中国科学院海洋研究所机构知识库

浮游植物（phytoplankton）是海洋生态系统的重要组成部分，是海洋初级生产力的重要来源，参与海洋碳循环、生物地球化学循环等重要生态过程。当海洋中某些有毒有害微型浮游植物、大型藻或蓝细菌等快速增殖或聚集，使得海水水体呈现明显的颜色改变，形成了有害藻华（harmful algal blooms，HABs）。探究浮游植物群落结构和多样性水平，跟踪HAB物种分布和动态变化是了解该海域生态系统状况的重要调查内容，帮助我们更全面地了解海洋生态系统的结构和功能，为保护海洋生态环境和可持续利用海洋资源提供重要依据。相比于传统形态学调查方法，基于高通量测序（high-throughput sequencing, HTS）的宏条形码分析（metabarcoding analysis）可以用于全面、深入解析物种组成和多样性，还可以基于各序列的丰度占比获得各物种的相对定量信息，以便跟踪特定浮游植物类群（如HAB物种）的动态分布规律和变化趋势。

本论文基于通用分子标记18S rDNA V4的宏条形码分析，对山东近岸海域HAB物种开展了全面调查，并针对海链藻和夜光藻这两个典型HAB类群及单细胞样本进行了分子多样性的深入研究。得到的主要结果如下：

（1）基于宏条形码技术的山东近岸海域有害藻华物种分析

利用基于18S rDNA V4区的宏条形码分析方法首次对2022年春季（5月）山东近岸海域HAB物种组成、相对丰度和地理分布格局展开了系统调查分析。共注释鉴定到浮游植物8个门，29个纲，77个目，131个科，213个属，其中绝大多数的ASVs注释为甲藻门。共检出95个HAB物种，其中40个为该海域未记录物种，显示出宏条形码分析方法的优势。宏条形码分析表明山东近岸海域的HAB物种并非均匀分布，而是具有一定的区域性特征，同一种HAB物种分布于多个不相邻的海域。

针对山东近海浮游植物和HAB物种的宏条形码分析发现一些值得重视但是尚未得到很好解决的问题，推测宏条形码分析揭示的分子多样性（molecular diversity，即ASV数目）不等同于物种多样性（species diversity）。另外，ASV的相对丰度与其对应物种的细胞密度并不是简单的线性关系，即宏条形码分析揭示的分子丰度（molecular abundance，即ASV的相对丰度）不等同于物种相对丰度（species abundance）。

（2）基于环境宏条形码技术的典型有害藻华类群分析

为探讨宏条形码分析方法对浮游植物多样性的分析潜力，本论文针对两个典型HAB类群进行了深入探讨，包括硅藻门的海链藻属和甲藻门的夜光藻属。

针对胶州湾海域2019年全年逐月航次样本，共鉴定到20个海链藻物种，其中15个为胶州湾未记录物种。许多海链藻属ASVs在胶州湾海域表现出强烈的时间分布特征差异，分为春冬型海链藻和夏秋型海链藻。相比之下，这些海链藻属ASVs在胶州湾均未表现出较明显的空间分布特征差异。共鉴定到150个夜光藻属ASVs，且相对丰度和丰富度在夏季最低，在秋冬季最高，春季次之；相对丰度和丰富度在胶州湾外高于胶州湾内站位。

针对山东近岸海域样本，共检出18个海链藻物种，其中10个为山东近岸海域未记录海链藻物种。由于采样季节的限制海链藻物种在山东近岸海域的鉴定尚不能覆盖所有山东近岸海域的海链藻历史记录；共鉴定到111个夜光藻属ASVs，丰度最高的优势ASV_8集中分布在东营近海综合养殖区内。不论在山东近岸海域还是胶州湾海域，夜光藻ASVs整体皆呈现出一条相对丰度最高的优势ASV和大量丰度较低的非优势ASVs序列的一致结构。同样是基于18S rDNA V4为分子标记的宏条形码分析，进一步向属水平的聚焦，可以揭示在整体探究时被掩盖的属内多样性及属内物种的分布特征。

（3）典型有害藻华类群的分子多样性探究

为探讨宏条形码分析中分子多样性的特点及其来源，首次开展单个细胞或单个株系内的宏条形码分析。

针对26个海链藻属物种单株系，发现每个株系内都含有多个ASVs且与物种多样性（物种数目）具有较大差距。另外，各株系18S rDNA V4序列组成符合相同的整体趋势，即都具有一条相对丰度最高的优势ASV和大量丰度较低的非优势ASVs。同物种的非优势ASVs基本在不同株系之间共享，表明这些高水平分子多样性并不是由随机发生的扩增或测序错误主导。进一步在16个夜光藻属单细胞内开展研究，聚焦于基因组内的拷贝多样性。发现中国近岸海域的夜光藻不存在物种及种内多样性。每个夜光藻单细胞约有100个ASVs，优势ASV可确定夜光藻的物种及种内遗传多样性，并以此追踪夜光藻物种的动态分布特征。

