Ecophysiology, toxicology and genetic affinities of marine and freshwater epiphytic dinoflagellates from Tasmania, Australia

The dinoflagellates Ostreopsis siamensis, Coolia monotis, Prorocentrum lima and Prorocentrum rhathymum are commonly recognized as tropical, epiphytic species. The present work reports the occurrence of this assemblage from temperate seagrass habitat on the east coast of Tasmania, Australia. Molecular genetic sequencing, revealed little variance (- 0.2 %) within 0. siamensis, P. lima and P. rhathymum strains and high variance (-25%) within C. monotis strains where 3 major clades were seen, loosely based on geographical distribution. All species exhibited growth at lower temperature ranges (10-25 °C) than that described for tropical species (20-30°C). Ostreopsis siamensis survived 10 °C in the form of a vegetative resting stage. We conclude that this epiphytic assemblage is most likely indigenous to Tasmania, and not a recent range extension from tropical Queensland. Toxins were identified from all epiphytic species. Ostreopsis siamensis contained 0.011-0.05 pg palytoxin ceir 1, C. monotis extracts (1.5x106 cells) caused death in mice within 12 hrs, P. lima contained diarrhetic shellfish toxins (25 pg ceir 1 okadaic acid and 2 pg celr 1 DTX-1) and uncharacterised fast acting toxins were found in P. rhathymum extracts (1.8 x 107 cells) causing mouse death in 20 mins. Toxicity of P. rhathymum was further investigated following recognition of an association between seasonal oyster spat (Crassostrea gigas) mortalities and high (-12 OOO cells L\ 1) P. rhathymum density in a local estuary (Little Swanport). Oyster spat feeding assays revealed histopathological symptoms similar to those seen in affected field samples (thin dilated gut tubules and sloughing of gut cells) however no mortality was observed. In a comparable incident an unprecedented mortality of farmed Sydney rock oysters (Saccostrea glomerate) in New South Wales led to investigation of another Prorocentrum species (P. minimum) as a possible causative agent. Brine shrimp and intraperitoneal mouse bioassays were negative for toxic effects. However oyster spat bioassays evoked distinctive histopathological symptoms (large focal reactions in the digestive tubules and gills). These findings add an entirely new dimension to investigations into harmful Australian microalgae with implications for aquaculture not in terms ofbiotoxin contamination but as the cause of major shellfish mortalities. The little known freshwater Prorocentrum P. playfairi and P. foveolata were successfully cultured for the first time from Tasmanian coastal lagoons to elucidate their possible toxicity ecophysiology and genetic affinities with better-known marine congeneric species. Prorocentrum playfairi only grew in salinities of 0-5 psu whereas P. foveolata tolerated salinities of 0-30 psu. Both species were negative for diarrhetic shellfish toxins such as those found in marine Prorocentrum. Molecular sequencing suggested that these species are good Prorocentrum but more closely related to toxic benthic marine Prorocentrum (P. lima P. cf. belizeanum ) than pelagic marine Prorocentrum (P. gracile P. triestinum P. compressum etc.). We suggest a possible evolutionary origin from P. lima like dinoflagellates through the isolation of coastal lagoons and subsequent adaptation to freshwater."