海洋生物分类与系统演化实验室知识库

有孔虫是一类数量庞大，从赤道到两极广泛分布，并且对环境十分敏感的原生生物，常作为生态指示种用于环境监测工作。由于有孔虫的壳体具有长期保存在地层中的特点，也被应用于古气候和古环境的重建工作中。近些年来，随着分子生物学的快速发展，环境DNA（environmental DNA，eDNA）测序的方法也被用于生态调查。

当前地球面临的海洋酸化的速度比3亿年来的任何地质历史时期都要快，这将会对海洋生物特别是以有孔虫为代表的钙质生物产生影响。盐度是海洋环境重建的重要参数，特别是陆架到潮间带地区，海平面的变化会带来盐度的巨大改变。本研究采集黄海陆架和潮间带的沉积物样品，探究了黄海海域有孔虫分子多样性与环境关系，并进行盐度梯度和海洋酸化的实验室培养研究，利用传统形态学和eDNA测序相结合的方法对结果进行分析，探究盐度变化，或海洋酸化对底栖有孔虫群落丰度和多样性，以及代表物种如Cribrononion gnythosuturatum的影响。主要研究内容包括以下四部分：

1．黄海有孔虫的分子多样性和环境相关性研究

本研究基于eDNA对黄海25个站点（33~36°N）的有孔虫多样性进行了调查，并首次对该地区有孔虫分子多样性与环境条件进行了相关性评估。本研究结果表明，相比于传统方法，利用分子方法可以获得更多的物种信息。控制底栖有孔虫群落多样性的主要环境因素为温度、深度和沉积物碳的含量。该海域底栖有孔虫的群落多样性随着水深的增加，温度的降低而减少。底栖有孔虫的群落多样性与沉积物中有机碳含量呈显著负相关，与无机碳含量呈显著正相关。沉积物中的浮游有孔虫eDNA能够在一定程度上反应海流情况。由于eDNA方法在物种层面建立关系式需要更多的调查来支持，我们建议使用群落参数进行环境指示。本研究填补了黄海地区有孔虫eDNA研究的空白，探究了黄海区域影响底栖有孔虫生态分布的主要因素，揭示了有孔虫eDNA多样性在环境指示方面具有的潜力。

2．海洋酸化对底栖有孔虫分子多样性的影响

本研究采集黄海陆架4个站位点的沉积物样品，在400 ppm、800 ppm、1200 ppm、1600 ppm共4个CO₂分压梯度下培养八个月（由低到高依次培养两个月），利用高通量测序的方法对沉积物中的有孔虫eDNA进行测序分析。实验过程中海水pH值与CO₂分压有显著负相关关系，海水酸性随着CO₂分压的上升不断增强。测序共获得共计获得212个OTUs和743,884条reads，其中有178个OTUs共计738,597条reads匹配到有孔虫。实验结果显示，底栖有孔虫群落总体丰度在400–1600 ppm呈现先下降后上升的趋势，而OTUs数与群落阿尔法多样性都呈先上升后降低的趋势，临界值出现于1200 ppm。我们推测在未来海洋酸化的情况下，有孔虫的物种数和多样性持续增加，而超过临界值（>1200 ppm）后，在较强的海洋酸化环境压力下，有孔虫多样性显著降低，但有些机会主义种在短时间内迅速繁衍，例如Elphidium、Nemogullmia、Allogromiid、Spiroplectammina等属，群落结构也因此发生改变。

3．基于eDNA宏条形码和形态学揭示底栖有孔虫对盐度变化的响应过程

盐度是海洋重要的理化因子，对底栖生物的生长和繁殖都有着显著的影响。Cribrononion gnythosuturatum是中国沿岸表层沉积和岩心沉积中的常见物种，但它对于盐度变化的适应性尚不明确。本研究采集黄海潮间带沉积物样品，在0–60 psu共计13个盐度梯度下平行培养10周，期间添加钙黄绿素对新钙化的房室进行标记。沉积物样品采用传统形态学和eDNA测序两种方法进行分析，探究底栖有孔虫群落对盐度的响应关系中形态学和eDNA测序方法的异同，以及C. gnythosuturatum对盐度的响应关系。实验前提取活体C. gnythosuturatum的SSU rDNA片段，加入本地数据库用于序列比对。

