IOC Science and Communication Centre on Harmful Algae University of Copenhagen

Harmful dinoflagellates, Gymnodiniales, page 1 of 7

Module III Section 5, Gymnodiniales and Noctilucales (4 hours)

Contents
5.1. Suggested reading 5.2. Introduction to the taxonomy of unarmoured dinoflagellates Identification of genera Identification of species 5.3. Harmful species 5.4. References References in bold are available as pdf files on the course CD.

5.1. Suggested reading

Present document and and the following papers: Daugbjerg, N., Hansen, G., Larsen, J.& Moestrup, O. 2000. Phylogeny of some major genera of dinoflagellates based on ultrastructure and partial LSU rDNA sequence data, including the erection of 3 new genera of unarmoured dinoflagellates. - Phycologia 39:302-317. Hallegraeff, G.M. & Fraga, S. 1998. Bloom dynamics of the toxic dinoflagellate Gymnodinium catenatum, with emphasis on Tasmanian and Spanish coastal waters. - In: Anderson, D.M., Cembella, A.D. & Hallegraeff, G.M. (eds), Physiological ecology of harmful algal blooms, NATO ASI Series, vol. G 41, Springer-Verlag, Berlin, pp. 59-80 , (see Books, pdf file NATO ASI). .

5.2. Introduction to the taxonomy of unarmoured dinoflagellates

The 'unarmoured' or 'naked' dinoflagellates lack cellulosic amphiesmal plates. Most unarmoured dinoflagellates belong to the orders Oxyrrhinales, Actiniscales, Noctilucales and Gymnodiniales, but others may have unarmoured stages in their life cycles (Desmomonadales, Dinotrichales, Dinamoebales, Dinococcales, Brachydiniales (Larsen & Sournia 1991). Compared to the armoured dinoflagellates, the taxonomy of the Gymnodiniales is still poorly known and not very robust. Most genera and species have until now been defined by classical-morphological characters, whereas fine structural features and molecular data are only now beginning to make their impact on modern dinoflagellate taxonomy.

Harmful dinoflagellates, Gymnodiniales, page 2 of 7 Fine structural studies of unarmoured species, though still few in number compared to the diversity of species, indicate that a well established genus as Gymnodinium is polyphyletic, and therefore can be expected to be divided into a number of genera in the future (Daugbjerg et al. 2000). Thus, the flagellar apparatus of Akashiwo sanguinea (Hirasaka) Hansen et Moestrup (= G. sangineum) shows greater similarity to Polykrikos kofoidii Chatton than to other species of Gymnodinium (Roberts 1991). Molecular studies also support that the Gymnodiniales as presently understood is a polyphyletic assemblage (Saunders et al. 1997, Daugbjerg et al. 2000, Shao et al. 2004). Several genera of unarmoured dinoflagellates, e.g. Amphidinium, Gymnodinium, and Gyrodinium comprise both photo- hetero-, and mixotrophic species, and even different types of chloroplasts occur in some genera (Larsen & Sournia 1991). This feature, which is highly unusual from a taxonomic point of view, does not occur in other groups of algae, and further supports the suspected polyphyletic nature of these genera. Identification of genera In the Gymnodiniales, generic assignment has traditionally been based on characters related to the cingulum (girdle), i.e. the position of the cingulum, the extent of possible displacement of the girdle ends, and the number of turns around the cell body. However, application of these features is not always straightforward as dislocation and displacement of the cingulum are relative characters. Thus, the boundary between e.g. Amphidinium and Gymnodinium is not clear and interpreted differently by different authors. In the type species of Amphidinium, A. operculatum, the cingulum is strongly dislocated and appears as a collar around the tongue-shaped epicone. Other species with less pronounced dislocation of the cingulum have been assigned either to Amphidinium or to Gymnodinium, even by the same author (compare A. pelagicum and G. achromaticum, Lebour 1925). Also the boundary between Gymnodinium and Katodinium is not clear. In the latter genus the cingulum is dislocated posteriorly, but Katodinium glaucum (Lebour) Loeblich III is assigned to Gyrodinium by some authors (e.g. Dodge 1982) because of the large displacement of the ends of the cingulum. The distinction between the largest genera Gymnodinium Stein and Gyrodinium Kofoid et Swezy is based on cingular displacement according to Kofoid & Swezy (1921) with a displacement larger than 1/5 of the cell length characterizing Gyrodinium. This feature, although it may seem an objective character, has been shown not to be conservative at least in some species (Kimball & Wood 1965), and the cingular displacement appears to be about 1/5 of the cell length in many species making assignment to Gymnodinium or Gyrodinium arbitrary. The weakness of the characters used to distinguish genera has long been obvious, but the lack of additional or alternative characters has impeded significant progress of the taxonomy of the Gymnodiniales. The recent studies of e.g. Daugbjerg et al. (2000), De Salas et al. (2003) and Jrgensen et al. (2004) have contributed to a clarification of generic boundaries in the Gymnodiniales. In addition to molecular features, morphology of the apical groove type has been proven useful to establish the following genera previously included in Gymnodinium: Akashivo, Gymnodinium, Karenia, Karlodinium and Takayama. Identification of species Species of unarmoured dinoflagellates are distinguished by morphological characters such as size, shape, position and morphology of the cingulum (displacement and/or overhang)

