海洋生物分类与系统演化实验室知识库

为探索不同海区类型沉积物中底栖有孔虫的分子多样性及与环境因子之间的相互关系，本研究从潮间带、海湾、陆架浅海一直到深海大洋区，共采集了81个站位或季节的共计243个样品。提取环境样品的总DNA，利用有孔虫特异性引物 (s14F3-s17) 扩增其18S rRNA 基因3’端的一个高变区 (大约300 bp)，使用Illumina HiSeq 2500测序平台进行双端250 bp测序。此外，为探索不同DNA提取和染色方法对有孔虫分子鉴定研究的影响，本论文进行了有孔虫单种实验方法学研究。具体工作包括如下：

1. DNA提取和染色方法对有孔虫分子鉴定研究的影响研究

以野外采集的钙质瓷质壳类底栖有孔虫五玦虫属Quinqueloculina spp. (Foraminifera，Miliolida) 为研究材料，研究经虎红染色后虫体的DNA质量，以此评估虎红染色对有孔虫分子实验的影响。通过方法学的摸索可知，经1 g/L虎红染色36 hrs的大部分瓷质壳类底栖有孔虫样本可以成功提取到DNA，并且可以用于下游的PCR等分子实验。

2. 青岛湾潮间带底栖有孔虫分子多样性月变动及与环境关系研究

14个月采集的样品经测序共得到605种底栖有孔虫。沉积物中底栖有孔虫物种数在冬季和夏季较多，秋季次之，春季最少。而分子多样性的季节变化整体上呈现夏季高，秋冬季节次之，春季少的规律。Rotaliida相对丰度在秋冬季节较高，春季其次，夏季较少，Miliolida相对丰度随季节的变化趋势与之相反。优势属为：Ammonia (卷转虫属)，Textularia (串珠虫属)，Epistominella (小上口虫属)，Operculina (盖形虫属)，Bulimina (小泡虫属)。Epistominella及Operculina相对丰度与水温和泥温均呈显著正相关。底栖有孔虫的群落多样性在春季和夏季与水温和泥温正相关，与盐度负相关；在秋季与水温、泥温和盐度均呈负相关；而在冬季与水温和泥温呈负相关，与盐度呈正相关。有孔虫种类会随着季节发生演替，在不同年份差别较大。相邻月份的样品之间群落相似性更高，同时有孔虫群落结构年际变化在很短的尺度上就有很明显的体现。

3. 胶州湾浅海陆架底栖有孔虫分子多样性季节变动及与环境关系研究

样品采集自3个站位，5个季度共15个样品，测序共得到781种底栖有孔虫。沉积物中底栖有孔虫物种数及分子多样性整体上在秋季和夏季较高，冬季较低。有孔虫种类在一年四个季度中基本稳定不变，而特有种类在秋季最多，并呈现一定的季节变化。底栖有孔虫物种数量在C1站位较多，D6站位次之，A5站位较少。A5、C1和D6站位沉积物中底栖有孔虫的种类在相邻年份相同月份发生了比较大的变化。这三个站位的共有的底栖有孔虫种类占比稳定在50%左右。优势属为：Ammonia，Bulimina，Epistominella，Cibicides (面包虫属)，Stainforthia以及未能鉴定到属的轮虫unidentified_Rotaliida，并且优势属在各个季节的分布是更替变换的。Epistominella属的相对丰度与水深和水温均呈显著负相关。此外，A5和C1站位沉积物中底栖有孔虫群落结构更相似，而与D6站位差别较大。底栖有孔虫的群落多样性在春季与底层水温呈正相关，与水深、盐度和pH呈负相关；在夏季与底层水温和盐度呈正相关，与水深和pH呈负相关；在秋季与底层水温呈正相关，与盐度和pH呈负相关；而在冬季与底层水温呈负相关，与盐度和pH呈正相关。