因此，在环境调查中高水平的分子多样性包括了物种间的多样性，物种内的遗传多样性和基因组内拷贝多样性，后者往往在宏条形码分析的部分属内举足轻重，反而常未被考虑在内。基于此揭露了环境调查宏条形码分析中获得的分子多样性与实际解读的物种多样性及物种内遗传多样性的距离，为环境样本的宏条形码分析调查提供正确的解读思路。

Phytoplankton is a crucial component of the marine ecosystem, contributing significantly to primary productivity in the oceans and participating in major ecological processes, such as the ocean carbon cycle and biogeochemical cycles. When some toxic and harmful micro-phytoplankton, macroalgae or cyanobacteria in the ocean proliferate or accumulate rapidly, the sea water body shows obvious color changes and forms harmful algal blooms (HABs). Exploring the community structure and diversity of phytoplankton and tracking the distribution and dynamic changes of HAB species are crucial for understanding the marine ecosystem status, helping us to understand the cycle and evolution process of the marine ecosystem more coherently, and to understand the structure and function of the marine ecosystem more comprehensively. Compared with traditional morphological methods, high-throughput sequencing (HTS)-based metabarcoding analysis offered a more comprehensive and detailed analysis of species composition and diversity in the samples, but also obtains the relative quantitative information of each species based on the abundance of each sequence, enabling the tracking of dynamic distribution patterns and trends of various phytoplankton species.

In this thesis, metabarcoding analysis based on 18S rDNA V4 was applied to systematically explore the HAB species in the Shandong coast, and then focused on two representative groups, the genus Thalassiosira of diatom and the genus Noctiluca of dinoflagellate, for in-depth research on molecular diversity.

The main contents and results of this thesis are as follows:

1. HAB species analysis in Shandong coast based on metabarcoding analysis

Based on the metabarcoding analysis of 18S rDNA V4, a systematic survey was conducted to determine the species composition, relative abundance, and geographic distribution of phytoplankton and HAB species in Shandong coast in the spring of 2022 (May) for the first time. A total of 8 phyla, 29 classes, 77 orders, 131 families and 213 genera of phytoplankton were identified, of which dinoflagellates were dominant phylum. A total of 95 HAB species were identified, including 40 new HAB species in Shandong coast. This demonstrates the advantages of the metabarcoding analysis. Furthermore, HAB species in Shandong coast were not evenly distributed and have certain regional characteristics, with the same HAB species present in multiple non-contiguous sea areas.

Several issues that require attention but have not yet been adequately addressed have been identified. The molecular diversity (i.e. number of ASVs) revealed by metabarcoding analysis was not equivalent to species diversity. Furthermore, it is important to note that the relationship between the abundance of ASVs and the cell densities of their corresponding species was not always linear. This means that the molecular abundance, as revealed by metabarcoding analyses, does not necessarily equate to the species abundance.

2. Typical HAB genera analysis based on environmental metabarcoding analysis

To analyse the metabarcoding results, explore the potential of metabarcoding methods, this thesis provided an in-depth discussion on typical HAB species, including the genus Thalassiosira of diatom and the genus Noctiluca of dinoflagellate.

For month-by-month samples throughout 2019 in the Jiaozhou Bay, a total of 20 Thalassiosira species were identified, of which 15 were new identified. Many Thalassiosira ASVs showed a strong temporal preference. In contrast, these Thalassiosira ASVs did not show obvious spatial preference in the Jiaozhou Bay. A total of 150 Noctiluca ASVs were identified in the Jiaozhou Bay. The relative abundance and richness of Noctiluca ASVs were the lowest in summer, the highest in autumn and winter, and the second highest in spring. Moreover, the relative abundance and richness of Noctiluca ASVs were higher outside the bay than inside.

For Shandong coast samples, a total of 18 Thalassiosira species were identified, of which 10 were unequivocally identified. Due to the limitations of the sampling season, not all historical records of Thalassiosira in Shandong coast could be identified. A total of 111 Noctiluca ASVs were identified in Shandong coast. The dominant ASV_8 had the highest relative abundance in the Dongying coastal comprehensive culture area. The ASVs in Shandong coast and Jiaozhou Bay all exhibited a structure of one dominant ASV with the highest relative abundance and a large number of non-dominant ASVs with lower abundance. Also based on the metabarcoding analysis of 18S rDNA V4, further focusing on the genus level can reveal the hidden species diversity and distribution preferences within the genus.

3. Molecular diversity dissection of typical HAB genera

To investigate the nature of molecular diversity in metabarcoding analyses, we conducted metabarcoding analyses within individual cells or strains for the first time.

