IOCAS-IR  > 海洋生物分类与系统演化实验室
西太平洋典型生境中有孔虫遗传多样性的初步研究
李青霞
Subtype博士
Thesis Advisor类彦立
2020-08-19
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学博士
Keyword有孔虫 西太平洋 分子技术 遗传多样性
Abstract

有孔虫是一种古老的原生动物,体外覆盖着钙质、胶结质或有机质外壳。在早期寒武纪便已出现,发展至今种类繁多。有孔虫分布极为广泛,能够生活在不同的海洋生境甚至淡水环境里,按照生活方式可分为浮游和底栖两大类,绝大多数属于底栖有孔虫,浮游有孔虫仅大约50种。传统上,通过在显微镜下观察壳体的形态特征来对有孔虫进行分类。分子技术的应用使有孔虫研究从形态特征观察深入到遗传序列分析,也加深了对有孔虫分类演化、系统发育、生态分布及其与环境关系的研究。有孔虫是研究古海洋学最重要的生物指标,探究现代有孔虫分子多样性及进化关系对生物地层学、古气候与古环境研究具有重要的科学意义。

本论文以西太平洋为目标海区,以有孔虫为研究对象,利用2016年热带西太平洋共享航次、2017年西北太平洋航次和2017年三沙永乐龙洞科学考察航次的浮游拖网样本和沉积物样本,应用第一代测序技术和第二代测序技术(即高通量测序技术),结合形态学观察,分析了西太平洋海区多种典型生境(深海平原、海洋蓝洞、半封闭海湾)中浮游有孔虫的基因型多样性、进化关系及底栖有孔虫的分子多样性、群落组成和空间分布,以期检验有孔虫的种内遗传多样性是否普遍存在以及有孔虫的多样性和群落特征在分子水平上是否存在地理差异,并尝试找出有孔虫群落与环境因子的相关关系。具体研究工作分为以下三部分:

1. 利用第一代测序技术研究热带西太平洋浮游有孔虫的基因型多样性

浮游有孔虫Globigerinoides ruber是生活在热带和亚热带海域的常见种类,也是古海洋和古气候研究的重要材料。本实验在热带西太平洋海域利用浮游生物垂直拖网收集了目标站位0 - 200米水体中的浮游生物,通过体视显微镜观察,识别、挑选出单个活体G. ruber(白色)个体。利用脱氧胆酸钠(DOC)裂解液提取G. ruber(白色)个体的DNA,利用巢式聚合酶链式反应和有孔虫特异性引物扩增有孔虫核糖体小亚基DNASSU rDNA)片段,应用第一代测序技术对目的片段进行测序。将获得的目的片段与NCBI数据库中的G. ruber遗传序列一起进行分析,研究了G. ruber基因型的多样性,并比较了不同基因型个体的生理生态和地理分布差异。基于SSU rDNA构建的系统进化树显示:G. ruber(白色)I型和II两种基因型,它们与G. ruber(粉色)、Globigeinoides conglobatus共同构成四条平行进化系。I型和IIG. ruber(白色)个体的形态特征和栖息环境存在明显差异:IG. ruber(白色)个体的三个房室是显著的球状,口孔大而高拱,主要生活在30 m以浅的水体中;IIG. ruber(白色)个体的三个房室是扁平的球状,口孔小而低矮,主要生活在30 m以下的水体中。此外,两种基因型个体的氧、碳稳定同位素组成、Mg/Ca比及SSU rDNA序列组成也明显不同。本工作证实了浮游有孔虫种内遗传多样性的普遍存在,是国内对浮游有孔虫基因型多样性进行的初次研究,也是为数不多的西太平洋热带海域浮游有孔虫遗传数据资料

2. 利用第二代测序技术研究西北太平洋深海底栖有孔虫的DNA分子多样性与群落特征

深海储藏着丰富的自然资源,是目前世界各国争相开发的战略要地。本工作以底栖有孔虫为研究对象,应用第二代测序技术(即高通量测序技术)调查了它在平均水深超5000 m的西北太平洋深海平原的分子多样性和群落特征。我们共获得1536个操作分类单元(operational taxonomic unitsOTUs),成功注释到有孔虫的OTUs1330个,其中721个有孔虫OTUs属于单房室软壳类有孔虫Monothalamiids,说明Monothalamiids是该海区底栖有孔虫群落的优势类群。OTU的注释结果显示,仅20.6%的有孔虫OTUs能在数据库中匹配到相似度大于或等于97%的参考序列,这说明西北太平洋深海底栖有孔虫的序列与目前检获到的有孔虫序列之间存在高度的遗传差异性,该海区极可能蕴藏着许多未知的“新物种”甚至新的高级分类阶元。