4. 黄海陆架区底栖有孔虫分子多样性对pH变化的响应研究

经测序，H02站位共得到348种底栖有孔虫，H07站位394种，H09站位460种，B21站位367种，SY02站位460种。因此，五个站位的沉积物中底栖有孔虫种数在H09和SY02站最多，H07和B21次之，H02站位最少。在H07和B21站位，海水pH的降低会导致底栖有孔虫物种数的减少，从而产生不利的影响。随培养时间增加，近岸站位B21有孔虫数目基本不变，而H02站则有所下降。相反，离岸站H07及SY02有孔虫数目则随着培养时间增加，H09站位有孔虫数目随培养时间先升高后降低。黄海各个站位之间的底栖有孔虫优势类群大部分相同，但存在少量的差异。这些优势类群相对丰度总和不超过25%。沉积物中底栖有孔虫的物种数与水深、水温和盐度没有明显的相关性。

5. 热带西太平洋海山区底栖有孔虫分子多样性及与环境关系研究

在热带西太平洋海山区17个站位采集的样品经测序共得到2238种底栖有孔虫，远远高于潮间带、海湾及陆架浅海区。另外，2016年度沉积物样品中底栖有孔虫物种数量明显高于2014和2015年。而三个年份共有的有孔虫种类仅有30种。海山与邻近大洋区沉积物中底栖有孔虫的群落结构差别较大。另外同一年份的样品群落结构更相似，而不同年份的样品中有孔虫群落结构差异较大。沉积物中底栖有孔虫的群落多样性与水深呈负相关，与水温和盐度均呈正相关。Cibicides，Epistominella及Streptochilus属的相对丰度均与水温呈显著正相关，而与水深呈显著负相关，并且后者与盐度呈显著负相关；另外，Bulimina属的相对丰度仅与水温呈显著正相关，而与其他环境因子没有显著相关性。

综合比较四个海区：在目水平，四个海区的优势类群组成相似，玻璃质壳类有孔虫轮虫目Rotaliida在沉积物中均占具绝对优势，占比平均值为85.1%-99.9%，其次瓷质壳类有孔虫为粟米虫目Miliolida，占比平均值为0-10.2%。四个海区的共有优势属为Ammonia属和Epistominella属。热带西太平洋海山区沉积物中底栖有孔虫多样性远远高于潮间带及近岸海域，而且前者的SY站位可能存在新的有孔虫类群。水深、水温、盐度及pH等环境因子会显著影响底栖有孔虫的群落结构，只是影响方式不尽相同。另外，用于进行序列比对的数据库中关于有孔虫的记录较少，以至于大部分测序序列无法比对到属水平，而被划归到“Others”的范畴。

本研究系对热带西太平洋海山区底栖有孔虫分子多样性的首次探索性研究，也是国内第一次对潮间带、海湾、陆架浅海到深海大洋区底栖有孔虫的分子多样性进行的系统性调查。但是由于在多个区域 (例如胶州湾、热带西太平洋海山区等) 没有开展过底栖有孔虫形态学方面的调查研究，因此本论文缺乏一些与形态学数据比对的工作，有待后期进行更深入的研究。本论文不仅可以为各种海区类型底栖有孔虫的多样性研究提供依据，而且可以为传统方法对底栖有孔虫多样性的调查结果进行校正提供参考。同时还研究了底栖有孔虫分子多样性与环境因子之间的关系，找出了一些会对有孔虫群落结构产生重要影响的因素，为构建底栖有孔虫群落结构与环境关系模型提供一些参考。

In order to explore the diversity of benthic foraminifera and the relationship with environmental factors in various sea area, this study collected sediments containing foraminifera and the high-throughput sequencing method was used. From intertidal zone, bay, shallow sea to deep-sea area, we collected a total of 243 samples from 81 stations or seasons. The total DNA of environmental samples was extracted with foraminifera specific primers (s14F3-s17) to amplify a high variable region (about 300 BP) at the 3’ end of the 18S rRNA gene, and the Illumina HiSeq 2500 platform was used for PE 250 sequencing. In addition, in order to explore the effects of different DNA extraction and dyeing methods on foraminifera molecular identification, a single species experiment of foraminifera was carried out in this paper. We have done the following work:

1. Effects of DNA extraction and dyeing methods on molecular identification of foraminifera

In this paper we used Quinqueloculina spp. (Foraminifera, Miliolida) as the research material, and studied the DNA quality of the foraminifera specimens after Rose Bengal staining to explore the effect of Rose Bengal staining on the molecular identification of foraminifera. The result showed that most of the porcelain benthic foraminifera samples stained with 1 g/L Rose Bengal for 36 hrs could be successfully extracted DNA and be used in the downstream PCR and other molecular experiments.

