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海山作为深海大洋中的主要生态景观，特殊海底地形和水文动力孕育形成了较为特殊且丰富的生物群落，是深海生物多样性的热点区。海山效应通常是指由海山地形引起的水文扰动及后续的营养盐富集和生物聚集现象。这种海山效应常见于浅水海山，深水海山对浮游微型生物群落是否存在海山效应以及海山效应的影响范围仍不得而知。本文基于对西太平洋麦哲伦海山链的一座深水平顶海山（Kocebu Guyot）的科学考察，结合高通量测序和环境DNA等研究手段，以海山周围水体中的微型生物群落（细菌、原生生物和真菌）为研究对象，围绕深水海山是否有明确的“海山效应”这一科学问题，重点解决深水海山对微型生物群落多样性和连通性的影响范围及机制。

本研究首先以吕宋海峡表层水体的真核微生物群落为研究对象，利用二代高通量测序技术，探讨了采样量对寡营养海域的真核微生物分子生态学研究的影响。研究发现，采样量对评估真核微生物多样性及群落结构存在影响。在10 L至20 L的采样量范围内，采样量越大，检获的真核微生物ZOTU（Zero-radius Operational Taxonomic Units，可操作分类单元）数量越多。单因素方差分析结果显示，10 L、20 L、30 L和40 L水所检获的ZOTU数量之间没有显著差异；各采样量分组的Shannon、Chao1和ACE指数也不存在显著差异。在10 L至20 L的范围内，随着采样量的增加，真核微生物群落的稀有ZOTU数量也在增加，但丰富ZOTU数量保持稳定。PCoA结果显示，10 L、20 L、30 L和40 L水所检获的真核微生物群落结构之间不存在显著差异。综合考虑样品可得性和数据真实性，20 L水的采样量可用于寡营养海域微型生物的分子多样性研究。

基于对麦哲伦海山链一座深水（山顶-1,198 m）平顶海山周围水体中的细菌、原生生物和真菌群落的多样性和连通性的解析，本文发现深水海山对其周围水体中的细菌、原生生物和真菌群落存在“海山效应”，且不同的微生物群落对海山效应的反应机制不同。在生物多样性方面，细菌丰富度呈单峰型分布，300 m层最高，3,000 m层最低；原生生物丰富度呈三峰型分布，底层最高，200 m层和表层次之，3,000 m处最低；真菌丰富度总体随水深的增加而减少。在群落连通性方面，细菌的连通性最高，同水层和不同水层之间的样品共发生关系更复杂，原生生物共发生关系基本发生在山顶以上深度的同水层样品之间，真菌的样品共发生关系较前两者明显较少；细菌、原生生物和真菌群落同站位的样品在山顶以下深度的共有ZOTU比例与山顶以上深度相比均明显增加，而非海山区原生生物同站位共有ZOTU比例在以山顶深度为分界点的两个水层的变化幅度明显小于海山区的原生生物群落。上述结果表明，海山促进了其周围水体中细菌和原生生物垂直方向上的扩散，但限制了原生生物水平方向上的扩散。在生态过程组成机制方面，细菌群落受到决定性过程和随机性过程的组合影响，原生生物群落结构受决定性过程影响较大，而真菌群落主要受到随机性过程的影响。整体上，原生生物对深水海山效应的反应最为敏感，深水海山增强了原生生物群落的垂向交换，但限制其水平扩散，这一过程对其多样性的分化具有重要促进作用。

本研究以纤毛虫这一原生生物的代表类群为研究对象，解析了深水海山效应的影响范围。基于海山区和非海山区纤毛虫群落的对比研究发现，深水海山促进了纤毛虫群落在垂直方向上的扩散，增强了群落的共发生关系，能够影响山顶以上1,000 m范围内的纤毛虫群落结构。海山区和非海山区的纤毛虫群落结构未见显著差异，但海山区的纤毛虫分类多样性（ZOTU丰富度）和系统发育多样性（Faith’s PD）在200 m处最高，该层的纤毛虫群落与其上的真光层相似度较高；而非海山区纤毛虫多样性最高值并非出现在200 m层，而是在通常的叶绿素最大层（Deep chlorophyll maximum，DCM），200 m层的群落则与其下的暮光层及无光层的相似度较高。推测造成海山区和非海山区差异的主要原因是海山上方存在涡旋使理化环境发生改变，增大了叶绿素最大层的深度，并增加了真光层与200 m层的交流。海山区纤毛虫群落的样品共发生关系网络和物种共发生关系网络相比非海山区更复杂，可能是因为海山区垂直混合增加，促进了纤毛虫群落在不同水层之间的扩散。PLS-PM分析结果显示，水深对非海山区纤毛虫群落的分类多样性和系统发育多样性具有显著影响，而对海山区则不显著；方差分解分析结果显示，深度对非海山区的群落分类和系统发育多样性的差异性的解释率高于海山区，这可能是因为海山促进了不同水层间纤毛虫群落的交流，从而降低了深度的影响。另外，海山区自底层水体向上至叶绿素最大层均能检获底栖纤毛虫类群（核残迹纲），且丰度由底层至上层逐渐减少。源汇分析显示DCM层的核残迹纲主要来自于下层水体，推测是海山区较强的垂直混合将底栖纤毛虫类群输送到了真光层。

