugose, with ridges and grooves; several oscules scattered on the surface (< 0.1-0.2 cm diameter).

Preserved material slightly compressible and fragile, colour light brown. Skeleton: (Fig. 4B) Ectosome formed by megascleres in confusion. Choanosome bearing an inconspicuous reticulation which sometimes forms triangular meshes, with multispicular tracts transversed by free spicules, without any evident orientation. Spicules: Megascleres: acanthostyles I: 342-438-494 / 1316-20 m (Figs. 4C-D); acanthostyles II: 76-104-142 / 4.05.0-6.0 m (Figs. 4E-F); tylotes: 238-271-304 / 7.0-9.0-12 m (Figs. 4G-H); isochelae: 20-25-30 m (Fig. 4I); sigmas: 41-50-71 m (Fig. 4J). Remarks: Strongyloid tylotes were observed in the studied samples, some of which without spines. Isochelae are smaller in comparison to the measurements supplied by Ridley and Dendy (1887) and Hentschel (1914). Another unexpected observation made here were the very small numbers of acanthostyles II. The comparative material studied has spicules identical to those observed in the specimen from Joinville Island, with only a few significant differences (remeasured, in m: acanthostyles I: 332-385-418 / 13-14-16; acanthostyles II: 103-139-193 / 8.0-10-14; tylotes: 209-244-275 / 6.0-7.08.0; isochelae: 17-29-49; sigmas: 34-44-57).

222

Fig. 3: Artemisina apollinis (Ridley and Dendy, 1886). A. Specimen. B. Skeleton. C. Styles I. D. Extremities of style I. E. Style II. F. Extremities of styles II. G. Isochela. H. Toxa I. I. Detail of extremity of toxa I. J. Toxa II.

Distribution: Subantarctic region (Marion I.), South America (Chile), Antarctica (Wilhelm II Land, Queen Mary Land, Joinville I.). Bathymetry: 82-385 m. Suborder Mycalina Family Mycalidae Mycale (Oxymycale) acerata (Kirkpatrick, 1907) (Figs. 5A-H) Mycale acerata Kirkpatrick, 1907: 281; Burton, 1934: 23, pl. VIII, figs. 1-4; 1938: 11; 1940: 103; Koltun, 1976: 169; Desqueyroux and Moyano, 1987: 48; Desqueyroux-Fandez, 1989: 111, pl. II, figs. 9a-e, pl. VII, fig. 43, pl. VIII, figs. 4447; Barthel et al., 1990: 122; 1997: 48; Pansini et al., 1994: 70; Cattaneo-Vietti et al., 1999: 540; Ros et al., 2004: 110, text-fig. 9A-E

Oxymycale acerata; Sar et al., 1990: 253; Gutt and Koltun, 1995: 231. Further synonymy see Desqueyroux-Fandez (1989). Material: MCNPOR 1983, Est. 4864: 6301 S - 5449 W, 275 m, 02.II.1986. Comparative material: BMNH 1908.2.5.171b - Mycale acerata Kirkpatrick, 1907. Description: (Fig. 5A) Erect and ramified specimen, dimensions: 9.0 x 7.0 x 9.5 cm; surface partially destroyed. Preserved material bears hard consistency and little flexibility, colour mostly white, with light brown shades in some regions. Skeleton: Ectosome a tangential reticulation composed by multispicular tracts, forming triangular meshes (Fig. 5B). Choanosome with thick and compact multispicular tracts, connected by secondary tracts (Fig. 5C). Both types of anisochelae, as well as the raphids, are distributed along the entire tracts.

223

Fig. 4: Myxilla (Ectyomyxilla) mariana Ridley and Dendy, 1886. A. Specimen. B. Skeleton. C. Acanthostyle I. D. Extremities of acanthostyle I. E. Acanthostyle II. F. Extremities of acanthostyle II. G. Tylote. H. Extremities of tylote. I. Isochela. J. Sigmas.

Spicules: Megascleres: oxeas: 650-806.4-890 / 12.5-17.1-20 m (Figs. 5D-E); raphids: 25-31-35 m (Fig. 5F); anisochelae I: 87.5-104.6-117.5 m (Fig. 5G); anisochelae II: 30-44.852.5 m (Fig. 5H). Remarks: The species is widely distributed in the Antarctic and subantarctic regions. Comparative material analysed only differs by the presence of larger raphids and absence of smaller anisochelae (remeasured, in m: oxeas: 760-830-920 / 17-19-24; raphids: 62-76-90; anisochelas: 90-102-117). Distribution: Subantarctic region (Macquarie I., Kerguelen I.), South America (Chile, Argentina, Falkland Is.), South Atlantic Ocean (Shag Rocks, South Georgia, South Orkneys), Antarctica (Victoria Land, Graham Land, Adelie Land, Wilhelm II Land, Banzare Land; McRobertson Land; Princess

Ragnhild Land, Enderby Land, Weddell Sea, South Shetland Is., Joinville I.). Bathymetry: 0-731 m. Family Isodictyidae Isodictya erinacea (Topsent, 1916) (Figs. 6A-E) Homoeodictya erinacea Topsent, 1916: 169 Isodictya erinacea; Koltun, 1964: 40, pl. VIII, figs. 4-7; 1976: 171; Desqueyroux, 1972: 52; 1975: 59, pl. II, figs. 18-20; Vacelet and Arnaud, 1972: 15; Desqueyroux-Fandez, 1989: 114, pl. III, figs. 12a-c, pl. IX, figs. 53-55; Barthel et al., 1990:

224

Fig. 5: Mycale (Oxymycale) acerata (Kirkpatrick, 1907). A. Specimen. B. Ectosome. C. Choanosome. D. Oxea. E. Extremities of oxea. F. Raphid. G. Anisochelas I, H. Anisochelas II.

122; 1997: 48; Sar et al., 1990: 253; Pansini et al., 1994: 72; Gutt and Koltun, 1995: 230; Cattaneo-Vietti et al., 1999: 540; Ros et al., 2004: 116, figs. 14a-g. Further synonymy see Koltun (1964). Material: MCNPOR 1961, Est. 4865: 6255 S - 5516 W, 82 m, 02.II.1986. Description: (Fig. 6A) Ramose fragment, dimensions: 14 x 3.5 x 2.0 cm; spiny surface, branching from the central axis. Preserved material of stiff consistency, colour light brown. Skeleton: (Fig. 6B) Ectosome absent. Choanosome composed of thick longitudinal multispicular tracts (400-820 m thickness), irregularly connected by megascleres in crisscross arrangement, forming rounded to polygonal meshes (370-810 m diameter). Isochelae dispersed along the tracts.

Spicules: Megascleres: oxeas: 600-718.4-800 / 18.8-27.131.3 m (Figs. 6C-D); isochelae: 42.5-52.7-57.5 m (Fig. 6E). Remarks: Desqueyroux-Fandez (1989) found a second category of smaller isochelae in her samples. In the samples studied by Ros et al. (2004), as well as in the present study, such spicules were also seen, but considered to be growth stages of the larger ones. Distribution: South Atlantic Ocean (South Georgia, Burdwood Bank), Antarctica (Graham Land, Palmer Archipelago, Victoria, Banzare Land; McRobertson Land, Enderby Land, Adelie Land, Weddell Sea, Joinville I., South Shetland Is., Bransfield Strait). Bathymetry: 20-920 m.

225

Fig. 6: Isodictya erinacea (Topsent, 1916). A. Specimen. B. Skeleton. C. Oxeas. D. Extremities of oxea. E. Isochela.

