IOC Science and Communication Centre on Harmful Algae University of Copenhagen

Harmful dinoflagellates; Dinophysiales, page 1 of 6

Module III
Section 3, Dinophysiales (3 hours)
Contents 3.1. Suggested reading 3.2. Introduction Taxonomy Morphology The species concept in Dinophysis and Phalacroma Life history 3.3. Harmful species 3.4. References

References in bold are available as pdf files on the course CD.

3.1. Suggested reading
Present document and the following paper: Maestrini, S.Y. 1998. Blooms dynamics and ecophysiology of Dinophysis spp. - In: Anderson, D.M., Cembella, A.D. & Hallegraeff, G.M. (eds), Physiological ecology of harmful algal blooms, NATO ASI series, Series G, Ecological sciences, vol. 41, Springer-Verlag, Berlin, pp. 243-265, (see Books, pdf file NATO ASI). .

3.2. Introduction
Taxonomy
The genera Dinophysis Ehrenberg 1839 (type species: D. acuta) and Phalacroma Stein 1883 (type species: P. porodictyum) both comprise potentially toxic species. The two genera are very similar and have been regarded as synonymous by many authors (Abé 1967, Balech 1967, 1988). However, recent studies based on molecular data suggest that the two general are phylogenetically well separated (Handy et al. 2009, Jensen & Daugbjerg 2009), and the latter authors offer an emended description of Phalacroma distinguishing this from Dinophysis by an elevated epitheca (but >1/4 of the cell length) and horizontal cingular lists. More than 200 species of Dinophysis and Phalacroma have been described. Taxonomic and floristic accounts include Kofoid & Skogsberg (1928), Schiller (1933), Abé (1967), Norris & Berner (1970), Taylor (1976), Balech (1988), Hallegraeff & Lucas (1988), and Steidinger

Harmful dinoflagellates, Dinophysiales, page 2 of 6 (1997); and for the potentially toxic species Larsen & Moestrup (1992), Balech (2002) and Taylor et al. (2004).

Morphology
Most species are strongly compressed and the cells are therefore usually seen in lateral view. The cingulum is displaced towards the apical end of the cell. In Dinophysis, the epitheca is not visible in lateral view and the upper cingular list is strong and funnel-shaped. In Phalacroma, the epitheca is elevated (but >1/4 of the cell length) and the cingular lists are directed horizontally (Jensen & Daugbjerg 2009). In both genera, the left sulcal list is particularly well developed and is supported by 3 ribs. The species concept in Dinophysis and Phalacroma The species concept in Dinophysis and Phalacroma is weak with only few characters available to distinguish the large number of species. Many species are poorly circumscribed, and the genera are badly in need of revision. The use of plate pattern has so far proven unfeasible for species identification (Norris & Berner 1970, Balech 1976, Hallegraeff & Lucas 1988). Optical pattern recognition systems have been applied on several occasions (e.g. Lassus et al. 1998) and may carry some potential for the development of more robust species concepts in Dinophysis, and also molecular techniques may eventually help to clarify the species concept (Edvardsen et al. 2000, Marin et al. 2001, Edvardsen et al. 2003). Currently, the most important diagnostic features are: cell size and shape, morphology of the left sulcal list, presence/absence of chloroplasts, and thecal ornamentation. Many species cannot be clearly distinguished on basis of these features, and with the intraspecific variation largely unknown in most species, it is sometimes not possible with confidence to identify certain cells in a sample to species. Examples include the pairs D. acuta – D. norvegica, D. caudata - D. tripos, D. acuminata – D. sacculus and P. mitra – P. rapa. Attempts to establish permanent cultures of species of Dinophysis were unsuccessful for many years, but recently Park et al. (2006) succeeded in establishing cultures of D. acuminata from Korean waters fed with Myrionecta rubra; and several species of Dinophysis (e.g. D. acuminata, D. acuta, D. caudata, and D. fortii) are now successfully cultivated, see also below. The recent availability of cultures constitutes an important breakthrough that will facilitate progress in clarification of the taxonomy of this genus Life history The life history of Dinophysis is poorly known, but there is circumstantial evidence that sexual reproduction does occur, and several species studied so far seem to have a polymorphic life cycle (Reguera & Gonzáles-Gil 2001, Reguera et al. 2004, 2007, Escalera & Reguera 2008 and reference therein). Because the small stages of the life cycle have a different size and shape, they have often been classified as different species. Therefore, a reduction in the number of species may be expected in the future

