海洋生态与环境科学重点实验室知识库

由于人类活动导致的富营养化以及气候变化，长江口海域是我国有害藻华（harmful algal blooms, HABs）高发区之一。为了探索长江口海域有害藻华的暴发规律和暴发机制，人们对长江口海域的有害藻华物种及其相关浮游植物开展了大量的航次调查研究。在对长江口海域浮游植物以及有害藻华物种的调查研究中，绝大多数研究是利用基于显微镜观察的形态学方法，获得了大量的调查数据。尽管如此，这些传统的研究方法不能充分区分形态相近的隐存物种，并且经常会漏掉尺寸比较小以及难以固定的物种，不能获得对长江口海域浮游植物特别是有害藻华物种的准确认识。本研究采用宏条形码分析方法，对长江口海域样本进行扩增子序列变异（amplicon sequence variant, ASV）分析，通过生物信息学分析，获得了长江口海域中浮游植物特别是有害藻华物种的组成及分布规律，揭示长江口海域有害藻华物种的多样性可能是该海域有害藻华频发的一个重要原因。历史航次分析表明角毛藻属（Chaetoceros）在长江口具有很高的多样性，并且其中包括多个物种有害藻华物种。本研究对长江口及我国其他近岸海域的角毛藻属物种进行了系统宏条形码研究。首先，以胶州湾为生态模型系统分析了角毛藻属物种的生物多样性以及不同物种的遗传多样性、时空分布的动态变化以及与环境因子的关系。其次，为了进一步分析角毛藻属物种的系统演化，从中国近岸海域分离了角毛藻属株系，通过全基因组测序和组装，首次构建了角毛藻属物种的线粒体基因组，并利用比较线粒体基因组学的方法，分析了角毛藻属物种的多样性和系统演化。主要的研究结果有：

1. 长江口海域浮游植物及有害藻华物种研究的统计分析

系统整理和分析了过去40年间长江口海域历次航次调查的研究结果，比较分析了52个航次调查鉴定的浮游植物组成，重点探索了有害藻华物种的组成。长江口海域与渤海、南海等海域相比具有很高的浮游植物多样性，物种以硅藻和甲藻为主，硅藻多样性最高。在航次调查报道中出现次数最多的是中肋骨条藻（Skeletonema costatum），其次是东海原甲藻（Prorocentrum donghaiense）。

虽然大多数报道认为在近几十年长江口海域的甲藻组成逐渐增加，硅藻的组成逐渐降低，但是对历史资料的总结和分析结果并不支持硅藻和甲藻组成的这种变化。本研究发现有害藻华物种也存在较高的生物多样性，共统计整理出125个有害藻华物种，包括硅藻69种、甲藻49种、蓝藻3种、金藻2种和隐藻1种。值得注意的是，尽管这些有害藻华物种存在于长江口海域，并不是所有的物种都在长江口海域暴发过藻华。藻华暴发需要特定的环境因子和营养状态。其中，中肋骨条藻、东海原甲藻以及米氏凯伦藻（Karenia mikimotoi）等是长江口海域主要的有害藻华物种。以往对长江口海域的调查绝大多数是采用显微镜镜检的方法进行物种鉴定，但是这种方法要求研究人员具有熟练的经验来判断物种的种类，对于不同浮游植物种内形态多样性的判断，形态相似的隐存种、对尺寸较小以及难以固定的浮游植物细胞的鉴定并不准确。因此，对于长江口海域有害藻华物种多样性的认识，以及有害藻华暴发机制的了解，尚需要结合分子分析方法进行更加深入的研究。