Order Haplosclerida Suborder Haplosclerina Family Chalinidae Haliclona (Gellius) rudis (Topsent, 1901) (Figs. 7A-G) Gellius rudis Topsent, 1901: 14, pl. I, fig. 9, pl. III, fig. 4; Desqueyroux-Fandez, 1989: 127, pl. IV, figs. 24a-b, pl. XV, fig. 86; Barthel et al., 1990: 123; Pansini et al., 1994: 80; Cattaneo-Vietti et al., 1999: 540. Further synonymy see Desqueyroux-Fandez (1989).

Material: MCNPOR 1984, Est. 4866: 6253 S - 5627 W, 194 m, 03.II.1986. Description: (Fig. 7A) Digitiform specimen, dimensions: 8.0 x 5.5 cm; surface hispid to the touch, with ridges and grooves; oscules 0.1-0.2 cm diameter, positioned on top of conules; small pores observed on surface (< 0.1 cm diameter). Preserved material with friable consistency, colour light brown. Skeleton: (Fig. 7B) Ectosome without specialization. Choanosome formed by a dense arrangement of multispicular tracts, interconnected by isolated megascleres. Part of the skeleton is halichondrioid, confused and irregular, with tracts oriented in several directions. Sigmas are seen at the nodes of megascleres.

226

Fig. 7: Haliclona (Gellius) rudis (Topsent, 1901). A. Specimen. B. Skeleton. C. Oxea I. D. Extremities of oxea I. E. Oxea II. F. Extremities of oxea II. G. Sigmas.

Spicules: Megascleres: oxeas I: 330-439.6-530 / 12.5-17.420 m (Figs. 7C-D); oxeas II: 240-322-420 / 2.5-5.5-8.8 m (Figs. 7E-F); sigmas: 17.5-32.5-55 m (Fig. 7G). Remarks: The spicules in the present material differ from those reported in most previous studies. In the present study two size classes of oxeas were found, a feature previously reported only by Boury-Esnault and van Beveren (1982). Distribution: Subantarctic region (Kerguelen I.), Antarctica (Bellingshausen Sea, Graham Land, Victoria Land, Weddell Sea, Joinville I., South Shetland Is., Bransfield Strait). Bathymetry: 20-500 m. Haliclona (Rhizoniera) dancoi (Topsent, 1901) (Figs. 8A-D) Reniera dancoi Topsent, 1901: 12, pl. II, fig. 1, pl. III, fig. 3.

Haliclona dancoi; Koltun, 1964: 95, pl. XV, figs. 5-6; 1976: 196; Barthel et al., 1990: 123; Gutt and Koltun, 1995: 231; Cattaneo-Vietti et al., 1999: 540. Further synonymy see Koltun (1964). Material: MCNPOR 1986, Est. 4867: 6257 S - 5650 W, 95 m, 03.II.1986. Description: (Fig. 8A) Partially broken specimen, arborescent, dimensions: 5.2 x 1.2 x 1.4 cm; surface hispid to the touch, with protruding spicules; oscules 0.1-0.2 cm in diameter. Preserved material with friable consistency, colour beige. Skeleton: (Fig. 8B) Ectosome formed by the ends of primary tracts, partially arranged in discrete bouquets. Choanosomal network composed by multispicular primary tracts (65-140 m thickness), connected by uni to paucispicular secondary tracts, forming polygonal to triangular meshes. Spicules: Megascleres: oxeas: 380-468.2-590 / 16.3-23.6-30 m (Figs. 8C-D).

227

Fig. 8: Haliclona (Rhizoniera) dancoi (Topsent, 1901). A. Specimen. B. Skeleton. C. Oxea. D. Extremities of oxea.

Remarks: Comparing the measurements of spicules from previous records (Topsent 1901, 1908, Kirkpatrick 1908, Hentschel 1914, Koltun 1964, 1976) with measurements obtained in the present study, some variation was observed; however this particularity is interpreted as intraespecific variation. Distribution: South Atlantic Ocean (South Orkneys), Antarctica (Bellingshausen Sea, Graham Land, Victoria Land, Wilhelm II Land, Princess Elisabeth Land, McRobertson Land, Enderby Land, Adelie Land, Sabrina Land, Weddell Sea, Joinville I., South Shetland Is.). Bathymetry: 18-2267 m.

Comparative material: BMNH Haliclonissa verrucosa Burton, 1932.

1928.2.15.723a

Description: (Fig. 9A) Cylindrical sponge; dimensions: 5.0 x 1.9 x 1.2 cm; surface verrucose and hispid to the touch. Oscules 0.1-0.3 cm in diameter. Preserved material showing very friable consistency, colour beige. Skeleton: (Fig. 9B) Ectosome formed by the ends of choanosomal tracts, in varied positions. Choanosome composed by longitudinal multispicular tracts, which protrude through the surface, irregularly connected by secondary tracts. Both types of oxeas form the tracts, although few oxeas II are present in the specimens studied. Spicules: Megascleres: oxeas I: 351.5-412.1-503.5 / 10.412.4-15 m (Figs. 9C-D); oxeas II: 256.5-288.2-323 / 2.54.3-6.9 m (Figs. 9E-F). Remarks: Desqueyroux-Fandez and Valentine (2002) added new information concerning the spicular content of this species, recording a second category of oxeas which were also observed in the present study. Distribution: South America (Uruguay, Argentina), Antarctica (Palmer Archipelago, Victoria Land, Weddell Sea, Joinville I., South Shetland Is.). Bathymetry: 25-194 m.

Family Niphatidae Haliclonissa verrucosa Burton, 1932 (Figs. 9A-F) Haliclonissa verrucosa Burton, 1932: 270, pl. LI, fig. 3, textfig. 8; 1940: 100; Koltun, 1964: 102; Desqueyroux, 1972: 54; Barthel et al., 1990: 123. Material: MCNPOR 1990, Est. 4866: 6253 S - 5627 W, 194 m, 03.II.1986.

228

Fig. 9: Haliclonissa verrucosa Burton, 1932. A. Specimen. B. Skeleton. C. Oxea I. D. Extremities of oxea I. E. Oxea II. F. Extremities of oxea II.

Microxina benedeni (Topsent, 1901) (Figs. 10A-E) Gelliodes benedeni Topsent, 1901: 16, pl. II, fig. 3, pl. III, fig. 5. Microxina benedeni; Burton, 1934: 11; Koltun, 1976: 196; Desqueyroux, 1975: 73, pl. IV, figs. 55-57; Barthel et al., 1990: 123; 1997: 49; Pansini et al., 1994: 80; Gutt and Koltun, 1995: 230; Cattaneo-Vietti et al., 1999: 540. Further synonymy see Desqueyroux (1975).

Material: MCNPOR 1982, Est. 4866: 6253 S - 5627 W, 194 m, 03.II.1986. Description: (Fig. 10A) Cylindrical specimen; dimensions: 7.3 x 2.2 x 2.4 cm; surface densely spiny due to the presence of stiff conules; oscules 0.1-0.3 cm diameter. Preserved material very firm and incompressible in consistency, colour light brown. Skeleton: (Fig. 10B) Ectosome without tangential specialization. Choanosome composed by longitudinal multispicular tracts (320-700 m thickness), forming tufts

229

Fig. 10: Microxina benedeni Topsent, 1901. A. Specimen. B. Skeleton. C. Oxea. D. Extremities of oxea. E. Sigma.

which characterize the superficial texture; between the tracts the spicules occur in an irregular arrangement which can bear irregular meshes. Spicules: Megascleres: oxeas: 408.5-804.1-902.5 / 27.5-30.355 m (Figs. 10C-D); sigmas: 20-30.4-55 m (Fig. 10E). Remarks: The present sample has only sigmas as microscleres, which occur in low frequency. Distribution: South America (Falkland Is.), South Atlantic Ocean (South Georgia), Antarctica (Bellingshausen Sea, Graham Land, Victoria Land, Palmer Archipelago, Banzare Land; Princess Elisabeth Land, McRobertson Land, Enderby Land, Weddell Sea, Joinville I., South Shetland Is.). Bathymetry: 81-1266 m. Microxina phakelloides (Kirkpatrick, 1907) (Figs. 11A-F) Sigmaxynissa phakelloides Kirkpatrick, 1907: 272. Gellius phakelloides; Barthel et al., 1990: 123.

