海洋生物分类与系统演化实验室知识库

珊瑚礁生态系统因其高度的生物多样性和复杂的生物间相互作用而备受关注。线虫，作为珊瑚礁沉积物中的关键底栖动物，以及它们的共附生微生物，在维持珊瑚礁生态系统健康和稳定中起着至关重要的作用。当前，珊瑚礁正面临退化危机，保护珊瑚礁至关重要，生物多样性监测能够评估珊瑚礁健康状况，为科学管理和保护措施提供数据支撑。本研究在南海海盆以北区域（西沙群岛和中沙群岛）以及以南区域（南沙群岛）分别设定了8个采样站位，针对采集得到的珊瑚礁沉积物样品，利用eDNA宏条形码测序技术研究线虫生物多样性，利用宏基因组测序技术研究微生物多样性，通过对沉积物单条线虫的分离鉴定，利用eDNA宏条形码测序技术研究其共附生微生物多样性，并通过与沉积物微生物的比较，对线虫共附生微生物进行初步探究。主要结果如下：

（1）南海珊瑚礁沉积物中，鉴定出的线虫门共2纲、10目、37科、51属，优势类群为色矛纲（56.53%）和嘴刺纲（41.87%）。Alpha多样性分析揭示北部区域的线虫群落丰富度和均匀度高于南部区域。Beta多样性分析表明北部区域和南部区域线虫群落结构具有一定相似性。LEfSe分析进一步指出北部区域在双胃科、尖口科、海织科及对应属水平相对南部区域有显著性差异，而南部区域的显著性差异主要体现在索目、索科和六索属。冗余分析表明环境因子（纬度、总氮、总磷、铜、锌、镉、铅、砷）对群落结构的影响不显著。

（2）南海珊瑚礁沉积物中，鉴定出的微生物共4界、72门、185纲、371目、861科、3573属，优势类群为细菌界的变形菌门（45.68%）和放线菌门（8.08%）。Alpha多样性分析表明南部区域的生物量整体高于北部区域，但丰富度和均匀度大致相似。Beta多样性分析表明北部区域和南部区域微生物群落结构具有一定相似性，差异物种丰度柱状图表明两区域差异较显著的为Cloacibacillus属、Maricaulis属和Thermostichus属等。KEGG和eggNOG数据库比对分析表明代谢相关的基因作为主导。Beta多样性分析揭示北部区域和南部区域微生物功能基因具有一定相似性，差异功能基因丰度热图表明了北部区域氨基酸代谢、氮代谢、蛋白质合成相关基因的丰度较高，而南部区域则核苷酸代谢相关基因的丰度较高。微生物元素循环分析表明微生物群落在碳、氮、磷和硫元素循环中分别以厌氧碳固定、氮同化、嘧啶代谢和甲烷生成过程为主。

（3）南海珊瑚礁沉积物中分离的单线虫经DNA条形码鉴定，有效鉴定出249条线虫，共2纲、7目、16科、37属，均属于色矛纲（79.52%）和嘴刺纲（20.08%）。线虫共附生微生物，共鉴定出2界、52门、134纲、427目、904科、2028属，优势类群为变形菌门（39.56%）、拟杆菌门（17.84%）和厚壁菌门（10.72%）。同属不同站位线虫和同站位不同属线虫两种分组方式的Beta多样性分析表明，大部分线虫共附生微生物群落存在一定程度的相似性，仅少部分线虫如CQB、GQC、MJ2A、MJ2B和BJ4B等显示出较大的群落结构差异。功能基因预测分析表明，不同线虫共附生微生物群落的功能基因组成和丰度大致相同，主要包括代谢途径、次生代谢产物、抗生素的生物合成等。线虫共附生微生物与沉积物微生物的比较分析显示，两者共有微生物为295科，丰度高的共有微生物有红杆菌科和黄杆菌科；两者丰度存在显著差异的微生物为根瘤菌科，推测可能和线虫存在共生关系。

综上，通过线虫生物多样性及其共附生微生物的研究结果，为理解南海珊瑚礁生态系统的生物和功能多样性提供了重要的基础数据，并为评估生态系统健康状况、制定有效的保护和管理措施提供了科学依据。