2. 长江口海域浮游植物分布格局的宏条形码分析

利用宏条形码方法对包括长江口门内和长江口门外的18个站位的浮游植物以及有害藻华物种的组成和分布进行了分析。共获得4,161个注释为浮游植物的ASVs，属于7个门，30个纲，共注释到785个物种（包括433个可能存在的物种），与渤海、东海等海域相比长江口海域的浮游植物具有很高的生物多样性。另外，浮游植物的组成有明显的地理分布格局，长江口门内与长江口门外海域的浮游植物的组成有一个清晰的分界，棕鞭藻门（Ochrophyta）和隐藻门（Cryptophyta）主要分布在长江口门内，而甲藻门（Dinoflagellata）主要分布在长江口门外海域，此外还有一些物种主要分布在淡水和海水的交汇区。785个浮游植物中，有86个有害藻华物种（包括37个可能存在的物种），大部分有害藻华物种具有相似的分布格局，主要分布在长江口门外海域，说明长江口门内的有害藻华物种对长江口有害藻华频繁暴发的贡献不大。另外，长江口以往报道中最具优势的物种是中肋骨条藻，而在我们的研究中发现，骨条藻属（Skeletonema）物种具有较高的生物多样性，其中以玛氏骨条藻（Skeletonema marinoi）的丰度最高。在浮游植物及有害藻华物种与环境因子的相关性分析中

发现，盐度和温度对浮游植物的影响最大。另外，球形异帽藻、剧毒卡尔藻、多米环沟藻（Gyrodinium dominans）和Karenia papilionacea等有害藻华物种，与环境因子之间有显著的相关性。

3. 胶州湾角毛藻属物种的生物多样性及时空动态变化

利用宏条形码方法对胶州湾2019年12个月12个站位的角毛藻属物种的多样性和时空分布进行了研究。研究发现胶州湾的角毛藻属物种具有很高的多样性，155个ASVs中49个ASVs鉴定到了22个物种，还有106个ASVs只鉴定到了属的水平。另外，角毛藻属物种的丰富度和丰度在胶州湾有明显的时间分布，不同藻的分布特征不同，比如作为胶州湾优势种之一的旋链角毛藻（C. curvisetus）主要分布在7-12月份，在9月份丰度最高。但在空间分布上，角毛藻属物种的丰富度和丰度在各站位较均匀，没有明显的差异。在与环境因子的分析中发现温度、硅酸盐以及氮元素对角毛藻属物种的影响最大。

4. 角毛藻属物种线粒体基因组比较分析

目前，关于角毛藻的研究多以形态学和利用单个通用分子标记的研究为主。由于形态特征和单个通用分子标记分辨率的局限，对于角毛藻属的多样性及其系统演化缺少系统性的研究。近年来，细胞器基因组作为“超级条形码”被用于物种鉴定和进化分析。本研究探讨了利用线粒体基因组（mtDNAs）在研究角毛藻生物多样性及其演变中的意义。首次构建了18株角毛藻属物种的线粒体基因组，包括9个物种和7个角毛藻属未定种，说明角毛藻属还存在很多隐存种以及一些物种的分子标记还没有构建。与通用分子标记相比线粒体基因组可以很好的区分角毛藻株系，具有较高的分辨率。另外通过比较线粒体基因分析发现，虽然角毛藻属物种的多样性很高，但是线粒体基因组却很保守。通过分化时间的分析表明角毛藻属物种分化时间比较早。角毛藻属线粒体基因组的构建还可以为后续开发高分辨率的特异性分子标记提供支持，对角毛藻属物种特别是有害藻华物种的遗传多样性及生物地理分布格局提供有效的分子工具。

综上所述，本研究的主要创新点在于首次利用基于ASV的宏条形码方法对长江口海域的浮游植物及有害藻华物种的组成及分布进行了深入的分析，并结合生物信息学的方法探讨了环境因子与浮游植物间的相互关系。并首次利用基于ASV的宏条形码方法对胶州湾角毛藻属物种的多样性和时空动态变化进行了系统性的分析。另外，首次构建了18株角毛藻属物种的线粒体基因组，通过比较线粒体基因组学的方法对角毛藻属物种的多样性和系统演化进行了分析。本论文的研究成果为长江口海域有害藻华的监测、预警及防控提供了数据支持，同时也为角毛藻属物种的生物多样性、遗传多样性、时空分布动态变化及系统演化研究提供了重要的参考。