西北太平洋深海底栖有孔虫呈明显的斑块性分布,且站位间有孔虫的物种组成和多样性也存在较大差异,我们推测沉降到海底的有机物通量是导致有孔虫斑块性分布的重要因素。根据各个站位的有孔虫群落结构特征,11个样品可分为两组,一组样品采自纬度较高、水深较深的区域;另一组样品采自纬度相对较低、水深相对较浅的区域,说明纬度和水深是影响有孔虫群落结构的重要因素。本工作是国内利用高通量测序技术研究西北太平洋底栖有孔虫DNA分子多样性与群落组成的初次尝试,旨在探索有孔虫群落与环境因子之间的相关关系,并为揭示底栖有孔虫的全球地理分布模式、开发深海资源等工作提供分子生态数据支持。

3. 利用第二代测序技术研究世界最深海洋蓝洞–三沙永乐龙洞中有孔虫的DNA分子多样性和空间分布特征

海洋蓝洞是罕见的、珍贵的自然地质遗迹,具有极高的科学研究价值。它们的物理和化学性质独特,通常具有陡峭的内壁和孤立的水体,能够在限定的地理区域内形成相对独立的生物群落与生态系统。三沙永乐龙洞是世界上已知最深的海洋蓝洞,但目前人类对它仍知之甚少。本研究从三沙永乐龙洞洞口至洞底收集了12个沉积物样品,利用第二代测序技术研究了有孔虫在这一特殊生境中的DNA分子多样性、群落组成和空间分布特征,并将其与西太平洋的另外两种典型生境——黄海胶州湾(半封闭海湾)、西北太平洋(深海平原)进行了比较。

研究结果显示:三沙永乐龙洞中有孔虫的多样性远高于胶州湾和西北太平洋;三种生境中的有孔虫优势类群不同,胶州湾以单房室软壳类群Monothalamiids为主,西北太平洋以Monothalamiids和多房室胶结质类群Textulariida为主,而三沙永乐龙洞以多房室钙质类群Rotaliida为主。三沙永乐龙洞内有孔虫群落与水体理化因子的相关性分析结果表明:同一理化因子对不同有孔虫种类的影响不同,受理化因子的垂直变化影响,有孔虫的多样性和群落组成都表现出明显的垂直分布差异。洞内无氧区生活着大量有孔虫,某些能够进行反硝化作用的有孔虫种类在无氧区站位的相对丰度甚至高于它们在有氧区站位的相对丰度,特殊的深水无氧环境造就了独特的有孔虫群落。本工作证明了底栖有孔虫的多样性和群落组成在分子水平上存在显著的地理差异,揭示了三沙永乐龙洞内底栖有孔虫的空间垂直分布特征,发现了洞内无氧区的特征属种,证实了底栖有孔虫可以在无氧环境下生存的推断

Other Abstract

Foraminifera are a group of eukaryotic organisms with organic, calcareous or agglutinated shells. They appeared in the early Cambrian and have developed into a wide variety of species. They can colonize various marine habitats and even freshwater environments. They could be divided into two types according to their lifestyle: planktonic species living in water bodies and benthic species living in sediments. Traditionally, foraminifera are classified by observing their shells under a microscope. The application of molecular methods has enabled the biological research of foraminifera from morphological characteristics to DNA sequences and has also deepened the study of foraminifera taxonomic evolution, phylogeny, ecological distribution and the relationship with environmental changes. Foraminifera are indispensable biological indicators for the study of paleoceanography. Exploring the molecular diversity and evolutionary relationship of modern foraminifera has important scientific significance for the study of biostratigraphy, paleoclimate and paleoenvironment.

The samples I studied were collected from three cruises: the tropical Western Pacific cruise in 2016, the Northwest Pacific Ocean cruise in 2017 and the Sansha Yongle Blue Hole cruise in 2017. I applied the first-generation sequencing technology and the next-generation sequencing technology (high-throughput sequencing technology), combined with morphological observations, and analyzed the genotype diversity and phylogenetic relationship of planktonic foraminifera, as well as the molecular diversity, community composition and spatial distribution of benthic foraminifera in various typical habitats (deep ocean plain, ocean blue hole, semi-enclosed bay) in the Western Pacific Ocean. The purpose of the research is to verify whether the genetic diversity of foraminifera is widespread and whether there are geographic differences in foraminiferal diversity and community characteristics at the molecular level, and to find the correlation between foraminiferal community and environmental factors. The specific research work can be divided into the following three parts:

1. Genotype diversity of planktonic foraminifera in the tropical Western Pacific Ocean using first-generation sequencing technology

