海洋生态与环境科学重点实验室知识库

大型藻藻华，如浒苔（Ulva prolifera）形成的绿潮（green tide）和马尾藻（Sargassum spp.）形成的金潮（golden tide）等，是一类全球性的海洋生态灾害，严重损害沿海地区社会经济发展，对近海生态安全构成了潜在威胁。目前，对于大型藻藻华的成因和演变机制研究较多，但对其生态效应的研究相对较少。2007年以来，黄海海域连年发生浒苔形成的大规模绿潮，其规模堪称世界之最，绿潮的连年暴发对近海生态环境的影响不容忽视。在绿潮后期，漂浮绿藻的沉降、死亡和分解会释放大量有机物和营养盐。这一过程会导致浮游植物群落发生哪些变化？其过程和机理如何？值得深入研究。

近年来，国内学者通过表层沉积物中石莼属绿藻（Ulva spp.）生物标志物——28-异褐藻甾醇的分析，将绿潮后期大量漂浮绿藻的沉降区（以下简称沉降区）范围圈定在山东半岛东南部海域（36-36.5°N，122-124°E）。本文重点针对这一区域，通过现场调查和模拟实验，采用扩增子高通量测序和宏转录组等手段，分析了南黄海绿潮后期微型及微微型浮游植物群落的变化情况，结合水文和营养盐等环境因子分析，重点解析了沉降区浮游植物群落对环境变化的响应过程，并初步探讨了浮游植物群落变动的机理，以期为系统阐明黄海绿潮的生态效应提供科学依据。

通过对2016年黄海海域浮游植物样品的扩增子高通量测序分析，解析了绿潮后期南黄海海域浮游植物群落的组成和变化情况，揭示了沉降区浮游植物群落的独特变化。2016年，在南黄海海域采集的浮游植物样本中平均获得156,461条浮游植物序列和1,884个浮游植物OTUs（operational taxonomic units）。绿潮后期南黄海近岸海域的浮游植物群落变化不明显，甲藻一直占有绝对优势。而在南黄海中部海域，浮游植物群落发生明显变化，海金藻类优势度增加，硅藻和绿藻比例下降，推测与秋季黄海暖流的形成有关。有别于其他海域，沉降区的浮游植物群落出现了一些独特的变化，甲藻优势度降低，而海金藻和绿藻的比例上升，海金藻纲的抑食金球藻(Aureococcus anophagefferens)丰度显著增加，推测与黄海暖流的形成和漂浮绿藻的沉降分解有关。

针对南黄海海域绿藻沉降区开展了多年调查，对比分析了2016、2017和2019年沉降区绿藻沉降前后浮游植物群落变化的年际变异特征。2017年，在南黄海海域采集的浮游植物样本中平均获得126,033条浮游植物序列和1,983个浮游植物OTUs，其中沉降区的浮游植物多样性较高，甲藻优势度下降，硅藻、绿藻和隐藻的相对丰度增加，而抑食金球藻相对丰度变化不明显。2019年，沉降区浮游植物的生物量和物种丰富度在绿藻沉降后有明显增加，甲藻优势度下降，硅藻和绿藻优势度上升，抑食金球藻丰度增加。对比三年里沉降区浮游植物群落的变化情况可以看出，沉降区浮游植物群落的变化具有年际差异。其中，浮游植物生物量和抑食金球藻相对丰度的变化与绿潮规模的年际变异状况基本一致。

应用宏转录组学手段，分析了2019年沉降区采集的浮游植物样品，并开展了现场模拟实验，对浮游植物群落的变化机制进行了分析，探讨了微型及微微型浮游植物对绿藻沉降分解的响应过程和机理。绿藻的沉降分解能够产生氨氮等无机态营养盐以及溶解有机物，从而刺激浮游植物生长。研究发现，沉降区浮游植物类群在营养物质代谢和能量利用方面的基因表达最为活跃，主要表现在光合作用、氧化磷酸化、氮代谢以及大分子物质转运等方面，其中硅藻对光合作用光反应过程相关基因的表达贡献最大。模拟实验结果发现，硅藻能够利用添加的氨氮快速生长，在浮游植物群落中占据优势。绿藻沉降前后沉降区样品中抑食金球藻组织蛋白酶基因表达上调，热休克蛋白基因表达下调，在一定程度上反映了抑食金球藻利用有机态营养物质的能力以及对沉降区环境的适应性。可见，硅藻和抑食金球藻有可能通过不同策略以适应沉降区的环境变化。此外，研究还发现一些未知浮游植物类群在多个信号通路上的基因表达上调，很可能反映了沉降区浮游植物与微生物及环境因子之间的复杂相互作用。