Haliclona phakelloides; Koltun, 1964: 101, pl. XIV, figs. 1113. Further synonymy see Koltun (1964). Material: MCNPOR 2046, Est. 4865: 6255 S - 5516 W, 82 m, 03.II.1986. Description: (Fig. 11A) Massive and amorphous specimen; dimensions: 7.8 x 6.3 x 1.1 cm; surface conulose; oscules 0.1 cm diameter. Preserved material with friable consistency, colour light brown. Skeleton: (Fig. 11B) Ectosome formed by thick multispicular tracts, perpendicular to the surface, where megascleres are positioned in tufts which render the surface hispid. Choanosome with uni- to paucispicular tracts forming polygonal meshes, diameter 300-380 m. Sigmas and toxas between the meshes. Spicules: Megascleres: oxeas: 627-707-779 / 25.3-32.7-36.8 m (Figs. 11C-D); sigmas: 52.9-83.9-128.8 / 2.5-3.9-5.0 m (Fig. 11E); toxas: 94.3-130-184 / 2.5-3.9-5.0 m (Fig. 11F). Remarks: The spicules of the species are very characteristic, with oxeas of great dimensions, sigmas with remarkable

230

Fig. 11: Microxina phakelloides (Kirkpatrick. 1907). A. Specimen. B. Skeleton. C. Oxea. D. Extremities of oxea. E. Sigmas. F. Toxas.

deformation in its contour and centroangulate toxas. The values registered by Kirkpatrick (1908), Hentschel (1914) and Koltun (1964) for the spicular meaurements are very similar to the samples of the present study. Distribution: Antarctica (Victoria Land, Knox Land, Banzare Land, Wilhelm II Land, Weddell Sea, South Shetland Is., Joinville I., Bransfield Strait). Bathymetry: 66-550 m.

Concluding remarks
The species dealt with here are all first records for Joinville Island. With the new occurrences of Iophon terranovae, Myxilla (Ectyomyxilla) mariana and Haliclona (Rhizoniera) dancoi for this island, their distribution is extended. The known bathymetric ranges for I. terranovae, M. (Ectyomyxilla)

mariana, Haliclonissa verrucosa and Microxina phakelloides were also extended in the present contribution. This new panorama of the sponges in Antarctica corroborates the ideas of Desqueyroux-Fandez (1989) and Ros et al. (2004), in revealing the necessity for new collections, mainly in the region comprising the Graham Land and Palmer Archipelago, along with South Shetland Is., South Orkneys and the vicinity of South Sandwich. Accomplishment of this task would permit a better understanding of the real geographic and bathymetric distribution of the species belonging to the Antarctic complex. All the species recorded for Jonville I. until the present also occur at continental antarctic areas, and the majority generally extends their occurrence for the southernmost tip of South America, to

231

South Atlantic localities (South Georgia and South Orkneys) and to Kerguelen I. in Subantarctic region.

Acknowledgements
We thank Clare Valentine (BMNH), Dr. Barbara Calcinai (Dipartimento di Scienze del Mare, Universit di Ancona, Ancona, Italy) and Dr. Carsten Lter (ZMB) for the loan of comparative material; Dr. Eduardo Hajdu (Museu Nacional, Universidade Federal do Rio de Janeiro, Brazil) and Dr. Ruth Desqueyroux-Fandez (Museum dHistoire Naturelle de Genve, Geneva, Switzerland) for help with bibliography. We also thank CAPES and CNPq (Brazil) for research grants.

References
Barthel D, Tendal O, Panzer K (1990) Ecology and taxonomy of sponges in the eastern Weddell Sea shelf and slope communities. Ber Polarforsch 68: 120-130 Barthel D, Tendal O, Gatti S (1997) The sponge fauna of the Weddell Sea and its integration in benthic processes. Ber Polarforsch 249: 44-52 Boury-Esnault N, van Beveren M (1982) Les dmosponges du plateau continental de Kerguelen-Heard. Com Nat Fr Rech Antarct 52: 1-175 Burton M (1929) Porifera. Part II. Antarctic sponges. Nat Hist Rep Brit Antarct Terra Nova Exped, 1910, Zool 6(4): 393-458 Burton M (1932) Sponges. Discovery Rep 6: 237-392 Burton M (1934) Sponges. Further Zool Res Swedish Antarctic Exped 1901-1903 3(2): 1-58 Burton M (1938) Non-calcareous sponges. Australas Antarct Exp 1911-1914, Sci Rep Ser C - Zool Bot 9(5): 3-22 Burton M (1940) Las esponjas marinas del Museo Argentino de Ciencias Naturales. An Mus Argent Cienc Nat Bernardino Rivadavia 40: 95-121 Calcinai B, Pansini M (2000) Four new demosponge species from Terra Nova Bay (Ross Sea, Antarctica). Zoosystema 22(2): 369381 Cattaneo-Vietti R, Bavestrello G, Cerrano C, Gaino E, Mazzella L, Pansini M, Sar M (1999) The role of sponges in the Terra Nova Bay ecosystem. In: Faranda F (ed). Benthic ecology of Terra Nova bay. Springer-Verlag, Berlin. pp. 539-549 Desqueyroux R (1972) Gubki (Porifera) sobranie u Iuynix Shetlandskix Ostrovof y Antarktichescovo poluostrovo. Issledovania Fauni Morei 11(19): 49-55 Desqueyroux R (1975). Esponjas (Porifera) de la region antartica chilena. Cah Biol Mar 16: 47-82 Desqueyroux R, Moyano H (1987) Zoogeografia de demospongias chilenas. Bol Soc Biol Concepcin 58: 39-66 Desqueyroux-Fandez R (1989) Demospongiae (Porifera) del litoral chileno antrtico. Ser Cient Inst Antrt Chil 39: 97-158 Desqueyroux-Fandez R, Valentine C (2002). Family Niphatidae. In: Hooper JNA, van Soest RWM (eds). Systema Porifera: a guide to the classification of sponges. Kluwer Academic, New York. pp. 874-889