Coral reef ecosystems are of great interest because of their high biodiversity and complex interactions among organisms. Nematodes, as the main benthic organisms in coral reef sediments, and their symbiotic and epiphyte microbe play a crucial role in maintaining the health and stability of coral reef ecosystems. Currently, coral reefs are facing a degradation crisis and their protection is crucial. Biodiversity monitoring can assess the health of coral reefs and provide data support for scientific management and conservation measures. In this study, eight sampling stations were set up in the northern (Xisha and Zhongsha Islands) and southern (Nansha Islands) region of the South China Waters Basin, respectively. Nematode biodiversity were characterized by using eDNA metabarcoding sequencing technology, while microbe diversity were illustrated by using metagenome sequencing technology. Based on the isolation and identification of single nematode in the sediment, the diversity of nematode symbiotic and epiphyte microbe was studied by using eDNA metabarcoding sequencing technology. The main results are as follows：

(1) In the coral reef sediments of the South China Sea, the nematodes identified were 2 classes, 10 orders, 37 families, 51 genera, and the dominant groups were Chromadorea (56.53%) and Enoplea (41.87%). Alpha diversity analysis showed that the richness and evenness of nematode communities were higher in the northern region than in the southern region; Beta diversity analysis showed that the nematode community structures were similar in the northern and southern region. LEfSe analyses further indicated that there were significant differences in the northern region at the level of Diplogasteridae, Oxystominidae, Haliplectidae and corresponding genera relative to the southern region, while the significant differences in the southern region were mainly Mermithida, Mermithidae and Hexamermis. Redundancy analyses showed that environmental factors (latitude, total nitrogen, total phosphorus, copper, zinc, cadmium, lead and arsenic) did not have a significant effect on community structure.

(2) In the coral reef sediments of the South China Sea, the microbe identified include 4 phyla, 72 orders, 185 classes, 371 orders, 861 families, 3573 genera, and the dominant groups were Proteobacteria (45.68%) and Actinobacteria (8.08%). The Alpha diversity analysis showed that the biomass in the southern region was higher than that in the northern region, but the richness and evenness were roughly similar. Beta diversity analysis showed that the microbe communities in the northern and southern region were similar in structure, and the histograms of the abundance of different species showed that Cloacibacillus, Maricaulis, and Thermostichus were significantly different in the two regions. Respectively comparing with KEGG and eggNOG databases, the data showed that metabolism-related genes were predominant in the two region. Beta diversity analysis showed that the microbe functional genes were similar in the northern and southern region, and the heat map of the abundance of different functional genes showed that the abundance of genes related to amino acid metabolism, nitrogen metabolism, and protein synthesis was higher in the northern region, whereas that of genes related to nucleotide metabolism was higher in the southern region. The analysis of microbe elemental cycling showed that the microbe community was dominated by Anaerobic C fixation, nitrogen assimilation, pyrimidine metabolism and methanogenesis during the cycling of carbon, nitrogen, phosphorus and sulfur elements, respectively.

249 nematodes were hand-picked from coral reef sediments of the South China Sea and identified by DNA barcoding, including a total of 2 orders, 7 families, 16 genera, 37 genera. All of them belong to Chromadorea (79.52%) and Enoplea (20.08%). Nematode symbiotic and epiphyte microbe were identified in 2 kingdoms, 52 phyla, 134 classes, 427 orders, 904 families, 2028 genera, and the dominant groups were Proteobacteria (39.56%), Bacteroidota (17.84%) and Firmicutes (10.72%). Beta diversity analyses of nematodes of the same genus in different stations、nematodes in the same station of different genera showed that most of the nematode symbiotic and epiphyte microbe communities were similar, only a few nematodes, such as CQB, GQC, MJ2A, MJ2B, and BJ4B, showed large differences in the community structures of symbiotic and epiphyte microbe. Functional gene prediction analysis showed that the functional gene composition and abundance of different nematode symbiotic and epiphyte microbe communities were roughly the same, mainly including metabolic pathways, Biosynthesis of secondary metabolites and Biosynthesis of antibiotics. Comparative analysis of nematode symbiotic and epiphyte microbe and sediment microbe showed that, they share 295 families of microbe, and the common microbe with higher abundance were Rhodobacteraceae and Flavobacteriaceae; the microbe with significant differences in abundance between the two were Rhizobiaceae, which were presumed to have a symbiotic relationship with nematodes.

