海洋生物分类与系统演化实验室知识库

  真核微生物包括原生生物和单细胞真菌，有关其生物多样性和地理分布一直是国内外研究的热点，并存在巨大争议。相较水体，海洋沉积物中孕育了更为丰富多样的真核微生物群落，然而由于方法学的局限，对海洋底栖真核微生物多样性和地理分布的研究十分稀少，对其在海洋沉积物中的多样性分布格局尚缺乏认知。本论文针对如下科学问题开展研究：环境条件、纬度梯度和水深梯度等如何影响海洋沉积物中真核微生物多样性和分布。

  本文首先研究了水体的环境DNA（eDNA）对沉积物中真核微生物分子多样性评估的影响，优化海洋底栖真核微生物的分子多样性研究体系。本研究选择了黄海冷水团中的两个站位，同时提取水体和沉积物中的DNA和RNA，且沉积物DNA的提取采用直接提取和洗脱两种方法，结合DNA和cDNA高通量测序，研究水体以及沉积物中纤毛虫分子多样性的关系。结果显示：较之深层水，浅层水中浮游类群对沉积物中纤毛虫多样性评价的影响较大。洗脱DNA与cDNA测序方法类似可显著降低浮游类群eDNA对沉积物中纤毛虫多样性评估的影响。在此基础上，对比分析基于DNA和cDNA测序的海洋沉积物中真核微生物多样性和群落结构发现：较之DNA测序，cDNA测序可以降低后生动物对真核微生物多样性评估的影响。两种方法揭示的同一站位沉积物中真核微生物群落结构显著不同，cDNA测序获得的硅藻最为优势，与基于形态学方法研究结果一致；而DNA测序显示甲藻最为优势。因此cDNA高通量测序对于研究海洋沉积物中真核微生物的分子多样性及分布特征更具技术优势。

  在方法学优化的基础上，采用cDNA高通量测序的方法，研究了黄海冷水团对沉积物中真核微生物多样性和分布的影响。结果显示：真核微生物的分布模式与小型和大型底栖生物类似，冷水团内外群落结构显著不同，并且在南、北黄海显著不同。冷水团外各站位的平均多样性高于冷水团内站位。31%的OTUs仅在冷水团内或者外围检获，此类OTUs大多相对丰度较低，易被形态学检测遗漏；且这些OTUs的代表序列与数据库中已知物种的序列相似性低，属于未知种属。扩布限制，即空间变量，对真核微生物总体和优势类群的分布影响较大；但环境变量（水深和底层水温度）对稀有类群群落构成的影响更大。

  采用cDNA高通量测序的方法，研究了黄海、东海和南海沉积物中真核微生物多样性和分布，探究纬度梯度及环境变量对其分布的影响。结果显示：三个海域总共获得8,785个真核微生物的OTUs，南海获得的OTUs数最高，其他依次为黄海和东海；但就单个站位所获得OTUs数而言，黄海最高，其次为南海和东海。这一分布模式与中国近海大型底栖生物的纬度梯度分布模式不同。Spearman相关性分析显示真核微生物OTUs数与纬度呈显著正相关关系，与底层水温度呈显著负相关关系。黄海、东海和南海沉积物中相对丰度最高的类群不同，分别为硅藻、丝足虫类和纤毛虫。三个海域OTUs数最高的类群均为丝足虫类。整体上，三个海域真核微生物群落结构显著不同，较之南海，黄海和东海的群落构成相似性更高。扩布限制对总体真核微生物、优势和稀有类群分布的影响均大于环境变量。

  采用cDNA高通量测序的方法，研究了北纬36°断面从潮间带到陆架海到深海（6,000 m）沉积物中真核微生物多样性和群落构成，剖析相同纬度海域中水深对底栖真核微生物多样性和分布的影响。结果表明：潮间带到深海的十个站位总共获得3,855个真核微生物的OTUs，在总体OTUs数和单个站位获得的OTUs上，以陆架海区（黄海）为最高、西北太平洋深海次之，青岛湾潮间带海区多样性最低。陆架海与深海的OTUs数与水深呈显著负相关关系，与沉积物叶绿素a和脱镁叶绿素a含量呈显著正相关关系。三种生境中真核微生物群落结构显著不同，陆架海区和潮间带的群落相似性高于其与深海群落的相似性。潮间带的最优势类群为硅藻，而陆架和西北太平洋深海最优势类群为丝足虫类。环境变量对总体真核微生物（水深、沉积物叶绿素a含量和总有机质含量）、优势（水深、沉积物叶绿素a含量和总氮含量）和稀有（水深和沉积物叶绿素a含量）类群的影响大于空间变量的影响。

  总体来讲，本论文优化了海洋沉积物中真核微生物分子多样性的研究方法，首次展示了黄海冷水团（中尺度）对沉积物中真核微生物地理分布的显著影响；并发现中国近海（大尺度）沉积物中真核微生物分布与纬度梯度相关，但与大型生物不同的分布模式；同时在大的空间尺度和水深差异下，初步探明了水深梯度对沉积物中真核微生物分布的控制作用。研究证实真核微生物，尤其是稀有类群的分布具有地理限定性，环境变量和扩布限制显著影响底栖真核微生物的分布，但相对重要性随研究尺度和生境的差异而变化。

  Microbial eukaryotes (=microeukaryotes) comprise of the commonly known protists and lower fungi. The estimation of the diversity and distribution of microbial eukaryotes has long been an ongoing debate and attracted considerable attention. Compared with pelagic seawater, marine sediments inhabit a much higher diversity of microbial eukaryotes. However, our understanding on their diversity and distribution in marine sediments is hampered by the methodological limitations. Hitherto, the questions about how environmental conditions, and latitudinal and depth gradients shap the diversity and distribution of microbial eukaryotes in marine sediments, are still unclear. The aim of this study is to answer the questions mentioned above.

