海洋生态与环境科学重点实验室知识库

角藻属（Tripos）属于甲藻门（Dinoflagellata）、甲藻纲（Dinophyceae）、膝沟藻目（Gonyaulacales）、角甲藻科（Ceratiaceae）。角藻属物种多样性很高，在全球范围内得到分类鉴定的角藻属物种～800个，其中被藻类数据库（AlgaeBase）收录的角藻物种有81个。角藻属物种含有叶绿体，是重要的生产者，由于角藻属物种可以进行混合营养，也是重要的消费者。角藻属物种形态特征复杂，不同物种具有不同的最适温度、盐度等环境因素，导致不同海域具有不同角藻物种组成，因此角藻属物种常被用作重要的生态指示物。虽然该属物种还没有被发现产毒，但是有些角藻物种可以形成有害藻华，消耗营养和氧气，使鱼类贝类等窒息死亡，给渔业带来巨大损失。迄今10种角藻属物种被发现可以形成有害藻华，包括T. brevis, T. deflexus, T. dens, T. furca, T. fusus, T. humilis, T. macroceros, T. massiliensis, T. muelleri和T. trichoceros。尽管大多数角藻属物种表现出从温带到亚热带再到热带的广泛的温度适应（10 - 30 ℃），基本上涵盖了所有中国海域，表明中国海域应该具有大多数角藻物种。然而迄今为止在中国海域只鉴定到了45个角藻物种。

角藻属物种的形态具有相当大的可塑性，影响物种的准确鉴定。中国海域的角藻属物种多样性研究主要基于形态鉴定，因此可以判断中国海域角藻物种的鉴定存在不准确情况，导致中国海域角藻物种组成及时空动态变化均可能被错估。近年来，基于分子标记的物种鉴定大大提高了对角藻属物种的鉴定准确性。为了检查我国海域角藻属物种的多样性及其时空动态变化，本研究开展了如下两个方面的分析。

（1）基于通用分子标记18S rDNA V4，首次利用宏条形码分析方法系统探索了位于山东近海典型海洋生态系统胶州湾角藻的物种组成及其时空动态变化。针对胶州湾2019年全年12个逐月航次样本的宏条形码分析鉴定了5个已知角藻物种（T. furca, T. fusus, T. eugrammus, T. falcatus and T. massiliensis），其中2个角藻物种（T. eugrammus 和T. falcatus）是胶州湾的未记录物种，显示了宏条形码分析的优势。此外，发现胶州湾还可能存在11个角藻物种，这些角藻物种的18S rDNA V4序列与其近缘物种相同而不能区分，显示通用分子标记18S rDNA V4分辨率的不足。胶州湾角藻物种显示出显著的季节差异，所有角藻物种在夏、秋季的丰度和丰富度均较高，显示出对较高温度的偏好。角藻物种的相对丰度与环境中的NH₄⁺浓度成正相关，与环境中的NO₃^-浓度成负相关，表明环境中的氨氮可能促进绝大多数角藻的生长。

（2）由于夜光藻等物种中存在基因组内分子标记序列多样性（intragenomics variations, IGVs），针对山东近海航次样本中的30个角藻属物种单细胞开展针对通用分子标记18S rDNA V4的靶向扩增和高通量测序，开展宏条形码分析。在每个角藻单细胞中都鉴定出了大量不同的18S rDNA V4序列（amplicon sequence variants, ASVs）（19 -172个）。每个角藻细胞都包括一个的优势ASV（相对丰度较高），以及大量的非优势ASVs（低相对丰度）。每个细胞内ASVs之间的序列差异主要是由于少量碱基的替换和一些碱基的插入/删除事件导致的。30个角藻细胞可根据其优势ASVs分为5组物种，每组对应一个角藻物种，或一组具有18S rDNA V4优势序列相同的多个近缘角藻物种。不同单细胞中优势ASVs的相对丰度变化范围较大（从52.8%到95.7%）。因此，IGVs中非优势ASVs数目占宏条形码分析得到的分子多样性（molecular diversity）的绝大多数。分析结果表明，宏条形码分析结果中揭示的ASVs不仅包括角藻物种间多样性和物种内多样性（即遗传多样性），而且存在大量的IGVs。角藻中IGVs的存在可能导致宏条形码分析中将IGVs错误地解读为角藻的物种多样性或遗传多样性，进而导致角藻多样性的高估。

The genus Tripos belongs to the phylum Dinoflagellata, Dinophyceae, Gonyaulacales, Ceratiaceae. Tripos has many species with ~800 species taxonomically identified globally, of which 81 are listed in AlgaeBase. Tripos species contain chloroplasts, which are important producers, and they can be mixotrophic, so they can also act as consumers. Tripos are often used as an important ecological indicator due to their complex morphological characteristics, and different species have their preferred environmental factors such as temperature and salinity, resulting in different Tripos species composition in different sea areas. Although Tripos species have not been found to be toxic, some Tripos species can form harmful algal blooms, depleting nutrients and oxygen, leading to fish and shellfish suffocation and significant fisheries losses. 10 species have been found to form harmful algal blooms, including T. brevis, T. deflexus, T. dens, T. furca, T. fusus, T. humilis, T. macroceros, T. humilis, T. macroceros, T. humilis, and T. fusus. Most Tripos species show a wide range of temperature preferences (10 - 30 °C) from temperate to subtropical to tropical, which essentially covers all Chinese waters, suggests that most Tripos species should be present in Chinese waters, but only 45 have been identified.