In conclusion, the results of the study of nematode biodiversity and its symbiotic and epiphyte microbe provide important basic data for understanding the biodiversity and functional diversity of coral reef ecosystems in the South China Sea, as well as a scientific basis for assessing ecosystem health and formulating effective conservation and management measures.

第1章 绪论

1.1 珊瑚礁概述

1.1.1 珊瑚礁功能

1.1.2 珊瑚礁分布

1.1.3 珊瑚礁退化与保护

1.2 海洋生物多样性研究技术概述

1.2.1 传统生态学调查方法

1.2.2 分子生物学方法

1.2.3 其它研究技术

1.3 珊瑚礁生物多样性研究概述

1.3.1 珊瑚礁动物多样性

1.3.2 珊瑚礁植物多样性

1.3.3 珊瑚礁微生物多样性

1.4 海洋线虫及共附生微生物

1.4.1 海洋线虫简介

1.4.2 共附生微生物简介

1.4.3 海洋线虫共附生微生物研究

1.5 研究内容、目的及意义

第2章 南海珊瑚礁沉积物线虫生物多样性研究

2.1 材料与方法

2.1.1 样品收集

2.1.2 环境因子参数获取

2.1.3 DNA提取和高通量测序

2.1.4 OTUs数据生成与分析

2.1.5 线虫生物多样性和群落结构分析

2.2 结果

2.2.1 有效序列及OTUs

2.2.2 Alpha多样性

2.2.3 物种组成和相对丰度

2.2.4 群落结构与差异种类

2.2.5 环境因子对群落结构的影响

2.3 讨论

2.3.1 南海珊瑚礁线虫生物多样性

2.3.2 群落差异与LEfSe分析

2.3.3 环境因子与群落结构

2.3.4 线虫生物多样性评估方法

2.4 小结

第3章 南海珊瑚礁沉积物微生物多样性研究

3.1 材料与方法

3.1.1 样品收集

3.1.2 DNA提取和高通量测序

3.1.3 宏基因组数据分析

3.1.4 微生物物种多样性和功能多样性分析

3.2 结果

3.2.1 微生物宏基因组测序数据统计

3.2.2 物种组成及丰度分析

3.2.3 微生物群落Alpha多样性

3.2.4 微生物群落比较分析

3.2.5 微生物功能基因组成及丰度分析

3.2.6 微生物功能基因比较分析

3.2.7 微生物元素循环分析

3.3 讨论

3.3.1 宏基因组数据的应用前景

3.3.2 南海珊瑚礁微生物物种多样性

3.3.3 南海珊瑚礁微生物功能多样性

3.3.4 南海珊瑚礁微生物元素循环与代谢途径

3.4 小结

第4章 南海珊瑚礁沉积物线虫共附生微生物研究

4.1 材料与方法

4.1.1 样品收集

4.1.2 单线虫DNA提取和分子鉴定

4.1.3 高通量测序与数据分析

4.1.4 线虫共附生微生物多样性分析

4.2 结果

4.2.1 单线虫分子鉴定

4.2.2 有效序列及OTUs

4.2.3 物种注释及分类学分析

4.2.4 群落结构比较分析

4.2.5 功能基因预测分析

4.2.6 共附生微生物与沉积物微生物比较分析

4.3 讨论

4.3.1 单线虫分子鉴定与eDNA监测

4.3.2 线虫共附生微生物物种多样性

4.3.3 南海珊瑚礁线虫共附生微生物功能多样性

4.3.4 线虫共附生微生物与沉积物微生物

4.4 小结

第5章 总结与展望

5.1 研究结论

5.2 主要创新点

5.3 不足与展望

参考文献

致 谢

作者简历及攻读学位期间发表的学术论文与其他相关学术成果