Globigerinoides ruber is one of the most common planktonic foraminiferal species in tropical-subtropical ocean, which is also an important proxy for paleoceanography and paleoclimatology research. Here, we collected planktonic foraminifera in the water body within 200 meters of the target stations in the tropical Western Pacific Ocean with plankton nets (76 μm mesh size). Globigerinoides ruber (white) individuals were picked out through stereoscopic microscopy and their total DNA was extracted by using DOC lysate. The ribosomal small subunit DNA (SSU rDNA) fragments were amplified with foraminiferal specific primers and were sequenced by the first-generation sequencing technology. The obtained target fragments were analyzed together with the genetic sequences of G. ruber in the NCBI database. The diversity of G. ruber genotypes was studied and the physiology, ecology and geographical distribution characteristics of individuals with different genotypes were compared. The phylogenetic tree constructed based on SSU rDNA showed that: G. ruber (white) had two genotypes, type I and type II, and they formed four parallel evolutionary lines together with G. ruber (pink) and Globigerinoides conglobatus. The individuals with genotype type I and type II were both found in our samples from the tropical Western Pacific Ocean, but they had different morphological characteristics and habitats. The individuals with genotype type I had three spherical chambers and a large aperture, mainly living in the shallow water (<30 m); the individuals with genotype type II had three compressed subspherical chambers and a small aperture, mainly living in the water layer deeper than 30 m. In addition, the oxygen and carbon stable isotopic composition, Mg/Ca ratio and SSU rDNA sequence composition of the two genotypes were also significantly different. This work confirmed the prevalence of genetic diversity within planktonic foraminifera. It is the first attempt to study the genotype diversity of planktonic foraminifera using first-generation sequencing technology in China and is an important supplement to the genetic data of planktonic foraminifera in the Western Pacific Ocean.

2. Study on molecular diversity and community characteristics of benthic foraminifera in the Northwest Pacific Ocean using next-generation sequencing technology

Deep sea contains a large amount of natural resources and has become a strategic area for exploration by countries around the world. This work took benthic foraminifera as the research object, and used next-generation sequencing technology to investigate their molecular diversity and community characteristics in the deep-sea plain of the Northwest Pacific Ocean with an average water depth of more than 5000 m. We obtained a total of 1536 operational taxonomic units (OTUs), and 1330 OTUs were assigned to foraminifera successfully. There were 721 foraminiferal OTUs (about 54%) belonging to the ancient lineages of soft-walled monothalamous foraminifera, indicating that Monothalamiids were the dominant group of benthic foraminifera community in this area. The results showed that only 20.6% of foraminiferal OTUs could match a reference sequence with a similarity greater than or equal to 97% in the database, indicating that the genetic sequences of the benthic foraminifera in the Northwest Pacific Ocean were different from that currently detected, and the Northwest Pacific Ocean might contain many unknown "new species" or even high-level taxonomic groups.

The distribution of deep-sea benthic foraminifera in the Northwest Pacific Ocean showed obvious spatial heterogeneity, and the community composition and diversity of foraminifera were different among stations, which might be caused by the flux of organic matter deposited on the seafloor. According to the structural characteristics of foraminiferal community at each station, eleven samples could be divided into two groups. One group of samples was collected from deep areas with high latitude; the other group of samples was collected from shallow areas with relatively low latitude. This result proved that latitude and water depth were important factors affecting the structure of foraminifera community. This work was the first attempt to study the molecular diversity and ecological distribution of benthic foraminifera in the Northwest Pacific Ocean using next-generation sequencing technology in China. It aims to explore the relationship between foraminiferal communities and environmental factors, and to provide molecular ecological data support for the work of revealing the global geographic distribution pattern of benthic foraminifera and the utilization of deep-sea resources.

3. Research on molecular diversity and spatial distribution characteristics of foraminifera in the world's deepest marine blue hole – Sansha Yongle Blue Hole using next-generation sequencing technology

Marine blue holes are rare and precious geological heritages and have extremely high scientific research value. They have unique physical and chemical characteristics because of the steep-walled structure and isolated water column which create isolated biological communities and ecosystems within a limited geographical area. The Sansha Yongle Blue Hole (SYBH), a marine blue hole in China, is known for having the deepest water depth in the world. Here, we collected twelve sediment samples from the top to the bottom of SYBH and investigated the molecular diversity and community structure of foraminifera in this special habitat by using high-throughput sequencing technology. We compared the foraminiferal diversity in SYBH with the foraminiferal diversity in the costal Jiaozhou Bay (JZB) and the abyssal Northwest Pacific Ocean (NWP).