此外，本文结合第三代高通量测序技术，解析了深水海山对原生生物种下水平多样性的影响。基于原生生物三代测序数据与单倍体网络分析的结果发现，海山区的Caecitellus spp.的单倍体多样性较高，尤其在500 m及以深的水层。不同水层之间，尤其是在500 m及以深的水层之间，种群间的基因流水平较高。遗传分化与变异分析显示，不同水层之间的遗传分化程度不明显，但同一水层内的遗传变异水平较高。海山处加强的垂直混合促进了不同水层的Caecitellus spp.的垂直扩散，异质化的环境提高了基因变异水平，从而促进了海山处Caecitellus spp.的分化。

总体来讲，本研究发现深水海山存在明确的“海山效应”，揭示了深水海山的“海山效应”对水体中的微型生物群落水平和垂直尺度的影响，发现细菌、真菌和原生生物具有不同的反应机制。相较细菌和真菌，原生生物群落对海山效应的反应最为敏感，深水海山通过增强不同水层之间的混合促进了其群落的垂直扩散，增加了连通性，影响范围可达真光层。海山区增强的垂直混合提高了不同水层之间的基因流水平，因此促进了深海生物的遗传变异与进化。

Seamounts are predominant ecological landscapes that distribute in deep ocean, their unique undersea topographies combining with hydrological dynamics harbour special and diverse biological communities and are hotspots of deep-sea biodiversity. Seamount effect is generally defined as hydrography disturbances caused by topography and subsequent nutrient enrichment and biological aggregations around seamounts. Seamount effect has been frequently observed in shallow seamounts, but has never been documented in deep seamounts. Whether and to what extent deep seamounts can have an imprint on planktonic community is still unknown. The dissertation is based on the scientific research of a deep flat-topped seamount of the Magellan Seamount Chain（MSC）in the western Pacific Ocean, utilizes the combination of high-throughput sequencing and environmental DNA, takes the planktonic microbial communities（bacteria, protists and fungi）as research objects, focuses on the unambiguous “seamount effects” of the deep seamount upon planktonic microbes and aims to unravel the influencing range of the “seamount effect” and underlying mechanisms shaping the diversity and connectivity of planktonic microbial communities.

The research firstly took the microeukayote community in the Luzon Strait as subject and evaluated the influence of sample size in exploring microeukayote molecular ecology in oligotrophic surface ocean using the second generation high-throughput sequencing technology. The research revealed that sample size had influence on evaluating the microeukayote biodiversity and community structure. In the range of 10 L to 20 L, the ZOTU（Zero-radius Operational Taxonomic Units）richness of microeukaryotes increased as the sample size increased. The results of analysis of variance indicated that there were no significant differences between ZOTU richness obtained from 10 L, 20 L, 30 L and 40 L water；there was no significant difference among Shannon, Chao1 and ACE indexes of all sample sizes. In the range of 10 L to 20 L, as the sample size increased, the number of rare ZOTU increased, while number of abundant ZOTU was steady. The results of PCoA showed that no significant difference was detected between community structure of 10 L, 20 L, 30 L and 40 L water. Taken together the sample availability and data validity, 20 L can be used as proper sample size for microeukayotes molecular ecology research in the oligotrophic surface ocean.