    In this study, we firstly evaluated the influence of the environmental DNA (eDNA) in waters on assessing the microbial eukaryotes diversity in sediments. Based on the sediment and overlying water samples collected from two stations in the Yellow Sea Cold Water Mass (YSCWM), we evaluated the ciliate molecular diversity in sediments in relation to that in the upper water layers by using DNA and cDNA (complementary DNA) high-throughput sequencing. The DNA in sediments was extracted by the direct extraction and elution methods, respectively. The results showed that the planktonic eDNA which affeced the evaluation of the molecular diversity of ciliates in sediments was mainly derived from the upper water layers. In contrast with DNA sequencing by the direct extraction of DNA from the sediments, DNA sequencing by the eluted DNA as well as cDNA sequencing could reduce the influences of planktonic eDNA on the evaluation of molecular diversity of ciliates in sediments. Furthermore, we compared the diversity and community compositions of microbial eukaryotes revealed by DNA and cDNA sequencing. The results showed that cDNA sequencing could reduce the influence of the metazoan DNA on the assessment of microbial eukaryote diversity in comparison with DNA sequencing. Moreover, the communities of microbial eukaryotes revealed by the two methods from the same station were significantly different. cDNA sequencing revealed Diatomea was the most dominant group, which was similar with the results based on morphological methods, while DNA sequencing showed Dinoflagellata was the most dominant group. The results indicate that the cDNA sequencing method is more practicable in detecting the diversity and the distribution pattern of microbial eukaryotes in marine sediments.

  Based on the results of methodological evaluation, we utilized the cDNA high-throughput sequencing method to investigate the influence of the Yellow Sea Cold Water Mass (YSCWM) on the diversity and distribution of microbial eukaryotes in sediments in the Yellow Sea and adjacent sea area. The results showed that a similar pattern with that of meio- and macrobenthos was detected for the microbial eukaryotes, whose communities were significantly different inside and outside the YSCWM, and were significantly different in the north and south Yellow Sea. The mean richness of microbial eukaryotes inside the YSCWM was lower than that outside the YSCWM. About 31% of the total OTUs were exclusively obtained inside or outside the YSCWM, where they were generally in low abundance and were easy to be neglected by traditional morphological methods. Moreover, these OTUs had low identities to known species in the databases, and thus belong to the novel taxa. The dispersal limitation (spatial factors) had stronger effects in shaping the total and abundant communities than environmental factors, while environmental factors (depth and bottom water temperature) were more important in shaping the rare communities.

 We utilized the cDNA high-throughput sequencing method to investigate the microeukaryotes diversity and their distribution in sediments along a latitudinal gradient in the Yellow Sea (YS), East China Sea (ECS) and South China Sea (SCS). A total of 8,785 microeukaryotes OTUs were obtained in the three sea areas, and the number of OTUs obtained at the SCS was the highest, followed by the YS and ECS. By contrast, the mean OTU richness at each station was the highest in the YS, followed by the SCS and ECS. This pattern is different from the latitudinal distribution pattern of macrobenthos in the sea areas of China. The Spearman analysis showed that the number of OTUs was positively correlated with the latitude, and negatively correlated with the bottom water temperature. In term of community composition, the most dominant group varied in the three sea areas. The Diatomea, Cercozoa, and Ciliophora predominated in the YS, ECS and SCS, respectively. Cercozoa had the highest OTUs richness in all the three seas. The communities of microbial eukaryotes in the three seas were significantly different, and the similarity between the communities in the YS and ECS was higher, compared to those with the SCS. The dispersal limitation had stronger effects in shaping the total, abundant and rare communities than environmental factors did.

   We further utilized the cDNA high-throughput sequencing method to investigate the influence of depth gradient on the diversity and distribution of microbial eukaryotes from intertidal through continental shelf to deep-sea sediments (depth up to 6,000 m) in the same latitude (36°N). A total of 3,855 microeukaryote OTUs were obtained from all the samples. The total and mean OTU richness of microbial eukaryotes in the continental shelf (YS) was the highest, followed by the habitats of the deep sea (Northwestern Pacific Ocean) and intertidal zone (Qingdao Bay). The number of OTUs was negatively correlated with the depth and positively correlated with chlorophyll a and pheophytin a content in the coastal and deep-sea sediments. The communities of microbial eukaryotes from the three habitats were significantly different, and the community from the continental shelf was more similar with that from the intertidal zone than that from the deep-sea sediments. The most dominant group in the intertidal zone was Diatomea, and Cercozoa predominated in both the continental shelf and deep-sea sediments. The environmental factors had stronger effects in shaping the total (depth, chlorophyll a content and total organic matter content), abundant (depth, chlorophyll a content and total nitrogen content) and rare (depth and chlorophyll a content) communities than spatial factors did.

  In sum, we improved and optimized the research protocal on the diversity of microbial eukaryotes in marine sediments, and illustrated, for the first time, the influences of YSCWM on the mesoscale distribution pattern of benthic microbial eukaryotes. We also revealed the distribution pattern of benthic microbial eukaryotes along the latitudinal gradients on a large scale (China seas), showing a different latitudinal pattern from macrobenthos. Meanwhile, we revealed the distribution pattern of benthic microbial eukaryotes along a large spatial and water-depth gradient. Overall, this study indicates that the distribution of microbial eukaryotes in marine sediments, particularly that of rare taxa, was geographically restricted and was shaped by both the environmental factors and dispersal limitation. The relative importance of the environmental factors and dispersal limitation in shaping the distribution of microbial eukaryotes varied with different sampling scales as well as multiple habitats.