2. Monthly variation of benthic foraminiferal molecular diversity and its relationship with environment in Qingdao Bay

After sequencing, 605 species of benthic foraminifera were obtained from 14 samples. The number of benthic foraminifera species in sediments was more frequent in winter and summer, followed by autumn and spring. The Rotaliida order was dominant (85.1%), followed by the Miliolida (10.2%). The relative abundance of Rotaliida was higher in autumn and winter, followed by spring and less in summer. The relative abundance of Miliolida order was opposite to that of Rotaliida. The largest relative abundance in all the samples ranked as the top 10 were: Ammonia, Textularia, Epistominella, Operculina, and Bulimina. In addition, the records of foraminiferal sequence database used for sequence alignment was less recorded about the order Miliolida, so that the related sequences could not be compared to the level of the genus, and were classified to the category of “Others”. The molecular diversity of benthic foraminifera was in summer, followed by autumn, winter, and low in spring. Foraminifera species vary with seasons and differ greatly in different years. The similarity between samples in adjacent months was higher, and the interannual variation of foraminiferal community structure was obvious at a very short scale. Epistominella showed a significant positive correlation between abundance and water temperature and mud temperature. The abundance of Operculina was highly positively correlated with water temperature and mud temperature. The community diversity of benthic foraminifera was positively related to water temperature and mud temperature, negatively related to salinity in spring and summer; and negatively related to water temperature, mud temperature and salinity in autumn; but negatively related to water temperature and mud temperature salinity, and positively related to salinity in winter.

3. Seasonal variation of molecular diversity of benthic foraminiferal and its relationship with environment in Jiaozhou Bay

15 samples were collected from 3 stations, and in 5 quarters. 781 benthic foraminifera were obtained. The number of benthic foraminifera species in sediments was more frequent in autumn and summer, but less in winter. Foraminifera species remained stable in the four quarters of a year, while endemic species were the most abundant in autumn and showed seasonal changes. The number of benthic foraminifera species was more in C1 station, than in D6 station and A5 station. The species of benthic foraminifera in A5, C1 and D6 stations changed considerably in the same month of the adjacent years. The proportion of common species at these three stations was about 50%, while the proportion of endemic species increased from spring to winter. Rotaliida occupied an absolute advantage in sediments. The largest relative abundance in all the samples ranked as the top 10 were: Ammonia, Bulimina, Epistominella, Cibicides, Stainforthia, and the unidentified_Rotaliida. But their relative abundance was not more than 8% in all samples. Cibicides, Ammonia and Stainforthia were more in November; Bulimina and Epistominella were more in February. The distribution of dominant genera in the sediments of Jiaozhou Bay was alternation in each season. The molecular diversity of benthic foraminifera was higher in autumn and summer, and lower in winter. It was indicated that in the three seasons of spring, summer and autumn in 2014, “time” was an important factor affecting the similarity of benthic foraminiferal community structure in the sediments of Jiaozhou Bay. In addition, the structure of benthic foraminifera community in A5 and C1 stations was more similar than that in D6. The relative abundance of Epistominella was negatively correlated with water depth and water temperature. The community diversity of benthic foraminifera was positively correlated with water temperature and negatively correlated with water depth, salinity and pH in spring; and was positively correlated with water temperature and salinity and negatively correlated with water depth and pH in summer; and it was positively correlated with water temperature and negatively correlated with salinity and pH in fall; but negatively correlated with water temperature in winter and positive correlation with salinity and pH.