培养实验在形态学方面共获得有孔虫C. gnythosuturatum1636只，其中活体955只，C. gnythosuturatum在0–60 psu各盐度梯度下均可存活和生长，高通量测序结果中57个OTUs共计17091条序列比对到C. gnythosuturatum。研究结果显示，在极端低盐（<15 psu）条件下，壳体破损率急速上升，并且伴随生长率下降和繁殖的停止。在极端的高盐度条件下（50–60 psu）原生质向房室中心收缩，生长率和繁殖率下降。此外，分子结果和形态学结果都显示，异常盐度条件下（15–25 psu or 40–50 psu），种群丰度、相对丰度和生长率达到最高，壳体保存良好。本研究揭示了C. gnythosuturatum对于盐度的响应，可以用于指示河口、潟湖、潮间带等盐度变化较大的沉积环境。

底栖有孔虫群落最适宜生存的盐度为30–35 psu，盐度的升高和降低都会使群落丰度和物种数降低。eDNA测序方法能够探测到数量庞大的OTUs和物种数目，取样量少，灵敏度高。eDNA方法能够探测到形态学中难以鉴别的隐藏物种，单房室有孔虫类群（Monothalamids），该类群对高盐度有较好的耐受性。传统形态学对于种类单一，但是生物量高的瓷质壳体多房室有孔虫（Miliolida）类群，能比较直观地看出该类群生物数量随盐度梯度的变化而发生的改变，该类群对高盐度有较好的耐受性。两种方法都表明玻璃质壳体多房室有孔虫类群（Rotaliida）对于低盐度有较好的耐受性。本研究表明在生态监测工作中可以使用eDNA测序方法作为形态学的很好补充，利用各种类群的组合变化来推测海水的盐度。

Foraminifera are one of the Protozoa which have huge abundant and widely distributed from the equator to the poles. They are extremely sensitive to environmental changes. Because of these characteristics, foraminifera are usually use as indicators to monitor the environment. Because the tests of foraminifera are preserved in strata for a long time, they are also used in the reconstruction of paleoclimate and paleoenvironment. With the development of molecular biology recent years, environmental DNA (eDNA) sequencing also was used in ecology investigation.

The speed of ocean acidification the planet currently faced faster than any period in geological history in 300 million years. This will have an impact on marine organisms, especially calcareous organisms represented by foraminifera. Salinity is one of the most important factors in marine environmental reconstruction. Especially the shelf area, the sea level change will bring about the huge change in salinity. In this study, we collected sediments from the intertidal zone and shelf area from the Yellow Sea. Firstly, we study on the molecular diversity of foraminifera and its relationship with environmental factors. Additionally, the salinity gradients and ocean acidification culture experiments were conducted in laboratory. Traditionally morphology and eDNA sequencing methods were used to analyze how benthic foraminifera community and representative species (such as Cribrononion gnythosuturatum) response to salinity change and ocean acidification. The main research is divided into the following four parts:

1．Molecular diversity and environmental correlation of foraminifera in the Yellow Sea

This study based on foraminiferal eDNA diversity investigation of 25 stations in the Yellow Sea, and firstly we evaluated the relationship between foraminiferal molecular diversity and environmental factors. Result showed that comparing with traditionally morphological approach, eDNA sequencing detected more species. The main factors control benthic foraminifera community in Yellow Sea are temperature, depth and carbon content in sediments. The diversity of benthic foraminifera community decreased with the increase of depth and decrease of temperature. The diversity of benthic foraminifera community was negative correlated with organic carbon content and positive correlated with inorganic carbon content. The eDNA of planktic foraminifera in sediments could reflect the situation of ocean current to a certain extent. This is the first study on relationship of foraminiferal eDNA and environmental factors in the Yellow Sea shelf region of the western Pacific Ocean, and it demonstrates that community parameters in foraminiferal eDNA could be a valuable proxy for environmental changes in the shelf sea.