Through metabarcoding analysis of 26 Thalassiosira strains obtained by single-cell isolation and culture, it was discovered that each strain contained a varying number of ASVs. This diversity greatly exceeds the diversity of species (the number of species). Furthermore, the 18S rDNA V4 sequences from strains all followed a consistent pattern. Each strain had one dominant ASV with the highest relative abundance and a large number of non-dominant ASVs with lower abundances. The non-dominant ASVs of the same species are widely shared between strains, indicating that these molecular diversities are not dominated by randomly occurring amplification or sequencing errors. In 16 Noctiluca cells, further studies were conducted within individual cells, focusing on copy diversity within the genome. The study revealed that there was no species or intraspecific diversity in the Noctiluca found in the Chinese waters. There were approximately 100 ASVs per single cell. The dominant ASVs can determine the species and intraspecific genetic diversity of Noctiluca and track its dynamic distribution characteristics.

Thus, the high molecular diversity observed in cruise surveys includes inter-species diversity, intra-species genetic diversity and intra-genomic copy diversity. The latter is often significant within some genera analysed by metabarcoding, but is often not taken into account. Based on this, the distance between the molecular diversity obtained in the metabarcoding analysis of environmental surveys and the actual interpretation of inter-species diversity or intra-specific genetic diversity was revealed, which provides a correct interpretation idea for the metabarcoding analysis of environmental samples.