Harmful dinoflagellates, Gymnodiniales, page 3 of 7 and sulcus, presence/absence of chloroplasts and pyrenoids, shape and position of the nucleus, shape of the apical furrow when present, possible surface structures e.g. longitudinal stripes. Observation of other features, e.g. eyespots, species appendages etc., is obviously also important. The unarmoured dinoflagellates do not - as opposed to the armoured species - maintain their shape in preserved samples, and also other important morphological features are often obliterated upon preservation or prolonged observation under the microscope. Therefore, species identification usually requires observation of live cells. Some species are quite robust and may persist for several minutes when observed under the microscope, while the most delicate ones deteriorate almost immediately after swimming has ceased. In the latter case only few cells in each preparation can be expected to be identifiable. An apical groove occurs in many species but is often difficult to observe in the light microscope (Takayama 1985). This furrow is usually very delicate and thus obliterated in preserved material, and it is one of the first features that disappear when live cells begin to deteriorate during examination under the microscope. Also surface striations are delicate in some species. Several descriptions of unarmoured dinoflagellates are undoubtedly based on preserved material (see for example Schiller 1933), and their 'non-preservable' nature is a major reason why the taxonomy of the unarmoured dinoflagellates is still inadequate with many poorly described species, which can probably never be identified. For identification of unarmoured dinoflagellates, the reader is referred to Kofoid & Swezy (1921), Schiller (1933), Hulburt (1957), Larsen (1994, 1996), Steidinger (1997).

5.3. Harmful species

A number of unarmoured dinoflagellates may cause harmful blooms. From the public health point of view, Gymnodinium catenatum and Karenia brevis are the most serious as they may cause PSP or NSP, respectively (Fukuyo et al. 1993, Hallegraeff & Fraga 1998, Negri et al. 2001, Steidinger et al. 1998). Several species of unarmoured dinoflagellates may cause fish kills. Most serious is Karenia mikimotoi, and other closely related species, see table below. There has previously been considerable confusion regarding the identity of K. mikimotoi, but much was clarified through the careful study by Hansen et al. (2000). The ecology of this species has been reviewed by Gentien (1998). Cochlodinium polykrikoides often causes severe fish kills particularly in Japan and Korea, but recently it has been reported to have caused problems in several other countries including Canada, China, Malaysia, Mexico, Puerto Rico, the Philippines, USA, and the Middle East (Yuki & Yoshimatsu 1989, Kim et al. 2000, Iwataki et al. 2008, Matsuoka et al. 2008). Noctiluca scintillans (Macartney) Kofoid is widely distributed and may form extensive, sometimes spectacularly coloured blooms. In South-East Asian waters it may attain a green colour due the presence of an endosymbiotic flagellate. It is not considered a toxic species, but even so blooms of N. scintillans may cause fish kills through extensive production of mucus which may damage fish gills or through changes of oxygen or ammonia concentrations in the water mass (see Landsberg 2002, and references therein). For identification of harmful species of unarmoured dinoflagellates, see Taylor et al. (2004).