2．Effects of ocean acidification on the molecular diversity of benthic foraminifera.

In this study, sediment samples were collected from 4 stations and cultured at 400 ppm, 800 ppm, 1200 ppm and 1600 ppm in a series of continuously increasing CO₂ gradients in eight months. Environmental DNA sequencing method was used in analyze benthic foraminifera in sediments. In the experiment, sea water pH had negative correlation with partial pressure of CO₂. In total, there are 202 OTUs and 743,884 reads in this result of sequencing, and 178 OTUs and 738,597 reads of them assigned to benthic foraminifera. Experimental result shows that the abundance of benthic foraminifera showing a downward and then upward trend during 400–1600 ppm, but the trend of OTUs number is opposite. The threshold value is 1200 ppm. We presume that in the situation of ocean acidification in the future, the diversity of foraminifera species will increase continuously. When the partial pressure of CO₂ reached 1200 ppm, under the pressure of more acidified ocean environment, the diversity of foraminifera community would decrease apparently. However, some opportunistic species (such as Elphidium, Nemogullmia, Allogromiid, and Spiroplectammina) would thrive in this situation, and the structure of community would change.

3．Response process of the benthic foraminifera Cribrononion gnythosuturatum to salinity changes revealed by eDNA macrobarcoding and morphology

Salinity is an important physicochemical factor in the ocean and has a significant impact on the growth and reproduction of benthic organisms. Cribrononion gnythosuturatum is a common species in surface sediments and core samples in the Chinese coastal area but the adaptability of this species is unclear. In this study, sediment samples were collected from the intertidal zone of the Yellow Sea and cultured in the laboratory at a total of 13 salinity gradients (0–60 psu). During the culture period, calcein was added to mark the newly calcified chambers of foraminifera. Morphology and eDNA sequencing approach were used to compare similarities and differences and explore the response mechanisms of the benthic foraminifera Cribrononion gnythosuturatum to salinity changes. The SSU rDNA of live foraminifera Cribrononion gnythosuturatum were extracted and added in local databases for assignment.

A total of 1636 foraminifera C. gnythosuturatum were obtained morphologically in culture experiments, of which 955 were alive. C. gnythosuturatum survived and grew at all salinity gradients from 0–60 psu. A total of 17,091 sequences from 57 OTUs in eDNA sequencing results were assigned to C. gnythosuturatum. As results showed that under extremely low salinity (<15 psu) conditions, the broken rate of test was rising rapidly with rate of growth decreasing and the reproduction ceased. Under extremely high salinity (50–60 psu), contraction of protoplasm towards the atrioventricular center, with the rate of growth and reproduction decrease. Furthermore, both of morphology and eDNA sequencing results showed that, under abnormal salinities (15–25 psu or 40–50 psu) the abundance, relative abundance and growth rate of this species were at highest value, and the preservation of tests were good. This study revealed the response process of the C. gnythosuturatum to salinity changes, and it can be used for indicating the sedimentary environments with highly variable salinity, such as estuary, lagoon and intertidal zone.

The optimal salinity for the benthic foraminiferal community in the intertidal zone of Qingdao Bay was 30–35 psu, and that both increasing and decreasing salinity reduced community abundance and species numbers. The eDNA sequencing method can detect a large number of OTUs and species numbers, with low sampling volumes and high sensitivity. The eDNA method can detect hidden species that are difficult to identify by morphology, the single–chambered foraminiferal taxa (Monothalamids), which are more tolerant of high salinity. For single species with large numbers, such as the porcelaneous test group Miliolida, the traditional morphology method provides a relatively visual indication of the changes in biomass of this taxon in response to the salinity gradient, and the group had good tolerance to high salinity. The results of both methods showed that the hyaline tests group Rotaliida tolerant to lower salinities. This study demonstrates that eDNA sequencing methods can be used as a good complement to morphology in ecological monitoring efforts using variations in the assemblage of various taxa to infer the salinity of seawater.

