海洋生物分类与系统演化实验室知识库

我国南海珊瑚礁区地处热带-亚热带地区，生物多样、资源丰富，近年来因全球气候变化、人为营养富集、过度捕捞等问题而陷入退化危机。自由生活海洋线虫是后生动物中的优势类群，其摄食活动和新陈代谢能够促进生态系统物质循环，常被用于评估生态系统健康和海洋环境污染程度。同时海洋线虫作为珊瑚礁生态系统的重要组成部分，其生物多样性也因珊瑚礁危机而遭到破坏。目前针对我国南海珊瑚礁地区的海洋线虫研究相对较少，这一区域海洋线虫种类与分布情况尚不清楚。有关海洋线虫的分类学研究主要围绕传统形态学分析、DNA条形码分析、环境DNA宏条形码分析展开。但是由于海洋线虫体型小且培养困难，基于这些分析方法能够获取的分子生物学信息十分有限。为进一步明确我国南海珊瑚礁地区海洋线虫的生物多样性与分类学地位，本研究围绕海南、中北暗沙及西沙、南沙进行了34个站位的沉积物采集。一方面通过沉积物环境DNA分析初步调查海洋线虫的群体结构，另一方面通过沉积物单线虫分离鉴定来补充海洋线虫物种信息，将两种结果互相结合从而分析我国南海珊瑚礁区海洋线虫的生物多样性及分布情况。与此同时，利用模式生物秀丽隐杆线虫，摸索出适用于单条沉积物海洋线虫全基因组扩增的实验路线，针对此次沉积物分离出的11科22属共28个个体的海洋线虫进行全基因组扩增并首次完成基因组调查。最后基于18S rRNA序列信息，对本次南海珊瑚礁区沉积物分离的海洋线虫进行系统发育关系分析。主要研究结果包括以下几部分：

1. 海南、中北暗沙及西沙、南沙海洋线虫生物多样性

（1）针对海南、中北暗沙及西沙、南沙珊瑚礁地区共34个站位的沉积物，通过环境DNA分析一共鉴定出海洋线虫2纲11目62科100属。三个海域线虫纲水平全部为色矛纲（Chromadorea）与刺嘴纲（Enoplea）。其中线虫目水平主要为小杆目（Rhabditida）；线虫科水平主要为头叶科（Cephalobidae）与孔咽科（Aporcelaimidae）；线虫属水平主要为丽突属（Acrobeles）与孔咽属（Aporcelaimellus） 。

（2）针对海南、中北暗沙及西沙、南沙珊瑚礁地区沉积物进行单线虫分离，共鉴定海洋线虫366条，分属于2纲8目19科45属（含4个未鉴定属）57种（或分类实体）。优势属为伪线属（Pseudonchus）、阿顿嘉属（Adoncholaimus）、折咽属（Ptycholaimellus）、美色属（Euchromadora）与新色矛属（Neochromadora）等。

（3）两种方法鉴定结果差别比较大，其中重合部分共计2纲8目14科8属。综合两种方法的鉴定结果，海南、中北暗沙及西沙、南沙珊瑚礁地区最终鉴定出海洋线虫2纲11目67科137属（含4个未鉴定属）。

2. 海南、中北暗沙及西沙、南沙海洋线虫分布特点

（1）在沉积物环境DNA分析工作中，南沙的海洋线虫物种丰富度最高，海南的物种丰富度最低；中北暗沙及西沙的群落分布均匀程度最高，海南群落分布均匀度最低；海南与中北暗沙及西沙的线虫群落组成更为相似。其中海南优势属为Litomosoides属、Tylenchus属；中北暗沙及西沙优势属为Poikilolaimus属、Criconemoides属；南沙优势属为Tarvaia属、Monoposthia属、Filenchus属、Longidorus属、Haliplectus属、Discocriconemella属、Teratodiplogaster属等。三片海域的共有优势属为Acrobeles属及Aporcelaimellus属。