3.3. Harmful species
There are 11 species of Dinophysis and Phalacroma which have been shown to produce DSP toxins. They are: Dinophysis acuminata, D. acuta, D. caudata, D. fortii, D. miles, P. mitra, D. norvegica, P. rotundatum, D. tripos, D. infundibulus and D. ovum; two other species are suspected toxin producers: D. hastata and D. sacculus (Lee et al. 1989, Fernández et al. 2001, Holmes et al. 1999, Taylor et al. 2004; Fernández et al. 2006; Raho et al. 2008;

Harmful dinoflagellates, Dinophysiales, page 3 of 6 Suzuki et al. 2008). Several of these species are widely distributed. Current advances in analytical methods (LC-MS) allow analyses of toxins from a small number of single-cell isolates (10-100 cells), and the list of toxin-producing species may be expected to grow (Reguera & Pizarro 2008, Suzuki et al. 2009). The potentially toxic species may cause harmful effects without forming dense blooms. Cell concentrations as low as 200 cells/litre of D. fortii have been reported to cause DSP outbreaks (Yasumoto et al. 1980), and low concentrations of D. caudata may cause persistent low concentrations of DSP toxins in mussels (Holmes et al. 1999, but see also Marasigan et al. 2001). Maestrini (1998) reviewed the ecophysiology and bloom dynamics of Dinophysis. Early studies have shown that several potentially toxic species of Dinophysis have chloroplasts of cryptophyte affinity (Schnepf & Elbrächter 1988, Hallegraeff & Lucas 1988,). Several species are mixotrophic (Jacobson & Andersen 1994) and one, D. rotundata, is heterotrophic feeding on ciliates (Hansen 1991). Subsequently, further studies have shown that D. acuminata, D. acuta, D. fortii, D. norvegica, and D. tripos possess plastids that specifically originates from the cryptophytes T. amphioxeia or Geminigera cryophila (Janson & Granéli 2003, Janson 2004, Minnhagen & Janson 2006). Park et al. (2008, and further references therein) suggest that the growth of the photosynthetic Dinophysis caudata depends on the prey-predator interactions among Dinophysis, Myrionecta rubra and Teleaulax, but a more recent study indicates that the situation may be more complex (Park et al. 2010). Also the prey-predator-chloroplast relationship in D. acuminata appears to be more complex than previously believed as molecular and ultrastructural data are here in conflict (Janson & Granéli 2003, Janson 2004, Minnhagen & Janson 2006, Park et al. 2006, Garcia-Cuetos et al. 2010).

3.4 References
Abé, T.H. 1967. The armoured Dinoflagellata: II – Prorocentridae and Dinophysidae (B). Dinophysis and its allied genera. – Publ. Seto mar. biol. Lab. 15(1): 37-7. Balech, E. 1967. Dinoflagelados nuevos o interesantes del Golfo de Mexico y Caribe. - Rev. Mus. Argent. Cienc. Nat. Bernardino Rivadavia Inst. Nac. Invest. Cienc. Nat. 2:77–126. Balech, E. 1976. Some Norwegian Dinophysis species (Dinoflagellata). - Sarsia 61: 75-94. Balech, E. 1988. Los Dinoflagelados del Atlántico Sudoccidental. - Publ. Espec. Inst. Esp. Oceanogr. 1, 310 p. Balech, E. 2002. Dinoflagelados tecados tóxicos Del Cono Sur Americano. - In: Sar, E. A ., M. E. Ferrario & B. Reguera (eds.), Floraciones Algales Nocivas en el Cono Sur Americano. Instituto Español de Oceanografia, pp. 125-144. Edvardsen, B., Shalchian-Tabrizi, K.,.Brubak, S., Dahl, E., Jakobsen, K. & Paasche, E. 2000. Genetic analyses of Dinophysis species isolated from Norwegian waters. – Abstract of the ‘Ninth International Conference on Harmful Algal Blooms, Tasmania 2000, p.14. Edvardsen, B., Shalchian-Tabrizi, K., Jakobsen, K.S., Medlin, L.K., Dahl, E., Brubak, S. & Paasche,E. 2003. Genetic variability and molecular phylogeny of Dinophysis species from Norwegian waters inferred from single cell analyses of rRNA. – J. Phycol. 39: 395408. Escalera, L. & Reguera, B. 2008. Planozygote division and other observations on the sexual cycle of several species of Dinophysis (Dinophyceae, Dinophysiales). - J. Phycol. 44(6): 1425-1436.