Gutt J, Koltun WM (1995) Sponges of the Lazarev and Weddell Sea, Antarctica: explanations for their patchy occurrence. Antarct Sci 7 (3): 227-234 Hentschel E (1914) Monaxone Kieselschwmme und Hornschwmme der Deutschen Sdpolar-Expedition 1901-1903. Deutsche Sdpolar-Expedition, 1901-1903 15(1): 35-141 Kirkpatrick R (1907) Preliminary report on the Monaxonellida of the National Antarctic Expedition. Ann Mag Nat Hist (7) 20(117): 271-291 Kirkpatrick R (1908) Porifera (Sponges). II. Tetraxonida, Dendy. Nat Antarct Exped 1901-1904 4: 1-56 Koltun WM (1964) Sponges of the Antarctic. I. Tetraxonida and Cornacuspongida. Biological Reports of the Soviet Antarctic Expedition (1955-1958). Issledovania Fauni Morei 2: 6-116 Koltun WM (1976) Porifera - Part I: Antarctic Sponges. B.A.N.Z. Antarct Res Exped Rep - Ser B Zool Bot 9(4): 147-198 Mothes-de-Moraes B (1978) Esponjas tetraxonidas do litoral sulbrasileiro: II. Material coletado pelo N/Oc. Prof. W. Besnard durante o programa RS. Bol Inst Oceanogr 27(2): 57-78 Mothes B, Lerner CB (1995) Ectyonanchora ruthae sp. n. (Myxillidae) e outras esponjas detectadas na 1 Expedio Antrtica Brasileira (Porifera: Hexactinellida e Demospongiae). Biocincias 3(1): 155-171 Mothes B, Silva CMM (2002) Stelletta ruetzleri sp. nov., a new ancorinid from the Southwestern Atlantic (Porifera, Astrophorida). Sci Mar 66(1): 69-75 Mothes B, Campos MA, Lerner CB, Ferreira-Correia, MM (2004) Esponjas (Demospongiae, Halichondrida) da costa do Maranho, Brasil. Iheringia, Sr Zool 94(2): 149-154 Pansini M, Calcinai B, Cattaneo-Vietti R, Sar M (1994) Demosponges from Terra Nova bay (Ross Sea, Antarctica): 1987/88 and 1989/89 P.N.R.A. Expeditions. Nat Sci Com Antarct 3: 67-100 Ridley SO (1881) XI. Spongida. Horny and siliceous sponges of Magellan Straits, S.W. Chili, and Atlantic off S.W. Brazil. Proc Zool Soc London, 1881: 107-137 Ridley SO, Dendy A (1886) Preliminary report on the Monaxonida collected by H.M.S. Challenger. Ann Mag Nat Hist (5) 18(107): 325-351, 470-493 Ridley SO, Dendy A (1887) Report on the Monaxonida collected by H.M.S. Challenger during the years 1873-1876. Rep Sci Res Voy H.M.S. Challenger, Zool 20: 1-279 Ros P, Cristobo FJ, Urgorri V (2004) Poecilosclerida (Porifera, Demospongiae) collected by the spanish antarctic expedition BENTART-94. Cah Biol Mar 45: 97-119 Sar M (1978) Demospongiae di acque superficiale della Terra del Fuoco (Spedizioni AMF Mares - GRSTS e SAI). Bol Mus Ist Biol Univ Genova 46: 7-117 Sar M, Balduzzi A, Barbieri M, Bavestrello G, Bianchi CN, Boero F, Cattaneo-Vietti R, Corriero G, Morri C, Pansini M (1990) Hard bottom zoobenthos: an analysis of its composition, distribution and of the adaptative strategies of the species. Nat Sci Com Antarct 2: 249-260 Sar M, Balduzzi A, Barbieri M, Bavestrello G, Burlando B (1992) Biogeographic traits and checklist of Antarctic demosponges. Polar Biol 12: 559-585

232

Sollas WJ (1888) Report on the Tatractinellida collected by H.M.S. Challenger during the years 1873-1876. Rep Sci Res Voy H.M.S. Challenger, Zool 25: 1-458 Thiele J (1905) Die Kiesel- und Hornschwmme der Sammlung Plate. Zool Jahrb 6: 407-496 Topsent E (1901) Spongiaires. Rs Voy S.Y. Belgica 1897-1898-189, 6-9: 1-54 Topsent E (1908) Spongiaires. Exp Antarct Fr (1903-1905) comm Dr. Jean Charcot 4: 1-37 Topsent E (1913) Spongiaires de lExpdition Antarctique Nationale cossaise. Trans Roy Soci Edinburgh 49(3): 579-643

Topsent E (1916) Diagnoses dponges recueillies dans lAntarctique par le Pourquoi-Pas? Bull Mus Nat Hist Nat (1) 22(3): 163-172 Topsent E (1917) Spongiaires. In: Joubin L (ed). Deuxime Expdition Antarctique Franaise (1908-1910) commande par le Dr. Jean Charcot. Sciences Physiques: Documents Scientifiques (Paris). 4. Masson & Cie, Paris. pp. 1-88 Vacelet J, Arnaud F (1972) Invertbrs marins des XIIme et XVme expditions antarctiques franaises en Terre Adlie. 2. Dmosponges. Tethys Suppl 4: 9-24 von Lendenfeld R (1907) Tetraxonia der Deutschen SdpolarExpedition 1901-1903. Deutsche Sdpolar-Expedition, 1901-1903 9(1): 303-342

Porifera research: Biodiversity, innovation and sustainaBility - 2007

233

Demospongiae (Porifera) of the shallow coral reefs of Macei, Alagoas State, Brazil
Victor Ribeiro Cedro(1), Eduardo Hajdu(2), Hilda Helena Sovierzosky(1), Monica Dorigo Correia(1*)
Setor de Comunidades Bentnicas, Instituto de Cincias Biolgicas e da Sade, Universidade Federal de Alagoas. vrcedro@gmail.com, mdc@fapeal.br (2) Museu Nacional, Departamento de Invertebrados, Universidade Federal do Rio de Janeiro. hajdu@acd.ufrj.br
(1)

Abstract: Sponges occurring at Alagoas State reefs, in north-eastern Brazil, are poorly known. This work reports on the sponges of Macei coral reefs, the states capital. A total of 29 species were identified so far. These were Agelas dispar, Amphimedon aff. complanata, A. viridis, Biemna microacanthosigma, Chalinula molitba, Chondrilla aff. nucula, Chondrosia collectrix, Cinachyrella apion, C. alloclada, Cliona aff. celata, C. varians, Dragmacidon reticulatum, Dysidea etheria, Echinodictyum dendroides, Geodia corticostylifera, G. papyracea, Haliclona curacaoensis, H. manglaris, H. melana, Iotrochota birotulata, Ircinia strobilina, Mycale diversisigmata, Niphates erecta, Placospongia aff. melobesioides, Scopalina ruetzleri, Spirastrella coccinea, S. hartmani, Tedania ignis and Tethya sp. The numbers of species in each station varied from 16 to 24. Mycale diversisigmata is a first record for the southern Atlantic. These preliminary results indicate the occurrence of a moderately rich sponge fauna at the Macei reefs area, arguing for stricter control on human impact on these urban reefs. Keywords: Alagoas, Demospongiae, coral reef, SW Atlantic

Introduction
Sponges are among the most important components of coral reef benthic communities, frequently exceeding hermatypic corals in diversity and biomass. Each species abundance and distribution within a coral reef sponge community is related to biotic and abiotic parameters such as recruitment, spatial competition, luminosity, sedimentation and substrate type (Wiedenmayer 1977, Zea 1987, Daz and Rtzler 2001, Rtzler 2002, Valderrama and Zea 2003). Brazil has a large coastline, with a faunistic component which is still poorly studied in general, and Porifera stands out as one of the least studied taxa. Very few coastal states in Brazil have a well-known sponge fauna (e.g. Hajdu et al. 1996, 1999, Muricy and Silva 1999), and comprehensive knowledge of species distributions, habitat requirements, reproduction and all sorts of ecological interactions are virtually unknown for the entire coast. The taxonomy of marine sponges of north-eastern Brazil (geographic north-east coastal states Bahia to Cear; not regional, geopolitical north-east coastal states Bahia to Maranho) has been the focus of a very few studies, and even fewer described species from Alagoas, mostly from deep-waters. The latter date back to the H.M.S. Challenger expedition, three collecting stations having been set off the state of Alagoas (st. 122b, 122c, 123). The species reported from this material were Cacospongia levis (Poljaeff, 1884) and Stelospongus longispinus Duchassaing and Michelotti, 1864 [= Ircinia strobilina (Lamarck, 1816)], reported by Poljaeff (1884); Pheronema carpenteri Schulze, 1887, reported by Schulze (1887); and Geodia neptuni (Sollas, 1886)