Harmful dinoflagellates, Gymnodiniales, page 4 of 7 Species of Karenia, and related species

Species
Karenia asterichroma de Salas, Bolch & Hallegraeff 2004 K. bicuneiformis Botes et al. 2003 K. bidigitata Haywood et al. 2004

Comments and references

Described from Tasmania associated with salmonid mass mortality although its ichthyotoxicity is not confirmed yet (De Salas et al. 2004a) Described and so far reported only from South Africa, not associated with harmful events (Botes et al. 2003) Described and so far reported only from New Zealand, presumably synonymous with K. bicuneiformis (Haywood et al. 2004) Occurring regularly in the Gulf of Mexico, but reported also from other parts of the world, see Taylor et al. (2004), it may cause NSP, fish kills, and asthma-like symptoms in humans through aerosolization of toxins (Landsberg 2002) Described from New Zealand, where it has caused extensive fish kills (Chang 1999) Described from New Zealand, associated with toxic outbreaks causing abalone and fish kills (Chang & Ryan 2004) Only known from South Africa, it may produce aerosol toxins, and it appears to be toxic to some invertebrates (Botes et al. 2003) Reported from Hong Kong and Japan, where it has been associated with extensive fish fills (Yang et al. 2000) Described from Hong Kong, not associates with harmful effects (Yang et al. 2001) This species is widespread and it has caused fish kills in many parts of the world, it may produce both hemolytic and ichthyotoxins (Landsberg 2002, Taylor et al. 2004) Described and so far reported only from New Zealand (Haywood et al. 2004) Described from New Zealand, and reported also from Kuwait (Haywood et al. 2004); may produce gymnodimine toxins (Miles et al. 2003, Haywood et al. 2004) Described from Tasmania, Australia, ichthyotoxic (de Salas et al. 2004) Described from the Mediterranean Sea (Bergholtz et al. 2005), probably toxic but this needs to be confirmed Described from Australia (de Salas et al. 2005), ichthhyotoxic syn: K. micrum (Leadbeater et Dodge) Larsen 2000; presumably a widespread ichthhyotoxic species, several similar species, e.g. K. vitiligo, and Gymnodinium corsicum (Daugbjerg et al. 2000, Deeds et al. 2002, Kempton et al. 2002, Landsberg, 2002, Taylor et al. 2004) Reported from Australia (Larsen 1996, de Salas et al. 2003) Reported from Australia (Larsen 1996, de Salas et al. 2003) Reported from Australia and Japan (de Salas et al. 2003) Reported from Australia and Florida, USA, possibly ichthyotoxic (Larsen 1994, de Salas et al. 2003) Described and so far reported only from Tasmania, Austra-lia, not associated with harmful events (de Salas et al. 2003)

K. brevis (Davis) Hansen et Moestrup 2000

K. brevisulcata (Chang) Hansen et Moestrup 2000 K. concordia Chang & Ryan 2004 K. cristata Botes et al. 2003 K. digitata Yang et al. 2000 K. longicanalis Yang et al. 2001 K. mikimotoi (Oda) Hansen et Moestrup 2003 K. papilionecea Haywood et al. 2004 K. selliformis Haywood et al. 2004 K. umbella de Salas et al. 2004 Karlodinium armiger Bergholtz et al. 2005 K. australe de Salas et al. 2005