第1章 绪论... 1

1.1 有孔虫... 1

1.2 研究背景... 2

1.2.1 有孔虫分子生物学的发展... 2

1.2.2 有孔虫的实验室培养研究... 3

1.2.3 海水盐度... 4

1.2.4 海洋酸化... 5

1.3 研究目标及意义... 5

第2章 黄海有孔虫的分子多样性和环境相关性研究... 7

2.1 引言... 7

2.2 材料与方法... 8

2.2.1 样品采集... 8

2.2.2 环境DNA提取... 9

2.2.3 PCR扩增... 10

2.3 结果... 10

2.3.1 环境特征... 10

2.3.2 测序数据概况... 13

2.3.3 底栖有孔虫分类组成和相对丰度... 15

2.3.4 底栖有孔虫与环境因子的相关性分析... 18

2.3.5 黄海环流及浮游有孔虫... 23

2.4 讨论... 23

2.4.1 黄海底栖有孔虫各个类群分布特征... 23

2.4.2 底栖有孔虫对于水温和深度的响应及启示... 24

2.4.3 底栖有孔虫对于沉积物中碳的响应及启示... 25

2.4.4 底栖有孔虫不同类群对于环境因子的响应... 26

2.4.5 浮游有孔虫分布情况与海流的关系... 26

2.4.6 影响黄海底栖有孔虫分布的其他因素... 27

2.5 结论... 28

第3章 通过eDNA测序手段探究海洋酸化对底栖有孔虫多样性的影响... 29

3.1 引言... 29

3.2 材料与方法... 30

3.2.1 样品采集和培养... 30

3.2.2 环境DNA提取和PCR扩增... 31

3.2.3 数据质控... 32

3.3 结果... 32

3.3.1 环境参数... 32

3.3.2 环境DNA数据概况... 33

3.3.3 类群组成... 33

3.3.4 物种多样性... 35

3.3.5 相关性分析... 36

3.4 讨论... 37

3.5 结论... 38

第4章 基于eDNA宏条形码和形态学揭示底栖有孔虫Cribrononion gnythosuturatum（Ho, Hu & Wang, 1965）对盐度变化的响应过程... 39

4.1 引言... 39

4.2 材料与方法... 39

4.2.1 样品采集... 39

4.2.2 活体DNA的提取和分析... 40

4.2.3 盐度培养实验... 44

4.2.4 形态学样品... 44

4.2.5 高通量测序... 45

4.3 结果... 48

4.3.1 SSU rDNA序列特征与系统发育分析... 48

4.3.2 盐度培养实验结果... 49

4.4 讨论... 52

4.4.1 Cribrononion gnythosuturatum对盐度梯度的响应... 52

4.4.2 Cribrononion gnythosuturatum的生态地位及其古环境中的应用... 53

4.4.3 系统发育分析... 55

4.5 结论... 56

第5章 基于形态学和eDNA探究底栖有孔虫群落对海洋盐度变化的响应... 57

5.1 引言... 57

5.2 材料与方法... 58

5.2.1 样品采集与处理... 58

5.2.2 培养实验... 58

5.2.3 形态学处理方法... 59

5.2.4 环境DNA提取与PCR扩增... 59

5.2.5 数据质控和处理... 60

5.3 结果... 60

5.3.1 经典形态学结果分析... 60

5.3.2 分子生物学结果分析... 61

5.3.3 形态学和分子测序方法的比较... 62

5.4 讨论... 62

5.4.1 盐度对有孔虫不同物种的影响... 62

5.4.2 有孔虫不同壳质类型的生物矿化与盐度关系... 63

5.4.3 传统形态学与分子生物学... 64

5.5 结论... 65

第6章 总结与展望... 66

6.1 黄海有孔虫的分子多样性和环境相关性研究... 66

6.2 陆架底栖有孔虫对海洋酸化的响应... 66

6.3 Cribrononion gnythosuturatum对盐度的响应过程... 66

6.4 潮间带底栖有孔虫对盐度的响应... 67

6.5 研究创新点... 67

6.6 存在问题与展望... 67

参考文献... 69

致谢... 87

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    88