本研究通过对南黄海绿潮后期浮游植物群落变化情况的多年调查，揭示了南黄海海域浮游植物种类组成状况，以及沉降区绿藻沉降前后浮游植物群落的独特变化，并初步探讨了硅藻和海金藻等浮游植物类群对绿藻沉降分解的响应和机理。相关结果为全面揭示黄海海域大规模绿潮的生态效应，防控绿潮可能引发的生态安全问题提供了科学依据。

Macroalgal blooms, such as green tides of Ulva prolifera and golden tides of Sargassum spp., have become a global marine ecological disaster, which affected the socio-economic development of coastal areas and posed potential threat to marine ecosystems. Many studies have been performed on the formation mechanisms of the macroalgal blooms, while few studies focused on the ecological impacts of macroalgal blooms. In the Yellow Sea, green tides of Ulva prolifera have been recorded consecutively from 2007, and the scale of green tide could rank the first in the world. The ecological impacts of recurrent large-scale green tides on marine ecosystems become a big concern. After the decline of green tides, the settlement and decomposition of massive green algae will release huge amounts of organic matter and nutrients and affect phytoplankton communities. What kind of changes of phytoplankton communities will take place in response to the settlement and decomposition of green algae? What are the key processes and mechanisms accounting for the changes? These questions need to be elucidated.

Recently, the settlement region of mass drifting green algae in the Yellow Sea (36-36.5°N, 122-124°E) was identified in a region southeastern to Shandong Peninsula, based on the analysis of 28-isofucosterol, a biomarker for green algae in the genus Ulva, in surface sediment. To reveal the changes of phytoplankton community in the settlement region and their mechanisms, phytoplankton samples were collected in the southern Yellow Sea before and after the settlement of floating green algae, and a set of simulation experiments were carried out on board. Phytoplankton assemblages and the key physiological processes were resolved using the approaches of amplicon high-throughput sequencing (HTS) and metatranscriptome analysis. In combination with the data of temperature, salinity and nutrients, the mechanisms for phytoplankton community changes were analyzed. This study aims to provide a scientific basis to elucidate systematically the ecological consequences of green tides in the Yellow Sea.

Phytoplankton samples collected from the southern Yellow Sea before and after the settlement of green algae in 2016 were analyzed using HTS. The unique changes of phytoplankton communities were revealed in the settlement region. In 2016, a total of 156,461 phytoplankton tags on average and 1,884 phytoplankton operational taxonomic units (OTUs) were obtained in phytoplankton samples collected from the southern Yellow Sea. Phytoplankton communities in the coastal region exhibited no apparent changes, and the relative abundance of dinoflagellates remained to be the dominant group. In the central region of the SYS, however, phytoplankton communities had notable changes, with increasing proportion of pelagophytes and the decreasing proportion of diatoms and chlorophytes, probably related to the formation of the Yellow Sea warm current (YSWC) in autumn. In the settlement region of massive floating green algae, there were dramatic changes of phytoplankton community characterized by the increasing proportion of pelagophytes and chlorophytes and the decreasing proportion of dinoflagellates, the most remarkable feature is the increase of pelagophyte Aureococcus anophagefferens. It’s speculated that changes of phytoplankton community in the settlement region of green algae could be driven jointly by the formation of YSWC and the decomposition of deposited green algae.

Multiple-year investigations were carried out in the southern Yellow Sea, and the interannual variation pattern of phytoplankton community changes in the settlement region in 2016, 2017 and 2019 were compared and analyzed. In 2017, a total of 126,033 phytoplankton tags and 1983 phytoplankton OTUs were obtained from phytoplankton samples. The sample collected from the settlement region of floating green algae in September has a high diversity of phytoplankton taxa, and there were apparent increases in relative abundance of diatoms, chlorophytes and cryptophytes associated with dramatic decrease of dinoflagellates from June to September. The increase of pelagophyte A. anophagefferens was not obvious. In 2019, biomass and species richness of phytoplankton community in the settlement region increased after the decline of green tide, the relative abundance of dinoflagellates decreased, and the proportion of diatoms, chlorophytes and pelagophyte A. anophagefferens increased. Through the comparison of investigation results in 2016, 2017 ad 2019, it can be seen that the changes of phytoplankton community in the settlement region had strong interannual variation. The changes of phytoplankton biomass and pelagophyte proportion, in particular, coincide with the interannual variation of the scale of green tides.

Using the approach of metatranscriptomic analysis, phytoplankton samples collected from the southern Yellow Sea in 2019 were analyzed, and field simulation experiments were conducted to analyze the mechanism of phytoplankton community changes. The settlement and decomposition of green algae will release ammonium and other inorganic nutrients, as well as dissolved organic compounds, and promoted the growth of phytoplankton. Our study found active expression of genes related to nutrient and energy utilization, mainly in photosynthesis, oxidative phosphorylation, nitrogen metabolism, and macromolecule transport processes, in phytoplankton samples collected from the settlement region. Diatoms contributed the most to gene expression related to the photosynthetic light reaction process. The simulation experiment found that diatoms could use ammonia efficiently to grow. The up-regulated expression of the cathepsin gene and the down-regulated expression of the heat shock protein gene of A. anophagefferens reflected its ability to utilize organic compounds and its adaptation to environmental conditions in the settlement region. The diatoms and A. anophagefferens may have different nutritional strategies to proliferate in the settlement region. Moreover, the up-regulated expression of genes on multiple environmental signaling pathways in unidentified taxa may reflect the complex interactions among phytoplankton, microorganism and the environment in the settlement region.