[= Geodia vosmaeri (Sollas, 1886)] and Thenea fenestrata (Schmidt, 1880), reported by Sollas (1888). Further studies reporting on sponges of the north-eastern Brazilian coast were those of de Laubenfels (1956; without descriptions), Johnson (1971; essentially based on beach worn material), Boury-Esnault (1973), Hechtel (1976; without descriptions, 1983) and Sarmento and Correia (2002; without descriptions), among others. The high diversity of sponges on the Brazilian north-eastern coast has been best illustrated by Boury-Esnault (1973), Muricy and Moraes (1998) and Muricy et al. (2006), and of north-eastern Brazilian oceanic islands by Mothes and Bastian (1993) and Moraes et al. (2006). Sarmento and Correia (2002) reported the finding of 17 species of demosponges on Ponta Verde coral reef (Macei, AL), and correlated these to different habitat types. The present study reports on sponge assemblages found in four shallow urban reef areas of Macei (Alagoas State, north-eastern Brazil).

Materials and methods


Four sampling areas were chosen: (1) Pajuara reef (94100.27S / 354319.78W; Fig. 1A, 2A), (2) Piscina dos Amores (94009.24S / 354214.16W; Fig. 1B, 2B), (3) Ponta Verde (93956.87S / 354144.98W; Fig. 1C, 2C) and (4) Jatica reef (93914.56S / 354150.05W; Fig. 1D, 2D), in a SW - NE sequence. Distances were 2300 m between (1) and (2), 2000 m between (2) and (3) and 1200 m between (3) and (4). Collections were made in the years 2004 and 2005 by wading at low tide and snorkelling. Live specimens were observed underwater, and whenever possible photographed. Each specimen was individually

234

Results and discussion


A total of twenty-nine sponge species were identified (Table 1), comprising ten orders and nineteen families. The Haplosclerida, with seven species, was the richest order, followed by the Hadromerida, with six, and the Poecilosclerida, with five. The numbers of species found in each station varied from 16 in the Piscina dos Amores to 24 at Ponta Verde reef. This difference is likely to reflect the more extensive sampling at Ponta Verde, an easily accessible reef, coupled with its possession of a large sciaphilic and slightly eutrophic environment, where sponges constitute the dominant benthic taxon. Amphimedon viridis (Fig. 2E) was the sole species observed to be very common on all four sampled areas, thus confirming once more its abundance along the Brazilian coastline. The species has been previously reported as common along the south-eastern Brazilian coast (Muricy et al. 1991, Hajdu et al. 1999). Cinachyrella alloclada (Fig. 2F) was very common on three areas, and Chondrilla aff. nucula, Cliona aff. celata, C. varians, Haliclona manglaris (Fig. 2H), H. melana, Tedania ignis (Fig. 2G) and Tethya sp. on two stations only. Of these, all but H. manglaris were previously known to be common on distinct sectors of the Brazilian coastline (Muricy et al. 1991, Klautau et al. 1999, Muricy and Ribeiro 1999, Lazoski et al. 2001). Cinachyrella apion, Iotrochota birotulata and Spirastrella coccinea were all rare and each species was found in a single station. Mycale diversisigmata is a first record for the southern Atlantic. Tethya sp. is probably a new species and needs a formal description. Twenty-one out of the 28 species found are distributed in the Caribbean area too (e.g. Pulitzer-Finali 1986, van Soest et al. 2007), which indicates a marked Tropical western Atlantic affinity of the Macei reefs sponge fauna. Only three species found are provisional Brazilian endemics, viz. Echinodictyum dendroides, Biemna microacanthosigma and the likely new species of Tethya. Four species have widespread disjunct distributions and entitle for a morphogenetic revision study (Chondrilla aff. nucula, Chondrosia collectrix, Cliona aff. celata, Placospongia aff. melobesioides). Only 12 species reported here were listed for the neighbouring state of Pernambuco by Muricy and Moraes (1998), viz. Agelas dispar, Amphimedon viridis, Chondrilla aff. nucula (as Chodrilla nucula), Chondrosia collectrix, Cliona varians (as Anthosigmella varians), Dragmacidon reticulatum (as Pseudaxinella reticulata), Geodia corticostylifera, Geodia papyracea, Ircinia strobilina, Scopalina ruetzleri, Spirastrella coccinea and Tedania ignis. The similarity has not been found to be higher, probably because the reef strata sampled were

Fig. 1: Map showing the location of Macei (north-eastern Brazil) and the collecting stations analysed in this study. A. Pajuara coral reefs. B. Piscina dos Amores coral reef. C. Ponta Verde coral reef. D. Jatica coral reef. Scales: 400 km in map, 10 km in inserts. Maceis urban area is shaded.

packed and fixed in ethanol 70% soon afterwards. Some specimens collected earlier (2001) and deposited in the MNRJ (Museu Nacional/Universidade Federal do Rio de Janeiro Porifera collection) and UFALPOR (Setor de Comunidades Bentnicas/Universidade Federal de Alagoas Porifera collection) collections were also analyzed. Voucher specimens for each species studied are listed in Table 1, although not for every locality sampled. A complete list of localities sampled and collected specimens is available from the authors upon request. A semi-quantitative estimation of sponge abundance has been applied to each station considered, and consisted of the exhaustive compilation of visual records achieved on at least five full low-tide periods (between 2001 and 2006). These were written down immediately after leaving the sampling grounds when incoming rising tide forced a complete stop in the observations. Sponges were classified into dominant, common and rare. A fourth category, absent, has been added after comparison of data compiled from the four stations, and includes only those species found in at least one station. Specimens were identified by field observations and morphometry of the skeletal architecture. Microscopic slides of dissociated spicules and thick sections were made following the standard methods described by Hooper and van Soest (2002). A few specimens were further studied through scanning electron microscopy.

Fig. 2: Aerial views of the collecting localities, showing the reefs at low or nearly low tide, and some of the commonest species of demosponges found. A. Pajuara coral reefs. B. Piscina dos Amores coral reef. C. Ponta Verde coral reef. D. Jatica coral reef. E-H. Ponta Verde scyaphilic habitat. E. Amphimedon viridis. F. Cinachyrella alloclada (predominantly). G. Tedania ignis. H. Haliclona manglaris. Scales: 2 cm (E, G, H) and 4 cm (F).

