PHYLOGENETIC AND TAXONOMIC STUDY OF SUBTERRANEAN WATER PROTISTS.

用光镜和电子显微镜观察了沙质滩涂地下水原生动物，特别是甲藻，并用分子系统发育技术研究了它们的亲缘关系。抽样地点是日本北海道石垣市的石垣海滩，每月抽样一次。在采样时，还记录了水温和盐度。沙质海滩地下生境的盐度似乎同时受到来自内陆的海水和淡水的影响。尽管盐度条件不稳定，但发现那里栖息着许多种类的甲藻。通过分类调查，我发现了各种甲藻的存在，包括双鞭藻(11个种)、裸甲藻(5个种)、裸甲藻(3个种)、甲藻(4个种)、Oxyrvis(1个种)、Sinophysis(1个种)。和Roscoviia(1 sp.)其中近一半是新种，正式的分类描述将在其他地方公布。为了评估这些甲藻的系统发育亲缘关系，采用单细胞聚合酶链式反应(Single-cell PCR)方法扩增了18S rRNA基因。利用低温和较长的退火时间可以有效地扩增出该基因，但并不总是成功的。获得了绿色回甲藻的几乎完整的序列，并与从DDBJ下载的其他甲藻的序列以及我们自己的数据进行了比对，并用各种系统发育方法构建了系统发育树。结果表明，在光合作用的两性藻分支中含有G，绿色降落菌。这似乎表明，绿色革兰氏菌继而失去了其质体，后来获得了回旋型的形态。

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.