Based on the research exploring the diversity and connectivity of planktonic microbial communities around a deep flat-topped seamount（summit depth -1,198 m）in the MSC, we revealed that the deep seamount exerted “seamount effect” upon bacteria, protist and fungi communities and different microbial communities had different response mechanisms. For biodiversity, bacterial richness had a unimodal pattern with the highest value at the 300 m and the lowest at the 3,000 m；richness of protists had a trimodal pattern with the highest value at the bottom, 200 m and surface are subsequently lower, and lowest at the 3,000 m；fungal richness decreased as the depth increased. For community connectivity, bacteria had highest connectivity with more complex co-occurrence relationships between samples from same water layers and between sample from different water layers；the co-occurrence relationships of protists community were mostly detected between samples from same water layer, while the co-occurrence relationships of fungal community were much less than that of bacterial and protist communities；the proportions of shared ZOTUs among samples from same site of bacterial, protist and fungal communities below the summit depth were higher than that above the depth, in non-seamount area, the proportions of shared ZOTUs of protists from same site below the summit depth was also higher than that above the depth, but the increase degree in non-seamount area was lower than that in the seamount；the abovementioned results indicated that the seamount promoted the vertical dispersal of bacterial and protists communities, but impeded the horizontal dispersal of protists. For ecological processes, bacterial community was shaped by the combined action of deterministic and stochastic processes, protist community was mainly determined by deterministic processes, while fungal community was mainly influenced by stochastic processes. Overall, protists were most sensitive to the deep seamount effect. The deep seamount enhanced the vertical mixing of protist community, but impeded the horizontal dispersal. This impediment could promote the diversification of protists around the deep seamount.

The research chose ciliates, a representative protist group, to disentangle the influence range of deep seamount effect. The comparison between the ciliate communities around the seamount and in the non-seamount area revealed that the deep seamount promoted the vertical dispersal of ciliates and enhanced the co-occurrence relationships of ciliates community with a wide range of 1,000 m above the summit. There was no significant difference between ciliates communities in the seamount and non-seamount area. Nevertheless, in the seamount area, the taxonomic diversity（ZOTU richness）and phylogenetic diversity（Faith’s PD）of ciliates peaked at 200 m and had higher similarity with photic community；while in the non-seamount area, the taxonomic and phylogenetic diversity of ciliates were highest at normal DCM and had higher similarity with communities in twilight zone and aphotic zone. This distinction may owe to the ocean eddy influencing the physical-chemical environments around the seamount, thus moved the DCM water layer down and enhanced the interaction between photic water and 200 m water. The sample-sample and taxon-taxon co-occurrence relationships of ciliates community around the seamount was more complex than that in the non-seamount area, this may due to the enhanced vertical mixing at the seamount and thus promoted the dispersal of ciliates communities in different water layers. The results of PLS-PM showed that depth had significant influence on the taxonomic and phylogenetic diversity of ciliates community in the non-seamount area, while there was no significant correlation between depth and ciliates diversity around the seamount；the results of variance partitioning analysis indicated that depth had more explanation on variation of taxonomic and phylogenetic diversity of ciliates community in non-seamount area than that of seamount ciliates community, this distinction resulted from the enhanced vertical interaction of ciliates community around the seamount, and thus decreased the influence of depth. Moreover, a typical benthic ciliate taxon（Karyorelictea）was detected from bottom water layer to DCM water layer with a decreasing abundance；the SourceTracker analysis showed that the Karyorelictea in DCM water layer mainly came from lower water layers, thus leads to the conclusion that the enhanced vertical mixing around the seamount transported the benthic ciliates into the photic zone.

The research utilized the third-generation sequencing to unravel the deep seamount effect upon intra-species diversity of protists. The research based on the third-generation sequencing and haplotype network revealed that the Caecitellus spp. exhibited high haplotype diversity around the seamount, especially in water layers below 500 m. The gene flow was high between different water layers, especially between water layers deeper than 500 m. Genetic diversification and variation analysis suggested little genetic differentiation between water layers, but high level of genetic variation within each water layer. Hence, the seamount promoted the vertical dispersal of Caecitellus spp. in different water layers through the enhanced vertical mixing around the seamount. The heterogeneous environments boosted the genetic variation and thus facilitated the diversification of Caecitellus spp.

Overall, the research revealed a clear “seamount effect” of the deep seamount, and illustrated the “seamount effect” of the deep seamount upon surrounding planktonic microbes in horizontal and vertical aspects, and found that bacteria, protists and fungi had different responding mechanisms. Protists were the most sensitive group to the seamount effect compared with bacteria and fungi. The deep seamount significantly promoted the vertical dispersal and community connectivity of protists by enhancing mixing of different water layers, and the influential range could reach to the photic layer. The enhanced vertical mixing increased gene flow between different water layers, thus provided possibility of genetic variation and evolution of protists.