（2）在单线虫分离鉴定工作中，中北暗沙ZS13站位，以及南沙的AD、DM、Y6站位鉴定出5种及5种以上海洋线虫。西沙YZ、南沙ZX及CG站位的沉积物没有分离出海洋线虫。其余站位大部分都鉴定出了2~4种海洋线虫。其中Adoncholaimus sp.、Pseudonchus sp.、Epacanthion sp.、Viscosia sp.、Acanthopharynx sp.、Axonolaimus sp.、Catanema sp.、Desmodora sp.、Euchromadora sp.、Neochromadora sp.、Prochaetosoma sp.在5个及5个以上站位中均出现了分布。

3. 海南、中北暗沙及西沙、南沙海洋线虫全基因组扩增及系统发育关系

（1）摸索出适用于沉积物单条海洋线虫的全基因组扩增方法。基于沉积物分离得到的海洋线虫，从中选择不同科、同科不同属的个体进行全基因组扩增，共计2纲7目11科22属（包含1个未分类属）28种（或分类实体）。通过全基因组数据首次获取了这些海洋线虫的COI序列信息（1000bp左右），并首次完成基因组调查。

（2）对南海珊瑚礁区全基因组扩增的海洋线虫个体进行系统发育关系分析，结果显示链环科（Desmodoridae）、色矛科（Chromadoridae）与希阿利科（Xyalidae）是单系群，刺嘴科（Enoplidae）与瘤科（Oncholaimidae）的亲缘关系最近。

综上，针对海南、中北暗沙及西沙、南沙海洋线虫的种类、分布与系统发育关系分析，为厘清南海珊瑚礁区海洋线虫生物学信息提供了基础数据。针对沉积物中单条海洋线虫全基因组扩增提出了完整实验路线，这一工作弥补了海洋线虫传统形态分类方法的不足，为海洋线虫分子生物学分析提供了重要的实验方法和参考依据，有关海洋线虫的全基因组测序数据对于填补海洋线虫数据库空白以及促进海洋线虫分子生物学发展具有重要意义。这一实验路线对于海绵、珊瑚、藻类等不易采集、样品量少和提取DNA困难的生物研究工作同样具有参考意义。本工作为南海珊瑚礁区生态系统生物多样性保护、生物资源合理利用提供了科学依据。

The coral reef areas in the South China Sea are located in a tropical subtropical region with abundant biodiversity and resources. In recent years, it has fallen into a crisis of degradation due to global climate change, anthropogenic nutrient enrichment, overfishing, and other issues. Free living marine nematodes are a dominant group of metazoans, and their feeding activities and metabolism can promote ecosystem material cycling. They are often used to evaluate ecosystem health and marine environmental pollution levels. At the same time, marine nematodes, as an important component of coral reef ecosystems, have also suffered damage to their biodiversity due to the coral reef crisis. There is relatively little research on marine nematodes in the coral reef areas of the South China Sea, and it has been found that the number of species is far less than the actual number. At present, taxonomic research on marine nematodes mainly focuses on traditional morphological analysis, DNA barcoding analysis, and environmental DNA metabarcoding analysis. However, due to the small size and difficulty in cultivation of marine nematodes, the molecular biological information that can be obtained based on these analysis methods is very limited. To further clarify the biodiversity and taxonomic status of marine nematodes in the coral reef areas of the South China Sea, this study conducted sediment collection at 34 locations around Hainan, the Zhongsha, Xisha and Nansha Islands. On the one hand, preliminary investigation of the population structure of marine nematodes was conducted through DNA analysis of sediment environment. On the other hand, information on marine nematode species was supplemented through isolation and identification of sediment nematodes. The two results were combined to achieve analysis of the distribution pattern and biodiversity of marine nematodes. At the same time, using the model organism Caenorhabditis elegans, an experimental route suitable for the whole genome amplification of a single sediment marine nematode was explored. The whole genome amplification was carried out for 28 individuals from 11 families, 22 genera, and the genome survey was completed for the first time. Finally, based on the 18S rRNA sequence information, a phylogenetic analysis was conducted on the marine nematodes isolated from the sediments of the South China Sea coral reef area. The main research findings include the following parts:

1. Biodiversity of marine nematodes in Hainan, Zhongsha, Xisha, and Nansha Islands.

(1) Based on environmental DNA analysis, a total of 34 sediment samples were collected in Hainan, Zhongsha, Xisha, and Nansha coral reef areas, and 100 genera of marine nematodes were identified in 11 orders, 62 families, and 2 classes. All marine nematodes are classified at the level of Chromadoria and Enoplea. The main order is Rhabditida; the main family is Cephalobidae and Aporchellamidae; the main genera is Acrobeles and Aporchellamellus.

(2) A total of 366 marine nematodes were identified in the sediment of Hainan, Zhongsha, Xisha, and Nansha coral reef areas, belonging to 2 classes, 8 orders, 19 families, 45 genera (including 4 unidentified genera) and 57 species (or taxonomic entities). The dominant genera include Pseudonchus, Adoncholaimus, Ptycholaimellus, Euchromadora, and Neochromadora.

(3) There is a significant difference in the identification results between the two methods, with overlapping parts totaling 2 classes,8 orders, 14 families, and 8 genera. Marine nematodes were finally identified in 2 classes, 11 orders, 67 families, and 137 genera (including 4 unidentified genera) in the Hainan, Zhongsha, Xisha, and Nansha coral reef areas.

2. Distribution characteristics of marine nematodes in Hainan, Zhongsha, Xisha, and Nansha Islands.

(1) In the DNA analysis of sediment environments, the species richness of marine nematodes is highest in Nansha and lowest in Hainan. The evenness of community distribution is highest in the Zhongsha and Xisha, while the evenness of community distribution is lowest in Hainan. The composition of nematode communities in Hainan is more similar to that in the Zhongsha and Xisha. The dominant genera in Hainan are Litosoides and Tylenchus. The dominant genera of the Zhongsha and Xisha are Poikilolaimus and Criconemoids. The dominant genera in Nansha are Tarvaia, Monoposthia, Filenchus, Longidorus, Halippectus, Discocrisonemella, Teratodiplogaster, etc. The common dominant genera in the three sea areas are Acrobeles and Aporceaimellus.

(2) In the isolation and identification of single nematodes, 5 or more species of marine nematodes were identified at the ZS13 stations in the Zhongsha and the AD, DM, and Y6 stations in the Nansha, indicating a high biodiversity of marine nematodes. No marine nematodes were isolated from the sediments of YZ in Xisha, ZX in Nansha, and CG stations. Most of the other stations have identified 2~4 species of marine nematodes. Adoncholaimus sp., Pseudonchus sp., Epacanthion sp., Viscosia sp., Acanthopharynx sp., Axonoolaimus sp., Catanema sp., Desmodora sp., Euchromadora sp., Neochromadora sp., Prochaetosoma sp. were distributed in 5 or more stations.

3. Whole genome amplification and Phylogenetic relationships of marine nematodes in Hainan, Zhongsha, Xisha, and Nansha Islands.

(1) Explore a whole genome amplification method suitable for single marine nematodes in sediment. Based on sediment separation, marine nematodes were selected from individuals of different families and genera for whole genome amplification, totaling 28 species (or taxonomic entities) in 2 classes, 7 orders, 11 families, 22 genera (including 1 unclassified genus). The COI sequence information of these marine nematodes (around 1000bp) was obtained for the first time through whole genome data, and genome surveys were completed for the first time.

(2) The phylogenetic analysis of marine nematodes with whole genome amplification in the South China Sea coral reef area showed that the Desmodoridae, Chromadoridae, and Xyalidae families are monophyletic groups, while the Enoplidae family has the closest genetic relationship with the Oncholaiidae family.