K. veneficum (Ballantine) Larsen 2000

Takayama acrotrocha (Larsen) de Salas et al. 2003 T. cladochroma (Larsen) de Salas et al. 2003 T. helix de Salas et al. 2003 T. pulchella (Larsen) de Salas et al. 2003 T. tasmanica de Salas et al. 2003

Harmful dinoflagellates, Gymnodiniales, page 5 of 7

5.4. References
Bergholtz, T., Daugbjerg, N. & Moestrup, . 2005. On the identity of Karlodinium veneficum and description of Karlodinium armiger sp. nov. (Dinophyceae), based on light and electron microscopy, nuclear-encoded LSU rDNA, and pigment composition. - J. Phycol. 42, 170193. Botes, L., Sym, S.D. & Pitcher, G.C. 2003. Karenia cristata sp. nov. and Karenia bicuneiformis sp. nov. (Gymnodiniales, Dinophyceae): two new Karenia species from the South African coast. Phycologia 42: 563-571. Chang, F.H. 1999. Gymnodinium brevisulcatum sp. nov. (Gymnodiniales, Dinophyceae), a new species isolated from the 1998 summer toxic bloom in Wellington Harbour, New Zealand. Phycologia 38:377-384. Chang, F.H. & Ryan, K.G. 2004. Karenia concordia sp. nov. (Gymnodiniales, Dinophyceae), a new nonthecate dinoflagellate isolated from the New Zealand northeast coast during the 2002 harmful algal bloom events. - Phycologia 43:552-562. Daugbjerg, N., Hansen, G., Larsen, J. & Moestrup, O. 2000. Phylogeny of some major genera of dinoflagellates based on ultrastructure and partial LSU rDNA sequence data, including theerection of 3 new genera of unarmoured dinoflagellates. - Phycologia 39:302-317. Deeds, J.R., Terlizzi, D.E., Adolf, J.E., Stoecker, D.K. & Place, A.R. 2002. Toxic activity from cultures of Karlodinium micrum (=Gyrodinium galatheanum) Dinophyceae) a dinoflagellate associated with fish mortalities in an estuarine aquaculture facility. Harmful Algae 1: 169-189. De Salas, M.F., Bolch, C.J.S., Botes, L., Nash, G., Wright, S.W. & Hallegraeff, G.M. 2003. Takayama gen. nov. (Gymnodiniales, Dinophyceae), a new genus of unarmoured dinoflagellates with sigmoid apical grooves, including the description of two new species. J. Phycol. 39: 1233-1246. De Salas, M.F., Bolch, C.J.S., & Hallegraeff, G.M. 2005. Karlodinium australe sp. nov. (Gymnodiniales, Dinophyceae), a new potentially ichthyotoxic unarmoured dinoflagellate from lagoonal habitats of south-east Australia. Phycologia 44: 640-450. De Salas, M.F., Bolch, C.J.S., & Hallegraeff, G.M. 2004a. Karenia asterichroma sp. nov. (Gymnodiniales, Dinophyceae), a new dinoflagellate species associated with finfish aquaculture mortalities in Tasmania, Australia. Phycologia 43: 624-631. De Salas, M.F., Bolch, C.J.S., & Hallegraeff, G.M. 2004b. Karenia umbella sp. nov. (Gymnodiniales, Dinophyceae), a new potentially ichthyotoxic dinoflagellate species from Tasmania, Australia. Phycologia 43: 166-175. Dodge, J.D. 1982. Marine dinoflagellates of the British Isles. - Her Majestys Stationary Office. 303 p. Fukuyo, Y., Kodama, M., Ogata, T.,Ishimaru, T., Matsuoka, M., Okaichi, T., Maala, A.M. & Ordones, J.A. 1993. Occurrence of Gymnodinium catanatum in Manila Bay, the Philippines. In: Smayda, T.J, & Shimizu, Y. (eds), Toxic phytoplankton blooms in the sea, Elsevier, Amsterdam, pp.875-880. Gentien, P. 1998. Bloom dynamics and ecophysiology of the Gymnodinium mikimotoi species complex. - In: Anderson, D.M., Cembella, A.D. & Hallegraeff, G.M. (eds), Physiological ecology of harmful algal blooms, NATO ASI Series, vol. G 41, SpringerVerlag, Berlin, pp. 155-173. Hallegraeff, G.M. & Fraga, S. 1998. Bloom dynamics of the toxic dinoflagellate Gymnodinium catenatum, with emphasis on Tasmanian and Spanish coastal waters. -