235

236

Table 1: Occurrence and semi-quantitative estimation of abundance of the Macei reefs sponge fauna. Legend: (Dominant); (Common); (Rare); (Absent). Pajuara 17 Coral reefs Piscina dos Ponta Verde Amores 16 24 Jatica 24

Taxa Agelas dispar Duchassaing & Michelotti, 1864 (MNRJ 10294) - Agelasida Amphimedon viridis Duchassaing & Michelotti, 1864 (MNRJ 9033 ; UFAL/ POR 0027) - Haplosclerida Amphimedon aff. complanata Duchassaing, 1850 (MNRJ 4713) - Haplosclerida Biemna microacanthosigma Mothes, Hajdu, Lerner & van Soest, 2004 (UFAL/ POR 0043) - Poecilosclerida Chalinula molitba (de Laubenfels, 1949) (UFAL/POR 0042) - Haplosclerida Chondrilla aff. nucula Schmidt, 1862 (MNRJ 3146; UFAL/POR 0049) Chondrosida Chondrosia collectrix (Schmidt, 1870) (MNRJ 10279) - Chondrosida Cinachyrella alloclada (Uliczka, 1929) (MNRJ 10292; UFAL/POR 0004) Spirophorida Cinachyrella apion (Uliczka, 1929) (MNRJ 10290) - Spirophorida Cliona aff. celata Grant, 1826 (MNRJ 4650) - Hadromerida Cliona varians (Duchassaing & Michelotti, 1864) (MNRJ 3151) - Hadromerida Dragmacidon reticulatum (Ridley & Dendy, 1886) (MNRJ 10283;UFAL/POR 0053) - Halichondrida Dysidea etheria de Laubenfels, 1936 (UFAL/POR 0021) - Dictyoceratida Echinodictyum dendroides Hechtel, 1983 (MNRJ 4711; UFAL/POR 0025) Poecilosclerida Geodia corticostilyfera Hajdu, Muricy, Custdio, Russo & Peixinho, 2002 (MNRJ 10274) - Astrophorida Geodia papyracea Hechtel, 1965 (MNRJ 10285;UFAL/POR 0046) Astrophorida Haliclona curacaoensis van Soest, 1980 (MNRJ 10280; UFAL/POR 0040) - Haplosclerida Haliclona manglaris Alcolado, 1984 (MNRJ 10289) - Haplosclerida Haliclona melana Muricy & Ribeiro, 1999 (MNRJ 10277; UFAL/POR 0041) - Haplosclerida Iotrochota birotulata (Higgin, 1877) (MNRJ 10291) - Poeciloclerida Ircinia strobilina Lamarck, 1816 (MNRJ 4717; UFAL/POR 0017) Dictyoceratida Mycale diversisigmata van Soest, 1984 (MNRJ 4639) - Poecilosclerida Niphates erecta Duchassaing. & Michelotti, 1864 (MNRJ 10287) Haplosclerida Placospongia aff. melobesioides Gray, 1867 (MNRJ 4724) - Hadromerida Scopalina ruetzleri (Wiedenmayer, 1977) (MNRJ 10281) - Halichondrida Spirastrella coccinea (Duchassaing & Michelotti, 1864) (MNRJ 4629) Hadromerida Spirastrella hartmani Boury-Esnault, Klautau, Bzac, Wulff & Sol-Cava, 1999 (MNRJ 10286; UFAL/POR 0002) - Hadromerida Tedania ignis (Duchassaing. & Michelotti, 1864) (MNRJ 10293; UFAL/POR 0031) - Poecilosclerida Tethya sp. (MNRJ 4712) - Hadromerida Total of species

different. Muricy and Moraes (1998) focused mainly on the subtidal, while the data presented here were gathered in the intertidal. No sponge species has been hitherto recorded from the coast of Sergipe State, the adjacent state on the south, which is a clear sampling gap. On the other hand, most of the

species reported upon here also occur on Brazilian oceanic islands, as well as in the State of Bahia, further south (Moraes et al. 2006, Peixinho et al. unpubl. res.). These results, albeit preliminary, indicate the occurrence of a moderately rich demosponge assemblage in the Macei

237

intertidal coral reef areas. The new records found on these shallow urban reefs demonstrate the lack of comprehensive faunistic and taxonomic surveys of the benthic fauna of Alagoas coral reefs. Macei has a population quickly approaching 106 citizens. Findings presented here advise for stricter control on human impact on these shallow water urban reefs, some of which are usually visited at low tide by hundreds of people (local collectors of sea-food and tourists). The collected data increase significantly the knowledge concerning the sponge richness of Alagoas state coral reefs.

Acknowledgements
Authors are grateful to Alvaro Borba Junior (LABMAR/UFAL) for helping in the field trips. Marcia Atthias and Noemia Gonalves (Instituto de Biofsica Carlos Chagas Filho/UFRJ) kindly provided access to, and technical assistance on SEM operation. CNPq, FAPEAL and FAPERJ are thanked for the provision of grants and/or fellowships.

References
Boury-Esnault N (1973) Campagne de la Calypso au large des ctes atlantiques de lAmrique du Sud (1961-1962). I, 29. Spongiaires. Rs Scient Camp. Calypso, Paris, 10: 263-295 de Laubenfels, MW (1956) Preliminary discussion of the sponges of Brazil. Contr Avulsas Inst Oceanogr Univ So Paulo Oceanogr Biol 1: 1-4 Daz MC, Rtzler K (2001) Sponges: an essential component of Caribbean coral reefs. Bull Mar Sci 69(2): 535-546 Hajdu E, Berlinck RGS, Freitas JC de (1999) Porifera. In: Migotto A, Tiago CG (eds). Biodiversidade do Estado de So Paulo: sntese do conhecimento ao final do sculo XX (ser. eds. Joly CA, Bicudo CEM), vol. 3. Invertebrados Marinhos. Fapesp, So Paulo. pp. 20-30 Hajdu E, Muricy G, Berlinck RGS, Freitas JC (1996) Marine poriferan diversity in Brazil: through knowledge to management. In: Bicudo CE, Menezes NA (eds) Biodiversity in Brazil: a first approach. So Paulo: Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico. pp. 157-172 Hechtel GJ (1976) Zoogeography of Brazilian marine Demospongiae. In: Harrison FW, Cowden RR (eds), Aspects of sponge biology. Academic Press, New York. pp. 237260 Hechtel GJ (1983) New species of marine Demospongiae from Brazil. Iheringia, Zool, 63: 58-89 Hooper JNA, van Soest RWM (eds.) (2002). Systema Porifera: a guide to the classification of sponges, vol. 1. Kluwer Academic/ Plenum Publishers, New York Johnson MF (1971) Some marine sponges of northeast Brazil. Arq Cienc Mar 11(2): 103-116 Klautau M, Russo CAM, Lazoski C, Boury-Esnault N, Thorpe J, Sol-Cava A (1999) Does cosmopolitanism result from overconservative systematics? A case study using the marine sponge Chondrilla nucula. Evolution 53: 1414-1422