Due to serious eutrophication caused by human activities and climate change, the Changjiang Estuary is one of the areas with frequent harmful algal blooms (HABs) in China. In order to explore the regularity and mechanisms of the development of HABs in the Changjiang Estuary, a large number of expeditions have been conducted on HAB species, as well as other phytoplankton species that may also play a role in the development of HABs. In the investigation of phytoplankton and HAB species in the Changjiang Estuary, most of the researchs applied microscopic observation of morphological features, resulting in the accumulation nof a large quantity of valuable investigation data. However, morphological features-based methods usually have limited resolutions that can not accurately distinguish cryptic species with similar morphological features, and often misses species with small sizes and species that are difficult to fix. In this study, the composition and distribution of phytoplankton (including HAB species) in the Changjiang Estuary were analyzed using metabarcoding analysis. This study further characterized and analyzed Chaetoceros species, which are dominant phytoplankton uncovered in the metabarcoding analysis of samples collected in the Changjiang Estuary and in other coastal regions in China. First, the biodiversity, genetic diversity, and spatial-temporal distribution patterns of Chaetoceros species were analyzed using samples collected monthly in the Jiaozhou Bay. Second, phylogenetic evolution of Chaetoceros species were ascertained using common molecular markers and complete mitochondrial genomes (mtDNAs) of Chaetoceros strains isolated from various coastal regions of China. Main findings are as follows:

1. Comprehensive analysis of phytoplankton and HAB species in the Changjiang Estuary

Through statistical evaluation of 52 historical expeditions carried out in the Changjiang Estuary over the past 40 years, the diversity of various groups of 

phytoplankton in the Changjiang Estuary was compared. Diatom and dinoflagellates were found as the dominant species, with diatom species showing the highest diversity. Among all phytoplankton species reported, the most frequently occurring species was the diatom Skeletonema costatum, followed by the dinoflagellate Prorocentrum donghaiense. Although many reports suggested that the composition of dinoflagellates in the Changjiang Estuary gradually increased and that of diatoms gradually decreased in recent years, our quantitative comparison did not support such trends. High biodiversity of HAB species was also found. Among 125 HAB species uncovered in these expeditions, 69 were diatoms, 49 were dinoflagellates, three were cyanobacteria, two were Chrysophyta and one was Cryptophyta. It is important to note the presence of a HAB species in the Changjiang Estuary, does not mean that it causes HAB outbreaks. Algal blooms require specific environmental factors and nutritional status. Dominant HAB species in the Changjiang Estuary included Skeletonema costatum, Prorocentrum donghaiense, and Karenia mikimotoi. Methods applied in these historical expeditions of the Changjiang Estuary were predominantly morphological features-based methods that used microscope to identify the species. Such methods not only demandes skilled researchers with extensive traning, but also suffers from low resolution that cannot accurately distinguish cryptic species with similar morphology, species with small cell sizes, and species that are difficult to fix. Therefore, to gain accurate and in-depth understanding of species diversity and mechanism of the development of HABs in the Changjiang Estuary, molecular methods are needed to complement morphological features-based methods.

2. Metabarcoding analysis of HAB species in the Changjiang Estuary

The composition and distribution of phytoplankton and HABs at 18 sites inside and outside of the Changjiang Estuary were analyzed using metabarcoding analysis. A total of 4,161 ASVs annotated as phytoplankton were obtained, belonging to 7 phyla and 30 classes, and a total of 785 species were annotated (including 433 possible species). Phytoplankton had a high biodiversity. In addition, the phytoplankton has 

obvious geographical distribution pattern, with a clear boundary between the phytoplankton composition inside and outside the Changjiang Estuary, Ochrophyta and Cryptophyta are mainly distributed inside Changjiang Estuary, the Dinoflagellata are mainly distributed outside the Changjiang Estuary, and some species are mainly distributed in the intersection of freshwater and seawater. Among the 785 phytoplankton, there were 86 HAB species (including 37 possible species). Most of the HAB species had similar distribution pattern, mainly distributed outside the Changjiang Estuary, indicating that the HAB species in the Changjiang River contributed little to the frequent occurrence of harmful algal blooms. In addition, Skeletonema costatum is the most dominant species reported in the the Changjiang Estuary, and our study found that the species of Skeletonema has a high biodiversity in the the Changjiang Estuary and Skeletonema marinoi is the dominant species. In the correlation analysis between phytoplankton and HAB species and environmental factors, it is found that salinity and temperature have the greatest influence on phytoplankton. In addition, harmful algal bloom species, such as Isotherma globosa, Karlodinium veneficum, Gyrodinium Dominans and Karenia Papilionacea, were significantly correlated with environmental factors.