This study, through multi-year investigations of phytoplankton communities at the late stage of green tides in the southern Yellow Sea, reveals the composition of phytoplankton communities and the unique change of phytoplankton community in the settlement region. The responses and mechanisms of diatoms and pelagophytes to the settlement and decomposition of green algae were also analyzed primarily. The results provide a scientific basis to fully understand the ecological consequences of large-scale green tides in the Yellow Sea, and to prevent and control other ecological risks derived from green tides.

第1章 绪论... 1

1.1 大型藻藻华概述... 1

1.1.1 大型藻藻华发生概况及其危害效应... 1

1.1.2 黄海绿潮发生过程及其生态效应... 2

1.2 黄海海域环境及浮游植物类群概况... 8

1.2.1 黄海环境特征... 8

1.2.2 黄海浮游植物类群组成与变化情况... 9

1.3 宏基因组学及宏转录组学在浮游植物研究中的应用... 12

1.3.1 宏基因组学... 12

1.3.2 宏转录组学... 14

1.4 研究目的与意义... 15

第2章 南黄海绿潮后期浮游植物群落的组成与变化状况... 17

2.1 前言... 17

2.2 材料与方法... 18

2.2.1 样品采集... 18

2.2.2 DNA提取、扩增及测序... 20

2.2.3 生物信息学分析... 21

2.2.4 抑食金球藻的qPCR检测... 22

2.3 2016年绿潮后期南黄海浮游植物群落组成及变化情况... 22

2.3.1 数据质控和注释结果统计... 22

2.3.2 浮游植物群落组成及变化情况... 24

2.3.3 浮游植物群落多样性... 31

2.3.4 浮游植物群落与环境因素的关系... 32

2.3.5 基于qPCR方法对抑食金球藻的检测结果... 36

2.4 2017年绿潮后期南黄海浮游植物群落组成及变化情况... 36

2.4.1 数据质控和注释结果统计... 36

2.4.2 浮游植物群落组成及变化情况... 39

2.4.3 浮游植物群落多样性... 44

2.4.4 浮游植物群落与环境因素的关系... 45

2.5 讨论... 46

2.5.1 南黄海浮游植物群落的组成及动态变化... 46

2.5.2 南黄海浮游植物群落组成与变化的影响因素... 49

2.5.3 漂浮绿藻沉降区浮游植物群落变化的年际差异... 51

2.5.4 南黄海浮游植物群落变化的生态效应... 53

2.6 小结... 54

第3章 南黄海绿藻沉降区浮游植物群落变化机理... 55

3.1 前言... 55

3.2 材料与方法... 55

3.2.1 现场样品采集... 55

3.2.2 船载模拟实验方案... 57

3.2.3 DNA提取、扩增及测序... 58

3.2.4 RNA提取与宏转录组测序... 58

3.2.5 宏转录组生物信息学分析... 59

3.2.6 抑食金球藻的qPCR检测... 60

3.2.7 营养盐分析... 60

3.3 现场调查结果... 61

3.3.1 调查区域环境因子变化... 61

3.3.2 扩增子高通量测序结果... 61

3.3.3 2019年绿潮后期浮游植物群落的组成及变化情况... 63

3.3.4 宏转录组样品测序结果... 66

3.3.5 注释结果和基因表达情况... 67

3.3.6 差异表达基因分析... 70

3.3.7 特定代谢通路中的基因表达调控... 72

3.4 船载模拟实验结果... 78

3.4.1 营养盐分析结果... 78

3.4.2 扩增子高通量测序结果... 80

3.4.3 浮游植物群落组成及变化... 83

3.4.4 宏转录组样品测序结果... 87

3.4.5 注释结果和基因表达情况... 88

3.4.6 差异表达基因分析... 91

3.4.7 特定代谢通路中的基因表达调控... 92

3.5 讨论... 95

3.5.1 南黄海绿藻沉降区浮游植物群落变化... 95

3.5.2 绿藻沉降后硅藻和抑食金球藻的响应过程和机理... 96

3.5.3 绿藻沉降后浮游植物群落的其它响应过程... 98

3.6 小结... 100

第4章 结论与创新点... 101

4.1 主要结论... 101

4.2 创新点... 102

4.3 不足与展望... 102

参考文献... 103

附录 缩略语表... 117

致  谢... 119

作者简历及攻读学位期间发表的学术论文与研究成果    121