PHYLOGENETIC AND TAXONOMIC STUDY OF SUBTERRANEAN WATER PROTISTS.

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The subterranean water protists in sandy beach, especially dinoflagellates were investigated by means of light and electron microscopy and their phylogenetic affinities were studied using a molecular phylogenetic techniques. The sampling locality was at Ishikari Beach in Ishikari City, Hokkaido, Japan and samplings were made on a monthly basis. At the time of sampling, water temperature and salinity were also recorded. The salinity of the subterranean habitat in sandy beach seemed to be affected by both seawater and freshwater from inland. Despite it unstable salinity conditions, many species of dinoflagellates were found to inhabit there. By the taxonomic survey, I was able to demonstrate presence of various dinoflagellates, including Amphidinium (11 spp.), Gymnodinium (5 spp.), Gyrodinium (3 spp.), Katodinium (4 spp.), Oxyrrhis (1 sp.), Sinophysis (1 sp.) and Roscoffia (1 sp,). Nearly half of them were found to be new species and formal taxonomic descriptions will be published elsewhere.To assess the phylogenetic affinities of these dinoflagellate, single-cell PCR method was employed to amplify 18S rRNA gene. Use of low temperature and longer annealing time were found to be effective to successfully amplify the gene, but it was not always successful. Almost complete sequence of Gyrodinium viridescens was obtained and this together with those of other dinoflagellates downloaded from DDBJ as well as our own data were aligned and phylogenetic trees were constructed with various phylogenetic methods. The results indicated that G, viridescens was included in the photosynthetic Amphidinium clade. This seems to indicate that G. viridescens had secondarily lost its plastid and later acquired Gyrodinium-type morphology.