半咸水生境中典型原生动物类群的多样性及谱系生物地理学研究

Studies on the biogeography distribution have great significance for revealing the influence of ecological environment changing to the biomes and can help us to set out strategies to protect biodiversity. We had reached a consensus on the distribution pattern of metazoans long time ago. To that of protozoa, however, related studies were relatively poor and far from a consensus. To the best of our knowledge, previous studies on the genetic diversity and/or the biogeography distribution of protist mostly focused on the planktonic groups from biotopes of marine or freshwater, few research give insights to that of the groups inhabiting the transition regions of ocean and freshwater. .This project intends to study the biodiversity and phylogeography of typical groups of protozoa in brackish biotopes. The sampling biotopes include the coastal estuary, wetlands and inland lagoon. And we are going to focus on three typical groups of ciliated protozoa (pleurostomatids, peritrichs and colpodids) because they have relatively low distribution ability and theoretically have low gene flow and special genetic structures. .Firstly, we will investigate their species biodiversity in the sampling biotopes and select several (at least 2) cosmopolitan species for each group. Then, we will collect many populations (at least 50) for each cosmopolitan species, extract DNA and sequence several molecular genetic markers, including the mitochondria 16S rDNA, cox-1, and the nucleus SSU rDNA, ITS1-5.8S-ITS2 rDNA, beta-tubulin. Then their genetic diversity will be analyzed and compared. Based on the results of networks of gene markers, the phylogeography will be analyzed and discussed. .This research will give insights to the biodiversity and geography distribution pattern of sympatric ciliated protozoa inhabiting brackish biotopes. At the same time, it will also be a deep understanding of the genetic diversity of unicellular eukaryotes, their migration and/or transition between ocean and freshwater habitats, species formation, and their evolution and adaptive mechanism.

生物地理分布格局的研究对揭示生态环境变化对生物群落的影响和制定生物多样性保护策略具有重要意义。后生生物的分布格局人们早已达成共识，但对原生生物多样性地理分布格局的研究还相对较少，远未达成共识。已有的研究多关注海洋或者淡水原生生物浮游类群的遗传多样性和谱系地理分布，缺少对海洋—淡水过渡区类群的相关研究。本项目拟选择种群扩散能力相对偏低的漫游、周丛/底栖、土壤生纤毛虫原生动物典型类群（侧口类、缘毛类、肾形类）为研究对象，开展其在滨海的（河口、湿地）和内陆的（咸水湖）半咸水生境中的物种多样性和区系结构调查，筛选广布种，进行多分子标记（线粒体和细胞核）的遗传多样性和谱系生物地理学研究，解析较大地理尺度上半咸水生境纤毛虫原生动物的谱系分布格局及同域物种谱系分布规律，同时也将为深入了解单细胞真核生物的遗传多样性及其在海洋和淡水生境间的过渡迁移、物种形成过程、演化及适应性进化机制提供科学依据。

生物地理分布格局的研究对揭示生态环境变化对生物群落的影响和制定生物多样性保护策略具有重要意义。本项目围绕半咸水生境中典型纤毛虫原生动物类群的多样性和谱系/生物地理学开展了系列研究：基于现代分类学技术的物种多样性研究，基于条形码技术的谱系地理学研究，基于宏条形码技术的生物地理学研究。半咸水生境纤毛虫物种多样性调查方面的主要结果为：选择漫游、周丛/底栖、土壤生纤毛虫原生动物的典型类群开展了其在滨海和内陆半咸水生境的物种多样性调查，共采集鉴定100余种纤毛虫（包括侧口类26种、缘毛类11种、肾形类20余种和其他类群）。谱系生物地理学研究方面的主要结果为：筛选出广布种7种进行了多分子标记（18S、ITS、16S、COX1）的种群内个体间/种群间/相近种间的遗传多样性研究，结果发现纤毛虫单个个体内在多个基因均存在高度多样的单倍型，且同种群的个体间和不同种群间共享的单倍型并不多，说明了纤毛虫高度的基因多样性。我们PCR扩增中使用的DNA聚合酶（ExTaq酶）是之前同类研究中广泛采用的，但最新的研究发现由于该酶的保真度不够高会导致纤毛虫基因多样性会被高估，因此我们的结果出现了假阳性的可能，今后纤毛虫的基因多样性研究基因扩增时均应采用高保真酶。半咸水真核微型生物生物地理学研究的主要结果为：利用宏条形码eDNA对半咸水的河口（黄河口、长江口、珠江口）和潮间带（渤海、黄海、东海、南海）环境样品的微型真核生物多样性和群落结构进行了研究，分析了样品的阿尔法多样性和贝塔多样性，证明了虽然同属半咸水水体，但河口和潮间带具有各自不同的微型真核生物多样性与生物地理分布模式。本研究可为将来各类半咸水生境纤毛虫的多样性评估与生物地理分布格局研究提供科学依据，同时也将为深入了解单细胞真核生物的遗传多样性及其在海洋和淡水生境间的过渡迁移、物种形成过程、演化及适应性进化机制提供重要参考。