Morphological plasticity of Tripos species can affect accurate species identification, leading to inaccurate assessments of diversity. The diversity of Tripos species in the Chinese waters is mainly based on morphological identification, so it can be judged that there are inaccuracies in the identification of Tripos species in the Chinese waters, which may lead to the misestimation of the species composition and spatiotemporal dynamics of Tripos in the Chinese waters. In recent years, species identification based on molecular markers has greatly improved the accuracy of Tripos species identification. In order to examine the diversity and spatiotemporal dynamics of Tripos species in Chinese waters, the following two analyses were carried out in this study.

(1) In this study, based on the universal molecular marker 18S rDNA V4, the species composition and spatiotemporal dynamics of Tripos in Jiaozhou Bay, a typical marine ecosystem located in offshore Shandong Province, were systematically explored for the first time by using a metabarcoding analysis method. Analysis of 12 monthly voyage samples in Jiaozhou Bay in 2019 detected high diversity of Tripos, including 5 known Tripos species (T. furca, T. fusus, T. eugrammus, T. falcatus and T. massiliensis), including two species, which were first discovered in Jiaozhou Bay (T. eugrammus and T. falcatus), and potential 11 Tripos species. The 18S rDNA V4 sequences of these Tripos species were identical to their allied species and could not be confirmed, showing the inadequacy of the 18S rDNA V4 resolution. Tripos showed a strong seasonal preference, with all Tripos species had higher abundance and richness from June to December, showing a preference for higher temperatures. The relative abundance of the Tripos species was positively correlated with the concentration of NH₄⁺ and negatively correlated with the concentration of NO₃^- in the environment, indicating that ammonia nitrogen in the environment may promote the growth of most of Tripos species.

(2) Due to the presence of intragenomics variations (IGVs) in species such as Noctiluca scintillans, targeted amplification and high-throughput sequencing of the universal molecular marker 18S rDNA V4 were performed on 30 single cells of Tripos from the coastal voyage in Shandong Province, and metabarcoding analysis was carried out. A large number of different 18S rDNA V4 sequences (amplicon sequence variants, ASVs) (19-172) were identified in each Tripos single cell. Each Tripos cell had one dominant ASVs with high relative abundance, and a large number of ASVs that typically had a low relative abundance. Sequence differences in ASVs in each single cell are mainly due to a small number of base substitutions and some insertion/deletion events. The 30 Tripos cells could be classified into five groups of species based on their dominant ASVs, each group corresponding to one Tripos species, or a group of allied species with identical 18S rDNA V4 dominant sequences. The relative abundance of the dominant ASVs varied over a wide range (from 52.8% to 95.7%) from one cell to the next. As a result, the number of non-dominant ASVs in IGVs accounted for the vast majority of the molecular diversity obtained from metabarcoding analyses. This analysis showed that the ASVs revealed in the metabarcoding results included not only Tripos interspecies diversity and intraspecies diversity (i.e., genetic diversity), but also the presence of a large number of IGVs. The presence of IGVs in Tripos may lead to misinterpretation of IGVs as species diversity or genetic diversity in metabarcoding analyses, which in turn may lead to overestimation of Tripos diversity.

第1章 绪论 1

1.1 角藻概述 1

1.2 角藻的生态功能 1

1.2.1 生态指示价值 1

1.2.2 海洋碳循环的重要参与者 2

1.2.3 形成休眠孢囊 2

1.2.4 角藻赤潮物种 3

1.3 角藻赤潮事件 3

1.3.1 国际上重要的角藻赤潮事件 3

1.3.2 我国海域角藻赤潮事件 4

1.4 角藻物种鉴定 6

1.4.1 形态鉴定 6

1.4.2 分子鉴定 11

1.4.3 角藻物种的系统发育分析 12

1.5 基因组内分子标记多样性 15

1.6 科学问题与解决方法 16

1.6.1 存在的问题 16

1.6.2 本论文拟解决的科学问题 16

1.6.3 解决方法 16

1.7 研究内容 17

1.7.1 本论文的技术路线 17

第2章 胶州湾角藻物种的宏条形码分析 19

2.1 前言 19

2.2 材料与方法 20

2.2.1 胶州湾中的样本采集和环境因子测量 20

2.2.2 DNA提取、扩增和测序 21

2.2.3 数据分析 21

2.3 结果 22

2.3.1 胶州湾角藻物种的分子多样性 22

2.3.2 胶州湾角藻的物种多样性 23

2.3.3 胶州湾角藻物种的时间动态变化 24

2.3.4 胶州湾角藻物种的空间分布特征 25

2.3.5 环境因素对角藻物种的影响 26

2.3.6 角藻物种与其他物种的相互作用 27

2.4 讨论 28

2.5 小结 31

第3章 角藻物种的单细胞测序 33

3.1 引言 33

3.2 材料与方法 34

3.2.1 样品采集和单细胞分离 34

3.2.2 数据分析 34

3.3 结果 35

3.3.1 在角藻单细胞中鉴定出高水平的18S rDNA V4基因组内变异 35

3.3.2 不同角藻物种组中基因组内变异的比较分析 39

3.4 讨论 44

3.4.1 角藻物种存在高水平的基因组内变异 44

3.4.2 角藻物种中基因组内的分子变异和进化性质 45

3.4.3 单细胞测序可能揭示角藻的食性及物种的营养相互作用 46

3.4.4 正确选择用于宏条形码分析的分子标记 46

3.5 小结 46

第4章 结论与展望 49

4.1 主要结论 49

4.2 主要创新点 49

4.3 研究展望 49

参考文献 51

致 谢 63

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 65