4. Effect of pH variation on benthic foraminiferal molecular diversity in the Yellow Sea

After sequencing, 348 species of benthic foraminifera were obtained at H02 station; 394 species were obtained in H07 station; 460 in H09 station; 367 in B21 station and 460 in SY02 station. Therefore, the number of benthic foraminifera species at the five stations in the Yellow Sea was the largest in H09 and SY02 stations, followed by H07 and B21 stations, and H02 station was the least. For H07 and B21 stations, the decrease of seawater pH will lead to the decrease of benthic foraminifera species number and thus have an adverse effect. With the increase of incubation time, the number of foraminifera in offshore stations (B21) remained unchanged, while H02 stations decreased. On the contrary, the number of foraminifera in offshore stations (H07 and SY02) increased with the culture time, and the number of foraminifera at H09 station increased first and then decreased with incubation time. The dominant groups of benthic foraminifera among different stations in the Yellow Sea were mostly the same, but there were also a few differences. The sum of relative abundance of these dominant groups was not more than 25%, because of the unknown group “Others” occupied the majority. Incubation time and pH will have a great impact on the species of benthic foraminifera in the Yellow Sea sediments. The number of foraminifera species was not significantly correlated with water depth, water temperature and salinity.

5. Relationship between benthic foraminiferal molecular diversity and environmental factors in the tropical Western Pacific Ocean sediments

Samples from 17 stations have been sequenced and 2238 species of benthic foraminifera have been obtained, which were much higher than those in the intertidal zone, the Bay and the continental shelf. In addition, the species and relative abundance of benthic foraminifera in sediment samples from 2016 were significantly higher than those in 2014 and 2015. There may be a new foraminifera group in SY station. Except for SY station, Rotaliida dominated, followed by the Miliolida in the other stations. The community structure of benthic foraminifera in seamounts and the adjacent ocean region was quite different. In addition, the foraminiferal community structure of samples in the same year was more similar, but quite different in different years. There were only 30 species of foraminifera in the three years. The number, diversity, richness and species number of benthic foraminifera in all stations in 2016 were high. The relative abundance of Cibicides and Epistominella were significantly negatively correlated with water depth, but it had a significant positive correlation with water temperature. And the relative abundance of Bulimina was significantly positively correlated with water temperature. Moreover, the relative abundance of Streptochilus was significantly positively correlated with water temperature, and significantly negatively correlated with water depth and salinity. The community diversity of benthic foraminifera was negatively correlated with water depth and positively correlated with water temperature and salinity.

Comprehensive the four regions: Rotaliida occupied an absolute advantage in sediments. Ammonia and Epistominella belonged to the dominant group in all regions. The diversity of benthic foraminifera in the sediments of the tropical Western Pacific Ocean was much higher than that in the intertidal and nearshore waters. The environmental factors such as water depth, water temperature, salinity and pH will significantly affect the community structure of benthic foraminifera, but the modes of influence were different. In addition, due to the limitation of database, a large number of foraminiferal sequences could not be annotated to low taxonomic category.

This study was the first exploratory research of the molecular diversity of benthic foraminifera in the tropical Western Pacific Ocean. It was also the first systematic investigation of the molecular diversity of the benthic foraminifers in the intertidal zone, the Bay, the shallow sea and the deep ocean. However, due to the lack of research on the morphology of benthic foraminifera in many regions, such as the Jiaozhou Bay and the tropical Western Pacific Ocean, this paper lacks some work on comparison with morphological data, which needs to be further studied in the future work. Even so, this paper could not only provide a basis for the research of benthic foraminiferal diversity, but also could be used as a reference for correcting the results of the research on the benthic foraminiferal diversity studied by traditional methods. At the same time, the relationship between the molecular diversity of benthic foraminifera and the environmental factors was also studied, and some important factors that could affect the structure of the foraminiferal community were found out, and some references were provided for constructing the relationship model between the benthic foraminiferal community structure and environment.