Lazoski C, Sol-Cava AM, Boury-Esnault, N, Klautau M, Russo CAM (2001) Cryptic speciation in a high gene flow scenario in the oviparous marine sponge Chondrosia reniformis. Mar Biol 139: 421-429 Moraes FC, Oliveira MV, Klautau M, Hajdu E, Muricy G (2006) Biodiversidade de esponjas das ilhas ocenicas brasileiras. In: Alves RJV, de Alencar Castro JW. (Orgs.). Ilhas Ocenicas Brasileiras, da Pesquisa ao Manejo. Braslia: Ministrio do Meio Ambiente. pp. 147-177 Mothes B, Bastian MCKA (1993) Esponjas do Arquiplago de Fernando de Noronha (Porifera, Demospongiae). Iheringia Sr Zool 75: 15-31 Muricy G, Hajdu E, Custdio MR, Klautau M, Russo C, Peixinho S (1991) Sponge distribution at Arraial do Cabo, SE. Brazil. In: Magoon OT, Converse H, Tippie V, Tobie LT, Clark D (eds). Proc 7th Symp Coast Ocean Manag (Long Beach, USA). ASCE Publ. 2: 1183-1196 Muricy G, Moraes FC (1998) Marine sponges of Pernambuco State, NE Brazil. Rev Bras Oceanogr 46(2): 213-217 Muricy G, Ribeiro SM (1999). Shallow-water Haplosclerida (Porifera, Demospongiae) from Rio de Janeiro state, Brazil (Southwestern Atlantic). Beaufortia, 49(6): 47-60 Muricy G, Silva OC (1999) Esponjas marinhas do Estado do Rio de Janeiro: um recurso renovvel inexplorado. In: Silva SHG, Lavrado HP (eds). Ecologia dos Ambientes Costeiros do Estado do Rio de Janeiro. Sr Oecol Bras, vol. 2. PPGE-UFRJ. pp. 155-178 Poljaeff N (1884) Report on the Keratosa collected by H.M.S. Challenger during the years 18731876. Rep Sci Res Voy H.M.S. Challenger, Zool 11: 1-88 Pulitzer-Finali G (1986) A collection of West Indian Demospongiae (Porifera). In appendix, a list of the Demospongiae hitherto recorded from the West Indies. Ann Mus civ sto nat Giacomo Doria 86: 65-216 Rtzler K (2002) Impact of crustose sponges on Caribbean reef corals. Acta Geo Hisp 31(1): 61-72 Sarmento FJQ, Correia MD (2002) Descrio de parmetros ecolgicos e morfolgicos externos dos porferos no recife de coral da Ponta Verde, Macei, Alagoas, Brasil. Rev Bras Zooc 4(1): 215-226 Schulze FE (1887) Report on the Hexactinellida collected by H.M.S. Challenger during the years 18731876. Rep Sci Res Voy H.M.S. Challenger, Zool 21: 1-514 Sollas WJ (1888) Report on the Tetractinellida collected by H.M.S. Challenger, during the years 18731876. Rep Sci Res Voy H.M.S. Challenger, Zool 25(63): 1-458 Valderrama D, Zea S (2003) Esquemas de distribuicin de esponjas arrecifales (Porifera) del Noroccidente del Golfo de Urab, Caribe Sur, Colmbia. Bol Invest Mar Cost 32: 37-56 van Soest RWM, Boury-Esnault N, Janussen D, Hooper J (2007) World Porifera database. http://www.marinespecies.org/porifera/. Accessed on 2007-06-23 Wiedenmayer F (1977) Shallow-water sponges of the western Bahamas. Experientia Suppl 28: 1-287 Zea S (1987) Esponjas del Caribe Colombiano. Catlogo Cientifico, Bogot

Porifera research: Biodiversity, innovation and sustainaBility - 2007

239

How and why do sponges incorporate foreign material? Strategies in Porifera


Carlo Cerrano(1*), Barbara Calcinai(2), Cristina Gioia Di Camillo(2), Laura Valisano(1), Giorgio Bavestrello(2)
Dipartimento per lo studio del Territorio e delle sue Risorse, C.so Europa, 26, 16132, Genova, Italy. cerrano@dipteris.unige.it, valisano@dipteris.unige.it (2) Dipartimento di Scienze del Mare, Via Brecce Bianche, 60131, Ancona, Italy. b.calcinai@univpm.it, g.bavestrello@univpm.it, c.dicamillo@univpm.it
(1)

Abstract: The selection and incorporation of foreign materials in sponges is a complex phenomenon: it involves both a system of recognition of pinacocytes versus sand grain mineralogy and a system of coordination among cells, which transport and engulf particles in specific areas of the sponge surface. Concerning the mineralogical characteristic of the incorporated particles, it seems that quartz particles, when incorporated, could play an important role in collagen production. Among incorporating species, two different modalities can be defined, depending on the habit of the species: i) soft-bottom species (e.g. genera Oceanapia, Tectitethya, Cliona) engulf particles mainly from the base of their body and select mainly the size of particles independently from their mineralogical characteristics; engulfed particles, due to their weight, help the sponge to stabilize and to anchor to the soft substrate; ii) in hard-bottom species (e.g. genera Chondrosia and Ircinia) ectosome pinacocytes select particles, in relation to their size and mineralogy, and may incorporate them differently in some areas of their body according to their skeletal arrangement. Keywords: Porifera, selectivity, sediment incorporation, foreign inclusions, mineralogy

Introduction
Marine organisms, particularly in benthic environments, have to coexist with a continuous sediment rain and have adapted to this phenomenon in several ways (Miller et al. 2002). They can react cleaning their surface, more or less actively, or by trying to exploit sediments to feed or to build protective and/or structural elements. Several organisms like protists (Takahashi and Ling 1984), sponges (Teragawa 1986, Cerrano et al. 1999a), cnidarians (Haywick and Mueller 1997) annelids (Wilson 1974, Main and Nelson 1988), molluscs (Min-Da 1984), crustaceans (Dixon and Moore 1997, Krasnow and Taghon 1997), echinoderms (Massin and Doumen 1986), and tunicates (Kott 2006) are able to use foreign material as a cover, to protect or mask their body, building thecae, coats, tubes or other structures. Among Porifera and Cnidaria there are examples of species able to incorporate particles into their body. Its generally assumed that this strategy is performed to strengthen the skeleton but the real meaning of it and the related mechanisms are often unclear (Teragawa 1986). One of the most debated problems regarding this phenomenon is if organisms are able to select foreign bodies or if they utilise every kind of particle available in the surrounding environment. Generally this behaviour is regulated by the ability of organisms to handle particles so that a physical limit related to the particle size has to be always considered. The most intriguing aspect is the ability

of some species to recognise the mineral characteristics of the particles and therefore to select them (Bavestrello et al. 1996). The aim of this paper is to review the incorporation of foreign bodies in sponges, comparing the strategies of species living on soft and hard substrates and suggesting possible physical and biological explanations for this intriguing behaviour.

Soft-bottom sponges
Even if sponges typically live on hard substrates, there are several species more or less adapted to soft bottom environments. These may live loose on the sediment often partially or completely buried in it and survive well thanks to some very special adaptations, which limit sponge rolling and occlusion of aquiferous system by sand. On soft substrates it is possible to observe sponge fragments which occasionally may fall from coral or rocky reefs due to the production of asexual reproductive bodies and/or fragments, breakage during storms, localised infections by pathogens, or predator bites (Wulff 1985, Battershill and Bergquist 1990). The survival of unattached fragments depends on their ability to re-settle in a short span of time to avoid clogging by sediments (Ilan and Abelson 1995). For these fragments the incorporation of large amount of foreign bodies is crucial to assume a gravimetric polarity which allows them to stabilize and reorganize their aquiferous system.

240

We can classify soft bottom sponges into three main groups according to their adaptative strategies: i) sponges living on the sediment surface, ii) sponges partially buried, iii) sponges with the body completely buried, with particular anatomic adaptations.

Sponges on the sediment surface


Here we consider sponges that do not live exclusively on soft substrates but that can easily survive on soft substrates, fully regaining their vital functions. These non-sessile specimens have been generally found in shallow sub-littoral environments (Mercurio et al. 2006, Bell and Barnes 2002) and in the deep sea (Barthel and Tendal 1993). Examples of this habit can be found in lagoon environments (Ise et al. 2004, Mercurio et al. 2006), where several sponge fragments, often from species living typically on hard substrates, can be present. In studies performed in the Caribbean (Cerrano et al. 2004) and the Indonesian (Cerrano et al. 2002) lagoons, the comparison between environmental sediments and the particles incorporated by several sponge species shows that they mainly contain the fraction larger than 5 mm. Only a few species use the fractions available in the surrounding substrates without size preferences. The percentage of incorporated sediments can be highly variable, between 5 and 99% per sponge dry weight. A particular case concerns the gamma stage of Cliona nigricans, an excavating Atlanto-Mediterranean sponge living symbiotically with zooxanthellae that can grow with different shapes: endolithic, into coralligenous accretions, and massive, laid on detritic sediments (Fig. 1A). This species can engulf from the base (Fig. 1B) huge amounts of foreign material, up to 99% of its dry weight, being also able to store the fraction of sediment larger than 5 mm (Calcinai et al. 1999). Moreover, experimental data indicated that in this species the mineralogical features of the engulfed particles can affect morphogenetic processes, in particular quartz negatively affects the growth of C. nigricans specimens limiting the development of the oscula in the basal portion of the sponge that is in direct contact with the grains. On the contrary, oscula have been observed in specimens living on calcareous sand (Cerrano et al. 2007), highlighting once again the importance of substrate chemical composition on benthic organism distribution and development (Cerrano et al. 1999b, Bavestrello et al. 2003). In massive specimens of C. nigricans, the aquiferous system opens on the sediment using a water expulsion mechanisms similar to the one described for Spheciospongia cuspidifera in Belize (Rtzler 1997).