In summary, the analysis of the species, distribution, and phylogenetic relationships of marine nematodes in Hainan, Zhongsha, Xisha, and Nansha provides basic data for clarifying the biological information of marine nematodes in the coral reef areas of the South China Sea. A complete experimental route has been proposed for the amplification of the whole genome of a single marine nematode in sediment. This work fills the gap of traditional morphological classification methods for marine nematodes and provides important experimental methods and reference basis for molecular biology analysis of marine nematodes. The whole genome sequencing data of marine nematodes is of great significance for filling the gap in marine nematode databases and promoting the development of molecular biology of marine nematodes. This experimental route also has reference significance for biological research works of sponges, corals, algae, etc., which are difficult to collect, have small sample sizes, and are difficult to extract DNA. This work provides scientific basis for the protection of ecosystem biodiversity and rational utilization of biological resources in the South China Sea coral reef area.

第1章 绪论    1
1.1 珊瑚礁概述    1
1.1.1 珊瑚礁介绍    1
1.1.2 珊瑚礁生态危机    1
1.1.3 南海珊瑚礁介绍    2
1.2 海洋线虫概述    2
1.2.1 海洋线虫介绍    2
1.2.2 海洋线虫研究概况    3
1.2.3 我国海洋线虫研究现状    4
1.3 分子生物学研究方法概述    4
1.3.1 DNA条形码技术    4
1.3.2 环境DNA宏条形码技术    4
1.3.3 全基因组扩增测序技术    5
1.4 研究内容、目的及技术路线    7
第2章 海洋线虫群落结构与物种分布调查    9
2.1 材料与方法    9
2.1.1 样品采集    9
2.1.2 实验仪器、试剂及方法    10
2.1.3 DNA提取与高通量测序    10
2.2 结果    11
2.2.1 有效序列及OTUs    11
2.2.2 OTUs物种分类学信息注释    12
2.2.3 海洋线虫物种相对丰度    16
2.2.4 海洋线虫群落组成与分析    18
2.2.5 Alpha多样性分析    19
2.2.6 海域间差异显著性分析    21
2.3 讨论    22
2.3.1 海洋线虫生物分类学    22
2.3.2 海洋线虫群落结构差异    23
2.4 小结    23
第3章 海洋线虫生物多样性分析    25
3.1 材料与方法    25
3.1.1 样品处理    25
3.1.2 实验仪器、试剂及方法    25
3.1.3 单线虫分离与裂解    26
3.1.4 海洋线虫种类鉴定    26
3.2 结果    26
3.2.1 海洋线虫鉴定结果统计    26
3.2.2 海洋线虫分布情况统计    30
3.3 讨论    31
3.3.1 环境DNA分析与单线虫分离鉴定    31
3.3.2 海洋线虫生物多样性    32
3.3.3 海洋线虫物种分布及优势种属信息    32
3.4 小结    33
第4章 海洋线虫全基因组扩增及系统进化关系分析    35
4.1 材料与方法    35
4.1.1 沉积物处理    35
4.1.2 全基因组扩增实验方法优化    35
4.1.3 全基因组扩增及系统发育分析    35
4.2 结果    37
4.2.1 沉积物不同处理方式的比较    37
4.2.2 全基因组扩增实验方法探索    37
4.2.3 基因组调查及数据分析    38
4.2.4 海洋线虫系统发育关系分析    41
4.3 讨论    43
4.3.1 单条沉积物海洋线虫的全基因组扩增方法    43
4.3.2 基于全基因组扩增的海洋线虫基因组分析    43
4.3.3 海洋线虫线粒体基因组组装初步尝试与分析    44
4.3.4 南海珊瑚礁区海洋线虫系统进化关系    44
4.4 小结    44
第5章 总结与展望    47
5.1 研究结论    47
5.2 创新、不足与展望    48
参考文献    49
致 谢    57
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    59