第1章  引言... 1

1.1  海山效应及作用机制... 1

1.2  海山的生物多样性及连通性研究现状... 3

1.3  微型生物分子多样性及地理分布格局研究现状... 4

1.4  研究目的与意义... 5

第2章  采样量对寡营养海域真核微生物分子多样性评估的影响... 7

2.1  前言... 7

2.2  材料与方法... 8

2.2.1  采样点的选择和样品的采集... 8

2.2.2  DNA的提取和原生生物18S rDNA的扩增... 9

2.2.3  高通量测序与数据处理... 10

2.3  结果... 11

2.3.1  物种丰富度与采样量的关系... 11

2.3.2  群落结构与采样量的关系... 13

2.3.3  优势种和稀有种与采样量的关系... 14

2.3.4  采样量对不同粒级生物多样性的影响... 16

2.4  讨论... 16

2.4  小结... 18

第3章  深水海山对浮游细菌、原生生物和真菌多样性及群落连通性的影响... 19

3.1  前言... 19

3.2  材料与方法... 20

3.2.1  采样地与样品采集... 20

3.2.2  DNA提取、PCR扩增和高通量测序... 23

3.2.3  Alpha多样性和Beta多样性... 24

3.2.4  网络分析评估群落连通性... 25

3.2.5  环境选择、扩散和漂移对微型生物群落组成的影响... 26

3.2.6  环境因素对微型生物群落的影响... 26

3.2.7  海山的浮游微型生物群落的共有ZOTU比例及非海山区原生生物共有ZOTU比例... 31

3.3  海山的细菌、原生生物和真菌的生物多样性和群落结构... 31

3.3.1  细菌、原生生物和真菌的多样性... 31

3.3.2  细菌、原生生物和真菌的群落结构... 34

3.3.3  细菌、原生生物和真菌群落的连通性... 34

3.4  海山的细菌、原生生物和真菌群落组成的驱动因素... 36

3.4.1  环境选择、扩散和漂移对细菌、原生生物和真菌群落组成的影响... 36

3.4.2  环境选择对细菌、原生生物和真菌群落组成的影响... 37

3.4.3  扩散对细菌、原生生物和真菌群落组成的影响... 38

3.5  讨论... 42

3.5.1  水平方向上的“海山效应”. 43

3.5.2  垂直方向上的“海山效应”. 45

3.5.3  影响海山周围浮游微型生物分布及群落连通性的因素... 48

3.6  小结... 49

第4章  深水海山对浮游纤毛虫多样性及群落连通性的影响... 51

4.1  前言... 51

4.2  材料与方法... 52

4.2.1  采样地与样品采集... 52

4.2.2  DNA提取、PCR和高通量测序... 56

4.2.3  纤毛虫群落的分类多样性和系统发育多样性... 56

4.2.4  纤毛虫群落的生态过程组成... 58

4.2.5  样品共发生关系网络和物种共发生关系网络... 58

4.2.6  环境因子对纤毛虫群落结构的影响... 58

4.3  海山区和非海山区的纤毛虫生物多样性与群落结构... 59

4.4  纤毛虫群落组成的驱动因素... 64

4.4.1  影响海山区和非海山区纤毛虫群落的生态过程... 64

4.4.2  环境因素对海山区和非海山区纤毛虫多样性的影响... 64

4.4.3  扩散过程对海山区和非海山区纤毛虫群落的影响... 67

4.4.4  海山区和非海山区的物种共发生关系网络... 68

4.5  讨论... 75

4.5.1  深水海山效应的证据... 75

4.5.2  海山增强了垂直连通性和共发生关系复杂性... 77

4.6  小结... 77

第5章  三代测序揭示原生生物种下水平的水深分布格局... 79

5.1  引言... 79

5.2  材料与方法... 80

5.2.1  采样地和样品采集... 80

5.2.2  DNA提取和三代测序... 80

5.2.3  Swarm聚类和单倍体网络的构建... 80

5.3  结果与讨论... 81

5.3.1  Caecitellus spp.的进化关系... 81

5.3.2  Caecitellus spp.单倍体在不同水层的分布... 82

5.3.3  种群基因结构以及多样性... 82

5.4  小结... 86

第6章  结论与展望... 89

6.1  结论... 89

6.2  创新点... 90

6.3  问题与展望... 90

参考文献... 91

致  谢... 107

作者简历及攻读学位期间发表的学术论文与研究成果    109