3. Biological diversity and spatial-temporal dynamics of Chaetoceros species in the Jiaozhou Bay

The Jiaozhou Bay is an epitome of China’s coastal ecosystem and an ideal site for research on HABs with the accumulation of decades of historical investigation records. The species diversity and spatial-temporal distribution of Chaetoceros species at 12 stations in Jiaozhou Bay were studied by using metabarcoding analysis monthly. It was found that the species diversity of Chaetoceros in Jiaozhou Bay was very high. Among 155 ASVs, 22 species were identified in 49 ASVs, and 106 ASVs was identified to genus-level. In addition, the species richness and abundance of Chaetoceros had obvious temporal distribution in Jiaozhou Bay, and the distribution characteristics of different algae were different. For example, C. curvisetus, one of the 

dominant species in Jiaozhou Bay, mainly distributed from July to December, with the highest abundance in September. However, the spatial distribution of the species richness and abundance of Chaetoceros was relatively uniform at each site. Temperature and silicate had the greatest influence on Chaetoceros species.

4. Comparative analysis of mtDNAs of Chaetoceros species

At present, most of the researches on Chaetoceros are based on morphology and single molecular markers. Due to the limitation of morphological characteristics and limited resolution of single molecular markers, there is a lack of comprehensive studies on the diversity and phylogenetic evolution of Chaetoceros species. In recent years, organelle genomes have been recognized as super barcodes for species identification and evolutionary analysis due to their higher resolutions. In this study, we investigated the application of mtDNAs of Chaetoceros biodiversity and its evolution. Mitochondrial genomes of 18 species of Chaetoceros were constructed for the first time, including nine species and senven unknown (potentially novel) Chaetoceros species, suggesting that the biodiversity of Chaetoceros has been underestimated. Compared with common molecular markers, mtDNAs can also be used to distinguish Chaetoceros strains with high resolution. By comparing mitochondrial genes, it was found that although the species diversity of Chaetoceros is high, the mtDNA is relatively conserved. Divergence analysis showed that the species of Chaetoceros had an early divergence time and were probably still expanding. The construction of Chaetoceros mtDNAs can also provide support for the subsequent development of high-resolution molecular markers, and provide effective molecular tools for the genetic diversity and biogeographic distribution patterns of Chaetoceros species, especially harmful algal bloom species.

In summary, this is the first metabarcoding study of HAB species in the Changjiang Estuary. In this paper, the diversity and temporal-spatial dynamics of Chaetoceros species in Jiaozhou Bay were systematically analyzed for the first time using macrobarcoding method based on ASV. In addition, the mtDNAs of 18  Chaetoceros species were constructed for the first time, and the diversity and phylogenetic evolution of Chaetoceros species were analyzed by comparative genomics. The results of this study provide data support for the monitoring, warning and control of HABs in the Changjiang Estuary, and also provide important reference for the study of the biodiversity, genetic diversity, spatial-temporal distribution dynamics and phylogenetic evolution of Chaetoceros species.