Harmful dinoflagellates, Dinophysiales, page 4 of 6 Fernández, M.L., Reguera, B., Ramilo, I. & Martinez, A. 2001. Toxin content of Dinophysis acuminata, D. acuta, and D. caudata from the Galician Rias Bajas. - 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.360-363.

Fernández, M.L., B. Reguera, S. González-Gil and A. Míguez (2006). Pectenotoxin-2 in single-cell isolates of Dinophysis caudata and Dinophysis acuta from the Galician Rías (NW Spain). Toxicon 48(5): 477-490.
Garcia-Cuetos, L., Moestrup, Ø., Hansen, P.J. & Daugbjerg, N. 2010. The toxic dinoflagellate Dinophysis acuminata harbors permanent chloroplasts of cryptomonad origin, not kleptochloroplasts. – Harmful Algae 9: 25-38. Hallegraeff, G.M. & Lucas, I.A.N. 1988. The marine dinoflagellate genus Dinophysis (Dinophyceae): photosynthetic, neritic and non-photosynthetic, oceanic species. – Phycologia 27: 25-42. Handy, S.M., Bachvaroff, T.R., Timme, R.E., Wayne Coats, D., Kim, S. & Delwiche, C.F. 2009. Phylogeny of four Dinophysiacean genera (Dinophyceae, Dinophysilaes) based on rDNA sequences from single cells and environmental samples. – J. Phycol. 45: 1163-1174. Hansen, P.J. 1991. Dinophysis – a planktonic dinoflagellate which can act both as a prey and a predator of a ciliate. – Mar. Ecol. Prog. Ser. 69: 201-204. Holmes, J.H., Teo, S.L.M. & Khoo, H.W. 1999. Persistant low concentrations of diarrhetic shellfish toxins in green mussels Perna viridis from the Johor Strait, Singapore: first record of diarrhetic shellfish toxins from South-East Asia. - Mar. Ecol. Prog. Ser. 181: 257-268. Jacobson D.M. & Andersen, R.A. 1994. The discovery of mixotrophy in photosynthetic species of Dinophysis (Dinophyceae): light and electron microscopical observations of food vacuoles in Dinophysis acuminata, D. norvegica, and two heterotrophic dinophysoid dinoflagellates. - Phycologia 33: 97-110. Janson, S. 2004. Molecular evidence that plastids in the toxin producing dinoflagellate genus Dinophysis originate from the free-living cryptophyte Teleaulax amphioxeia. - Environ. Microbiol. 6:1102–6. Janson, S. & Granéli, E. 2003. Genetic analysis of the psbA gene from single cells indicates a cryptomonad origin of the plastid in Dinophysis (Dinophyceae). - Phycologia 42: 473477. Jensen, M.H. & Daugbjerg, N. 2009. Molecular phylogeny of selected species of the order Dinophysiales (Dinophyceae) – testing the hypothesis of a dinophysoid radiation. – J. Phycol. 45: 1136-1152 Kofoid, C.A. & T. Skogsberg.1928. The Dinoflagellata: the Dinophysoidea. - Memoirs of the Museum of Comparative Zoology of Harvard College 51: 1-766. Larsen, J. & Moestrup, Ø. 1992. Potentially toxic phytoplankton. 2. Genus Dinophysis (Dinophyceae). – ICES Identification Leaflets for Plankton, Copenhagen, International Council for the Exploration of the Sea, 12pp. Larsen, J. & Nguyen, N.L. 2004. Genus Dinophysis. – In: Larsen, J. & Nguyen, N.L. (ed.), Potentially toxic microalgae of Vietnamese coastal waters, Opera Bot. 140. Lassus, P. Seguel, M. & Truquet, P. 1998. Morphological study of atypical Dinophysis acuta Ehrenberg from Chilean coastal waters by a digital pattern-recognition system. – Bot. Mar. 41:435-41. Lee, J-S., Igarashi, T., Fraga, S., Dahl, E., Hovgaard, P. & Yasumoto T. 1989. Determination of diarrhetic shellfish toxins in various dinoflagellate species. – J. Applied Phycol. 1: 147-152.