This species incorporates all the granulometric size classes of nearby benthic sediments, using them in different ways. In the choanosome, sediments are sorted and distributed according to their size: fine sediments (40-60 m) are densely aggregated in the choanosome, whereas coarse particles are more evenly distributed in the lower portion of the body were they contribute to the stability of the sponge (Fig. 1C). Qualitatively, the choanosomal aggregations of fine sediment contain more siliceous material than the ambient sediment of the same size class. Microscopical analysis of the particles shows that this species selects and incorporates allocthonous sponge spicules, radiolarians and diatoms (Cerrano et al. 2004). Another interesting species is Biemna fortis, living in tropical lagoons in North Sulawesi (Indonesia). This sponge displays two different growth patterns depending on the thickness of unconsolidated sediments: when the sediment layer is thick, the sponge assumes a cylindrical form and incorporation is low; when there is a thin sediment layer the sponge adheres to the basal coral rock, developing a massive buried portion that is generally rich in embedded particles (Cerrano et al. 2002).

Sponges specialised to psammobiontic habit


All the known species of the genus Oceanapia live on soft substrates thanks to the ability of producing long fistules that anchor the sponge body to the loose substrate and discharge waste-water deep into the sediments (Werding and Sanchez 1991, Bavestrello et al. 2002). The specialisation of this genus to soft substrates is evidenced also by the differential production of secondary metabolites used as antipredatory that are synthesised exclusively in the exposed portions in O. sagittaria, suggesting that sediments are not just a mere substrate where sponges can live with low competition but also a refuge from potential predators (Schupp et al. 1999, Salomon et al. 2001). In lagoons O. amboinensis lives buried in unconsolidated sediments among sea grasses. The sponge develops a massive body and emerges from the sediment through numerous closed fistules. The sponge body is whitish, while the portions protruding from the sediment take an olive green colour. The buried portion of the body incorporates a high quantity of foreign materials, selecting particles larger than 2 mm, throughout the pinacoderm. Only exhalant areas, of 1-4 cm2, do not participate in this process (Cerrano et al. 2002, Bavestrello et al. 2002). Oceanapia fistulosa lives from 15-20 m depth down to at least 80 m, grows partially buried in detritic sediment (Fig. 1D). The globular sponge body, 5-15 cm in diameter, bears on its upper side several closed cylindrical fistules that emerge from the sediment generally covered by epibionts. On the other side, other buried closed fistules are strongly rooted in the sediments. The buried portion of the sponge incorporates a lot of foreign material such as sand, coral and shell fragments, particularly on the rooted fistules, which can reach a length of 15-20 cm and 1 cm in diameter, depending on the thickness and the granulometry of the unconsolidated sediments. In fine sediments this species, to

Sponges partially buried in sediments


Several species can live on soft substrates even without morphological adaptations to this environment. Tectitethya crypta is a massive, shallow-water sponge common in the Caribbean and frequently covered by a sediment and/ or algal coat, both on hard and soft bottoms. In lagoon environments this sponge can occur either loose or anchored, significantly varying its morphology (Cerrano et al. 2004).

241

Fig. 1: Examples from soft-bottoms sponges. A. Specimen of Cliona nigricans living on detritic substrates. B. Detail of the lower face of the sponge with several rocks having highly variable sizes. The fraction bigger than 5 mm is more abundant in the sponge than in the ambient sediments. Arrows indicate oscular openings. C. Half cut specimen of Tectitethya crypta. White arrows indicate aggregations of fine sediments, black arrow indicates coarse sediments. D. Drawing of Oceanapia fistulosa with the buried body mass covered by sand grains.

242

get stabilization, produces more rooted fistules, smaller in diameter but longer than those in coarse sediments.

Hard-bottom sponges
Burial/smothering, scour/abrasion, and changes in the physical characteristics of the substrate surface are the three main mechanisms by which sediments may affect benthic assemblages (Airoldi 2003). On hard substrates sedimentation is partly due to particles suspended in the water column and partly due to the detritus that rolls down vertical cliffs (Bavestrello et al. 1995a). This may cause mechanical damages in sponges and other benthic filter feeders, especially by clogging the aquiferous system impeding filtration. A solution to avoid sedimentation is generally represented by the colonisation of substrates under overhangs, but in this situation sponges have to compete with many other sessile animals that share the same strategy. Other organisms choose to grow vertically, limiting the surface available for sediments as happens for several species of the genera Axinella or Dysidea (Fig. 2A). Other species can clean their pinacoderm using superficial cellular movements (Bond 1992) that can easily either remove or take up several kinds of particles transforming the problem of sediments into an opportunity to providing a physical support to the skeletal development (Fig. 2B). According to Teragawa (1986) the sediment that settles on the surface of Dysidea etheria may follow different pathways being i) inhalated through ostia, ii) eliminated by transport or through dermal membrane oscillations or mucus sloughing, iii) incorporated into primary fibres, and iv) engulfed into secondary fibres in case the sponge is overloaded by sediments. When sediments are incorporated into spongin fibres it is possible to consider this localization as definitive but, on the contrary, a turnover was described for the sediments engulfed in the cortex of Chondrosia reniformis (Cerrano et al. 1999a). This species, presenting a collagenous structure and lacking its own spicules and spongin fibres, when anchored to a substrate, is able to incorporate foreign material, discerning from crystalline quartz sand grains and amorphous siliceous opaline spicules (Bavestrello et al. 1998a, 1998b). Laboratory experiments have shown that the cells of the sponge ectosome play a key role in the selection processes: quartz particles are incorporated while carbonatic particles are agglutinated and drop out from the sponge ectosome. In C. reniformis specimens anchored to the substrate, the upper ectosome can distinguish between silica and carbonates, ability lost in free, non-attached individuals, which incorporate both. This behavior indicates that specific receptors are present and can distinguish among the different mineralogical features of the sediment. Depending on the environmental conditions this mechanism can be switched on or off (Bavestrello et al. 1998b). The turnover of particles inside the body of C. reniformis is due to the ability of this sponge to dissolve quartz crystals releasing silicate (Bavestrello et al. 1995b). In C. reniformis the amount of incorporated sediment was used as a character to separate different species (Wiedenmayer 1977) while in dictyoceratid sponges the presence of sediment

in fibres or as a dermal crust is considered as a character to distinguish between different genera (Vacelet 1959). Nevertheless, Pronzato et al. (2004) considered the amount of mineral granules a specific character to distinguish Ircinia felix from I. variabilis. The evidence of a specific and fine tuned mechanism to select particles, according to their mineralogical features, suggests that a mineralogical and granulometric analysis of incorporated sediments may represent a tool for the classification of problematic taxonomic groups. The genus Ircinia is characterised by spongin fibres cored with fo