第1章 绪论... 1

1.1 海洋浮游植物及有害藻华概述... 1

1.1.1 海洋浮游植物概述... 1

1.1.2 有害藻华概述及分类... 2

1.1.3 有害藻华物种多样性... 4

1.2 海洋浮游植物的调查研究概述... 4

1.2.1 基于形态学以及特征物质的浮游植物鉴定方法... 5

1.2.2 基于分子标记的浮游植物鉴定方法... 6

1.2.3 基于宏条形码分析的浮游植物鉴定调查方法... 9

1.3 宏条形码分析技术的应用... 10

1.3.1 多样性与时空动态变化分析... 14

1.3.2 未知种及未记录种的发现... 14

1.3.3 浮游植物与环境因子相关性解析... 15

1.4 两类典型有害藻华概述及研究进展... 15

1.4.1 硅藻门海链藻属研究概述... 15

1.4.2 甲藻门夜光藻属研究概述... 17

1.5 本论文的研究内容与意义... 18

第2章 基于宏条形码技术的山东近岸海域有害藻华物种分析... 20

2.1 前言... 20

2.2 材料与方法... 21

2.2.1 样品采集处理... 21

2.2.2 DNA提取及扩增、测序... 24

2.2.3 宏条形码及生态学分析... 24

2.3 结果... 25

2.3.1 基于DADA2的浮游植物多样性分析... 25

2.3.2 山东近岸海域浮游植物群落组成... 28

2.3.3 山东近岸海域有害藻华物种的组成与分布... 30

2.3.4 基于形态学和宏条形码分析的浮游植物鉴定比较... 36

2.3.5 典型有害藻华物种的地理分布特征... 37

2.3.6 浮游植物多样性与环境因子的相关性... 39

2.4 讨论... 40

2.4.1 山东近岸海域的有害藻华物种... 41

2.4.2 宏条形码分析结果中的分子多样性... 41

2.4.3 宏条形码分析结果中的分子丰度... 42

2.4.4 分子标记及参考数据库的重要性... 43

2.5 小结... 43

第3章 基于环境宏条形码技术的典型有害藻华类群分析... 45

3.1 胶州湾海链藻属的多样性及分布特征... 45

3.1.1 前言... 45

3.1.2 材料与方法... 45

3.1.2.1 航次样本采集及处理... 45

3.1.2.2 DNA提取、PCR扩增及测序... 46

3.1.2.3 基于ASV的生物信息学分析... 46

3.1.2.4 生态学群落分析... 47

3.1.3 结果... 47

3.1.3.1 胶州湾海链藻属多样性... 47

3.1.3.2 胶州湾海链藻属的时空动态分布特征... 56

3.1.3.3 胶州湾海链藻属分布与环境因子相关性... 56

3.1.4 讨论... 58

3.1.4.1 胶州湾海链藻属丰富的多样性... 58

3.1.4.2 春冬型海链藻与夏秋型海链藻... 58

3.1.4.3 宏条形码分析作为新一代海链藻属生态学研究方法... 59

3.2 山东近岸海域海链藻属的多样性及分布特征... 60

3.2.1 前言... 60

3.2.2 材料与方法... 60

3.2.2.1 航次样本采集及处理... 60

3.2.2.2 DNA提取、PCR扩增及测序... 61

3.2.2.3 基于ASV的生物信息学分析... 61

3.2.2.4 生态学群落分析... 61

3.2.3 结果... 61

3.2.3.1 山东近岸海域海链藻属多样性... 61

3.2.3.2 山东近岸海域海链藻属地理分布特征... 67

3.2.3.3 山东近岸海域海链藻属分布与环境因子相关性... 68

3.2.4 讨论... 69

3.2.4.1 山东近岸海域海链藻属分子多样性... 69

3.2.4.2 海链藻属分类地位的不断校正... 69

3.3 胶州湾夜光藻的多样性及分布特征... 70

3.3.1 前言... 70

3.3.2 材料与方法... 70

3.3.3 结果... 70

3.3.3.1 胶州湾夜光藻多样性... 70

3.3.3.2 胶州湾夜光藻时空动态分布特征... 73

3.3.4 讨论... 76

3.3.4.1 胶州湾夜光藻丰富的分子多样性及来源... 76

3.3.4.2 胶州湾夜光藻强烈的时空分布特征差异... 77

3.4 山东近岸海域夜光藻的多样性及分布特征... 78

3.4.1 前言... 78

3.4.2 材料与方法... 78

3.4.3 结果... 78

3.4.3.1 山东近岸海域夜光藻多样性... 78

3.4.3.2 山东近岸海域夜光藻强烈的空间地理分布特征差异... 80

3.4.4 讨论... 83

3.5 小结... 84

第4章 典型有害藻华类群海链藻属物种的鉴定分析... 86

4.1 前言... 86

4.2 材料与方法... 87

4.2.1 样品采集及单株系分离培养... 87

4.2.2 DNA提取、测序及数据处理... 87

4.2.3 通用分子标记及细胞器基因组组装... 87

4.2.4 通用分子标记鉴定... 89

4.2.5 细胞器基因组注释... 89

4.2.6 细胞器基因组比较分析... 89

4.2.7 分歧时间估算... 90

4.3 结果... 90

4.3.1 海链藻单株系分离培养与鉴定... 90

4.3.2 海链藻线粒体基因组基本结构... 96

4.3.3 海链藻线粒体基因组的比较分析及系统发育分析... 97

4.3.4 海链藻叶绿体基因组基本结构... 100

4.3.5 海链藻叶绿体基因组的比较分析及系统发育分析... 104

4.4 讨论... 110

4.5 小结... 112

第5章 典型有害藻华类群的分子多样性探究... 113

5.1 基于单株系宏条形码分析的海链藻属分子多样性解析... 113

5.1.1 前言... 113

5.1.2 材料与方法... 114

5.1.2.1 样品采集与单细胞分离培养... 114

5.1.2.2 单株系DNA提取、扩增和测序... 114

5.1.2.3 单株系宏条形码分析... 114

5.1.3 结果... 115

5.1.3.1 26个海链藻株系的18S rDNA V4多样性... 115

5.1.3.2 海链藻株系间分子多样性差异... 119

5.1.3.3 海链藻株系ASV的系统发育网络... 120

5.1.3.4 非海链藻物种ASV的存在... 121

5.1.4 讨论... 121

5.1.4.1 海链藻属的分子多样性... 121

5.1.4.2 宏条形码技术的挑战... 122

5.1.4.3 非海链藻物种ASV的来源... 123

5.2 基于单细胞宏条形码分析的夜光藻的分子多样性解析... 124

5.2.1 前言... 124

5.2.2 材料与方法... 125

5.2.2.1 样品采集与单细胞分离... 125

5.2.2.2 单细胞DNA提取、扩增和测序... 125

5.2.2.3 单细胞宏条形码分析... 126

5.2.3 结果... 126

5.2.3.1 夜光藻中基因组内18S rDNA V4的高度变异... 126

5.2.3.2 单细胞测序揭示的非夜光藻序列... 132

5.2.4 讨论... 133

5.2.4.1 夜光藻单细胞内分子多样性... 133

5.2.4.2 分子标记的基因组内多样性... 134

5.2.4.3 宏条形码方法在单细胞的应用... 135

5.2.4.4 非夜光藻ASV来源... 135

5.3 小结... 136

第6章 总结与展望... 137

6.1 主要结论... 137

6.2 主要创新点... 138

6.3 研究展望... 138

参考文献... 141

附录一 我国海域硅藻门海链藻有害藻华暴发记录... 172

附录二 山东近岸海域航次采样细节信息... 176

附录三 山东近岸海域HAB物种ASV注释信息（明确鉴定到）... 179

附录四 山东近岸海域HAB物种ASV注释信息（可能存在）... 183

附录五 中国海链藻物种记录及修订名单... 189

附录六 胶州湾海域海链藻历史记录名单（经过Algaebase修订后）... 192

附录七 山东近岸海域海链藻历史记录名单（经过Algaebase修订后）... 195

致 谢... 197

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... 198