Harmful dinoflagellates, Dinophysiales, page 5 of 6 Maestrini, S.Y. 1998. Blooms dynamics and ecophysiology of Dinophysis spp. - In: Anderson, D.M., Cembella, A.D. & Hallegraeff, G.M. (eds), Physiological ecology of harmful algal blooms, NATO ASI series, Series G, Ecological sciences, vol. 41, Springer-Verlag, Berlin, pp. 243-265. Marasigan, A.N., Sato, S., Fukuyo, Y. & Kodama, M. 2001. Accumulation of a high level of diarrhetic shellfish toxins in the green mussel Perna viridis during a bloom of Dinophysis caudata and Dinophysis miles in Sapian Bay, Panay Island, the Philippines. – Fisheries Scieence 67: 994-996. Marin, I., Anguilera, A., Gonzáles-Gil, S., Reguera, B. & Abad, J.P. 2001. Genetic analysis of three species of Dinophysis causing diarrhetic shellfish outbreaks in Galicia (NWSpain). – 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. 222-225. Minnhagen, S. & Janson, S. 2006. Genetic analyses of Dinophysis spp. support kleptoplastidy. - FEMS Microbiol Ecol 57: 47–54. Norris, D.R. & Berner, L.D. 1970. Thecal morphology of selected species of Dinophysis (Dinoflagellata) from the Gulf of Mexico. - Contributions in Marine Science 15: 145192. Park M.G., Kim, S., Kim, H.S., Myung, G., Kang, Y.G. & Yih, W. 2006. First successful culture of the marine dinoflagellate Dinophysis acuminata. Aquat Microb Ecol 45:101–106. Park M.G., Kim, M., Kim, S. & Yih, W. 2010. Does Dinophysis caudata (Dinophyceae) have permanent plastids. – J. Phycol. 46: 236-242. Park, M. G., Park, J.S., Kim, M. & Yih, W. 2008. Plasted dynamics during survival of Dinophysis caudata without its ciliate prey. – J- Phycol. 44: 1154-1163. Reguera, B. & González-Gil, S. 2001. Small cell and intermediate cell formation in species of Dinophysis (Dinophyceae, Dinophysiales). - J. Phycol. 37:318-333. Reguera, B., González-Gil, S. & Delgado, M. 2004. Formation of Dinophysis dens and D. diegensis from laboratory incubations of Dinophysis acuta and D. caudata. - In: Steidinger, K.A., Landsberg, J.H., Tomas, C.R. & Vargo, G.A. (eds.). Florida Fish and Wildlife Conservation Commission and IOC of UNESCO, St Petersburg, Florida, USA, pp. 440-442. Reguera, B., S. González-Gil. S. & Delgado, M. 2007. Dinophysis diegensis Kofoid is a life history stage of Dinophysis caudata Kent (Dinophyceae, Dinophysiales). - J. Phycol. 43: 1083-109. Schiller, J. 1933. Dinoflagellatae (Peridineae). - In: Rabenhorst, L. (ed.), Kryptogamen Flora von Deutschland. 10(3). I Teil. Akademische Verlagsgesellschaft. Leipzig. 617 pp. Schnepf, E. & Elbrächter, M. 1988. Cryptophycean-like double membrane-bound chloroplasts in the dinoflagellate, Dinophysis Ehrenb.: evolutionary, phylogenetic, and toxicological implications. – Bot. Mar. 101:196-203. Steidinger, K.A. 1997. Dinoflagellates – In: Tomas, C.R. (ed.), Identifying marine phytoplankton, Academic Press, San Diego, pp. 387-584. Suzuki, T., Miyazono, A., Baba, K., Sugawara, R. & Kamiyama, T. 2009. LC–MS/MS analysis of okadaic acid analogues and other lipophilic toxins in single-cell isolates of several Dinophysis species collected in Hokkaido, Japan. Harmful Algae 8: 233–238 Taylor, F.J.R. 1976. Dinoflagellates from the International Indian Ocean Expedition. A report of material collected by the R.V. “Anton Bruun” 1963-64. – Bibliotheca Botanica, Stuttgart, 234 pp + 46 plates. 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

Harmful dinoflagellates, Dinophysiales, page 6 of 6 on harmful marine microalgae, Monographs on oceanographic methodology 11, Unesco, Paris, pp. 389-432. Yasumoto, T., Oshima, Y., .Sugawara, W., Fukuyo, Y., Oguri, H., Igarashi, T. & Fujita, N. 1980. Identification of Dinophysis fortii as the causative organism of diarrhetic shellfish poisoning. - Bulletin of the Japanese Society of Scientific Fisheries 46: 1405-1411.

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