Harmful dinoflagellates, Gymnodiniales, page 6 of 7 In: Anderson, D.M., Cembella, A.D. & Hallegraeff, G.M. (eds), Physiological ecology of harmful algal blooms, NATO ASI Series, vol. G 41, Springer-Verlag, Berlin, pp. 59-80. Hansen, G., Daugbjerg, N. & Henriksen, P. 2000. Comparative study of Gymnodinium mikimotoi and Gymnodinium aureolum comb nov. (= Gyrodinium aureolum) based on morphology,pigment composition, and molecular data. - Journal of Phycology 36: 394410. Haywood, A.J., Steidinger, K.A., Truby, E.W., Bergquist, P.R., Adamson, J. & MacKenzie, L. 2004. Comparative morphology and molecular phylogenetic analysis of three new species of the genus Karenia (Dinophyceae) from New Zealand. J. Phycol. 40: 165-179. Hulburt, E.M. 1957. The taxonomy of unarmoured Dinophyceae of shallow embayments on Cape Cod, Massachusetts. - Biol. Bull. 112: 196-219. Jrgensen, M.F., Murray, S. & Daugbjerg, N. 2004. Amphidinium revisited. I. Redefinition of Amphidinium (Dinophyceae) based on cladistic and molecular phylogenetic analyses. J. Phycol. 40: 351-365. Kempton, J.W., Lewitus, A.J., Deeds, J.R., Law, J.M. & Place, A.R. 2002. Toxicity of Karlodinium micrum (Dinophyceae) associated with a fish kill in a South Carolina brackish retention pond. Harmful Algae 1: 233-241. Kim, C.S., Lee, S.G. & Kim, H.G. 2000. Biochemical responses of fish exposed to a harmful dinoflagellate Cochlodinium polykrikoides. J. Exp. Mar. Biol. Ecol. 254: 131-141. Kimball, J.F. & Wood, E.J.F. 1965. A dinoflagellate with characters of Gymnodinium and Gyrodinium. - J. Protozool.12: 577-580. Kofoid, C.A. & Swezy, O. 1921. The free-living unarmoured dinoflagellates. - Mem. Univ.Calif. 5: 1-564. Landsberg, J. H. 2002. The effects of harmful algal blooms on aquatic organisms. Reviews in Fisheries Science 102: 113-390. Larsen, J. 1994. Unarmoured dinoflagellates from Australian waters. I. The genus Gymnodinium (Gymnodiniales, Dinophyceae). - Phycologia 33: 24-33. Larsen, J. 1996. Unarmoured dinoflagellates from Australian waters. II. Genus Gyrodinium (Gymnodiniales, Dinophyceae). - Phycologia 35, 342-49. Larsen, J. & Sournia, A. 1991. The diversity of heterotrophic dinoflagellates. - In: Patterson D.J. & Larsen, J. (eds), The biology of free-living heterotrophic flagellates, Systematic Association, Special Volume 45, Clarendon Press, Oxford, pp.313-332. Lebour, M. 1925. The dinoflagellates of the Northern Seas. - Mar. Biol. Ass. U.K. Plymouth, pp.1-250. Miles, C.O., Wilkins, A.L., Stirling, D.J. & MacKenzie, A.L. 2003. Gymnodimine C, an isomer of gymnodimine B, from Gymnodinium selliformis. - J. Agric. Food Chem. 51:4838-4840. Negri, A.P., Bolch, C.J.S., Blackburn, S.I., Dickman, M., Llewellyn, L.E. & Mendez, S. 2001. Paralytic shellfish toxins in Gymnodinium catenatum strains from six countries. In: Hallegraeff, G.M., Blackburn, S.I., Bolch, C.J. & Lewis, R.I. (eds), Harmful algal blooms 2000, Intergovernmental Oceanographic Commission of Unesco, Paris, pp. 210-213. Shao, P., Y-Q Chen, Zhou, H., Yuan, J., Qu, L-H., Zhao, D & Lin, Y.-S. 2004. Genetic variability in Gymnodiniaceae ITS regions: implications for species identification and phylogenetic analysis. - Mar. Biology 144:215-224. Roberts, K. 1991.The flagellar apparatus and cytoskeleton of dinoflagellates: organization and the use in systematics. - In: Patterson D.J. & Larsen, J. (eds), The biology of freeliving heterotrophic flagellates, Systematic Association, Special Volume 45, Clarendon Press, Oxford, pp. 285-302.