摘 要........................................................................................................... I
Abstract......................................................................................................V
目 录........................................................................................................ XI
第 1 章 绪论...............................................................................................1
1.1 有害藻华概述.....................................................................................................1
1.1.1 有害藻华的基本概念..................................................................................1
1.1.2 有害藻华的影响及危害..............................................................................2
1.2 长江口海域有害藻华研究进展.........................................................................2
1.2.1 长江口海域的水文结构和环境特征..........................................................2
1.2.2 长江口海域有害藻华的研究历程..............................................................4
1.3 有害藻华的宏条形码分析进展.........................................................................6
1.4 角毛藻属物种研究概述.....................................................................................7
1.4.1 角毛藻属物种的生态学研究概述..............................................................7
1.4.2 角毛藻属物种的分类学研究概述..............................................................9
1.5 本论文的主要研究内容...................................................................................11
1.6 本论文拟解决的科学问题...............................................................................13
第 2 章 长江口海域浮游植物及有害藻华物种研究的统计分析........15
2.1 数据来源及分析...............................................................................................15
2.1.1 数据来源....................................................................................................15
2.1.2 数据分析....................................................................................................15
2.2 结果...................................................................................................................16
2.2.1 长江口海域浮游植物的生物多样性........................................................16
2.2.2 长江口海域有害藻华物种的组成............................................................22
2.3 小结...................................................................................................................27
第 3 章 长江口海域浮游植物分布格局的宏条形码分析....................29
3.1 前言...................................................................................................................29
3.2 材料与方法.......................................................................................................30
3.2.1 航次样本的采集及保存............................................................................30
3.2.2 DNA 的提取、 PCR 扩增和测序 ..............................................................31
3.2.3 测序数据的生物信息学分析....................................................................32
3.2.4 浮游植物的分类注释分析........................................................................33
3.2.5 浮游植物物种与环境因子的相关性分析................................................33
3.3 结果...................................................................................................................34
3.3.1 长江口海域浮游植物的组成及相对丰度................................................34
3.3.2 长江口海域浮游植物地理分布特征........................................................38
3.3.3 有害藻华物种的组成及分布特征............................................................42
3.3.4 环境因子与浮游植物及有害藻华物种的相关性分析............................50
3.4 讨论...................................................................................................................52
3.4.1 长江口海域浮游植物的组成和分布........................................................52
3.4.2 长江口海域有害藻华物种的组成及分布................................................55
3.5 小结...................................................................................................................56
第 4 章 胶州湾角毛藻的生物多样性及其时空动态变化....................57
4.1 前言...................................................................................................................57
4.2 材料与方法.......................................................................................................59
4.2.1 航次样本的采集及保存............................................................................59
4.2.2 环境因子样本的采集及测定....................................................................59
4.2.3 DNA 的提取、 PCR 扩增及测序 ..............................................................60
4.2.4 基于 ASV 宏条形码的生物信息学分析..................................................60
4.3 结果...................................................................................................................60
4.3.1 本研究检测到的角毛藻属物种与历史资料的比较分析........................60
4.3.2 角毛藻属物种的遗传多样性分析............................................................63
4.3.3 角毛藻属物种的时空分布特征................................................................66
4.3.4 环境因子与角毛藻属物种的相关性分析................................................71
4.4 讨论...................................................................................................................72
4.4.1 角毛藻属物种较高的生物多样性............................................................72
4.4.2 角毛藻属物种的时空分布特征................................................................74
4.5 小结...................................................................................................................76
第 5 章 角毛藻线粒体基因组比较分析 ................................................77
5.1 前言...................................................................................................................77
5.2 材料与方法.......................................................................................................77
5.2.1 藻株的分离和培养....................................................................................77
5.2.2 DNA 的提取及测序...................................................................................78
5.2.3 角毛藻属物种的鉴定及线粒体基因组的组装........................................79
5.2.4 角毛藻线粒体基因组的注释及比较分析................................................79
5.2.5 角毛藻属物种分化时间的估计................................................................80
5.3 结果...................................................................................................................80
5.3.1 18 株角毛藻属物种的分子鉴定................................................................80
5.3.2 角毛藻线粒体基因组的结构特征和共线性分析....................................83
5.3.3 角毛藻属物种的系统发育组学分析........................................................85
5.3.4 角毛藻属物种的进化时间分析................................................................90
5.4 讨论...................................................................................................................92
5.4.1 角毛藻属物种的多样性及系统进化分析................................................92
5.4.2 角毛藻属物种对环境适应性的探讨........................................................94
5.5 小结...................................................................................................................95
第 6 章 结论与展望 ................................................................................97
6.1 主要结论...........................................................................................................97
6.2 主要创新点.......................................................................................................98
6.3 研究展望...........................................................................................................99
参考文献.................................................................................................101
附录一 长江口历史航次中的有害藻华物种 ......................................119
附录 二 长江口航次采样信息及环境因子信息 ................................131
致 谢.......................................................................................................133
作者简历及攻读学位期间发表的学术论文与研究成果....................135