The results showed that the molecular diversity of foraminifera in SYBH was much higher than that of JZB and NWP. The dominant taxa of foraminifera in the three habitats were different. The foraminiferal community in SYBH was dominated by multi-chambered calcareous taxa, while the foraminiferal community in the other two locations were dominated by agglutinated or organic groups. The correlation analysis between the foraminiferal community and the environmental factors in SYBH showed that the same factor had different effects on different foraminiferal species. The diversity and community composition of foraminifera showed obvious vertical variation under the influence of vertical changes in environmental factors. A large amount of foraminiferal DNA was detected in the anaerobic areas of SYBH. The relative abundance of some foraminiferal species capable of denitrification in the anaerobic zone was even higher than their relative abundance in the aerobic zone. The deep-water anaerobic environment created a unique foraminifera community. This work proved that there were significant geographic variations in the diversity and community composition of benthic foraminifera at the molecular level, revealed the vertical distribution characteristics of benthic foraminifera in SYBH, discovered representative species in the anaerobic zone of SYBH, and confirmed the inference that benthic foraminifera could survive in an anaerobic environment.

Language中文
Table of Contents

目 录

... I

ABSTRACT. V

1 绪论... 1

1.1 有孔虫简介... 1

1.2 研究背景... 2

1.2.1 浮游有孔虫及其分子生物学研究... 2

1.2.2 底栖有孔虫及其分子生物学研究... 5

1.3 研究目的... 8

2 利用第一代测序技术研究热带西太平洋浮游有孔虫的基因型多样性... 9

2.1 前言... 9

2.2 材料与方法... 11

2.2.1 样品采集与处理... 11

2.2.2 DNA提取和PCR扩增... 12

2.2.3 克隆和测序... 13

2.2.4 数据处理... 14

2.3 结果... 15

2.3.1 形态特征... 16

2.3.2 SSU rDNA序列特征... 17

2.3.3 基因型多样性... 18

2.4 讨论... 19

2.4.1 不同基因型G. ruber之间的差异... 19

2.4.2 基因型形成的可能机制... 20

2.4.3 基因型研究中存在的问题... 21

2.4.4 基因型多样性对古海洋学的意义... 21

2.5 结论... 22

3 利用第二代测序技术研究西北太平洋深海底栖有孔虫的DNA分子多样性与群落特征... 23

3.1 前言... 23

3.2 材料与方法... 24

3.2.1 样品采集和处理... 24

3.2.2 DNA提取... 24

3.2.3 PCR扩增... 25

3.2.4 文库构建和上机测序... 25

3.2.5 数据质控... 26

3.2.6 统计分析... 27

3.3 结果... 27

3.3.1 西北太平洋样品的数据概况... 27

3.3.2 西北太平洋的有孔虫多样性和群落结构... 30

3.3.3 西北、东北太平洋有孔虫群落特征的比较... 33

3.4 讨论... 36

3.4.1 西北太平洋深海底栖有孔虫的分子多样性... 36

3.4.2 西北太平洋深海底栖有孔虫群落结构的影响因素... 37

3.4.3 西北太平洋深海底栖有孔虫的空间分布特征... 38

3.4.4 有孔虫分子生态学研究中的不足... 39

3.5 结论... 40

4 利用第二代测序技术研究世界最深海洋蓝洞三沙永乐龙洞中有孔虫的DNA分子多样性和空间分布特征... 41

4.1 前言... 41

4.2 材料与方法... 42

4.2.1 样品采集... 42

4.2.2 DNA提取和PCR扩增... 45

4.2.3 文库构建和上机测序... 46

4.2.4 数据质控和统计分析... 47

4.3 结果... 48

4.3.1 数据概况... 48

4.3.2 群落组成的地理差异... 52

4.3.3 三沙永乐龙洞有孔虫多样性和分布特征... 55

4.3.4 环境因子相关性分析... 60

4.4 讨论... 64

4.4.1 有孔虫多样性的影响因素... 64

4.4.2 有孔虫群落组成的影响因素... 65

4.4.3 三沙永乐龙洞无氧区的有孔虫... 68

4.5 结论... 69

5 总结与展望... 71

5.1 主要结果与初步结论... 71

5.1.1 热带西太平洋浮游有孔虫代表种G. ruber的基因型多样性... 71

5.1.2 西北太平洋深海底栖有孔虫的DNA分子多样性和群落组成... 72

5.1.3 南海三沙永乐龙洞中底栖有孔虫的DNA分子多样性及空间分布特征... 72

5.2 存在问题... 73

5.2.1 浮游有孔虫基因型研究中存在的问题... 73

5.2.2 底栖有孔虫分子生态研究学中存在的问题... 74

5.3 展望... 74

参考文献... 75

作者简历及攻读学位期间发表的学术论文与研究成果... 91

 

Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/164789
Collection海洋生物分类与系统演化实验室
Recommended Citation
GB/T 7714
李青霞. 西太平洋典型生境中有孔虫遗传多样性的初步研究[D]. 中国科学院海洋研究所. 中国科学院大学,2020.
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