Harmful dinoflagellates, Gymnodiniales, page 7 of 7 Saunders, G., Hill, D.R.A., Sexton, J.P. & Anderson, R.A. 1997. Small-subunit ribosomal RNA sequences from selected dinoflagellates: testing classical evolutionary hypothesis with molecular systematic methods. - In: Bhattacharya, D. (ed.), Origins of Algae and their Plastids Springer-Verlag, Wien, pp. 237-259. Schiller, J. 1933. Dinoflagellatae (Peridineae) in Monographischer Behandlung. In: Rabenhorst, L. (ed.), Dr. L. Rabenhorsts Kryptogamenflora von Deutschland, sterreeich und der Schweiz 10.3(I), Akad. Verlag, Leipzig, pp. 1-590. Steidinger, K.A. 1997. Dinoflagellates In: Tomas, C.R. (ed.), Identifying marine phytoplankton, Academic Press, San Diego, pp. 387-584. Steidinger, K., Vargo, G.A., Tester, P.A. & Tomas, C.R. 1998. Bloom dynamics and physiology of Gymnodinium breve with emphasis on the Gulf of Mexico. In: Anderson, D.M., Cembella, A.D. & Hallegraeff, G.M. (eds), Physiological ecology of harmful algal blooms, NATO ASI Series, vol. G 41, Springer-Verlag, Berlin, pp. 133153. Takayama, H. Adachi, R. 1984. Gymnodinium nagasakiense sp. nov., a red-tide forming dinophyte in the adjacent waters of Japan. - Bulletin of the Plankton Society of Japan 31(1): 7-14. Takayama, H. 1985. Apical grooves of unarmoured dinoflagellates. - Bulletin of the Plankton Society of Japan 32: 129-140. Taylor, F.J.R., Fukuyo, Y., Larsen, J. & Hallegraeff, G.M. 2004. Taxonomy of harmful dinoflagellates. - In: Hallegraeff, G.M., Anderson, D.M., Cembella, A.D. (eds), Manual on harmful marine microalgae, Monographs on oceanographic methodology 11, Unesco, Paris, pp. pp. 389-432. Yang, Z.B., Takayama, H., Matsuoka, K. & Hodgkiss, I.J. 2000. Karenia digitata sp. nov. (Gymnodiniales, Dinophyceae), a new harmful algal bloom species from the coastal waters of west Japan and Hong Kong. - Phycologia 39: 463-470. Yang, Z.B., Hodgkiss, I.J. & Hansen, G. 2001. Karenia longicanalis sp. nov. (Dinophyceae): a new bloom-forming species isolated from Hong Kong, May 1998. Botanica Marina 44: 67-74. Yuki, K., Yoshimatsu, S. 1989. Two fish-killing species of Cochlodinium from Harima Naka, Seto Inland Sea, Japan. In: Okaichi, T., Anderson, D.M. & Nemoto, T. (eds), Red tides: biology, environmental science, and toxicology, Elsevier, New York, pp.451-454.