海洋生物分类与系统演化实验室知识库

有孔虫是一种绝大部分栖息在海洋环境中的单细胞原生动物，构成了现代海洋中最多样化的带壳微型生物类群。它们大部分能够分泌碳酸钙质的外壳，少部分细胞外会覆盖胶结质或有机质壳。其中绝大多数现代有孔虫是底栖有孔虫，只有大约40到50种为浮游有孔虫。底栖有孔虫不仅多样性更加丰富，它们的地质记录也比浮游有孔虫更长。底栖有孔虫最古老的化石出现在寒武纪，而浮游有孔虫的化石出现在侏罗纪。通过建立底栖有孔虫壳体元素指标与海洋环境因子（温度、盐度、pH等）的定量关系成为重建底层海水古环境最重要的方法之一。随着全球气候变暖，温度对生物的影响越来越多的受到人们的重视。有孔虫作为海洋古温度研究最重要的生物指标，探究底栖有孔虫对温度的响应对研究古气候与古海洋的重建具有重要的科学意义。

本文以底栖有孔虫为研究对象，利用对2017年中国胶州湾陆架区、2018年青岛湾潮间带、2018年中国胶州湾陆架区、2019年中国南海“海马”冷泉以及2019年亚北极沉积物样品采集，结合传统形态鉴定与高通量测序技术的方法，借助野外调查与实验室培养的途径，分析了低纬度到高纬度等不同区域条件下底栖有孔虫丰度、多样性、优势种以及壳质类型对温度的响应，尝试揭示温度对底栖有孔虫影响的内在机制，得到底栖有孔虫生物指标与温度的相互关系。具体研究内容分为以下五部分：

1.  实验室培养下探究陆架区底栖有孔虫群落对温度响应初探

底栖有孔虫作为最重要的海洋环境指示生物之一，在重建底层海水古温度中发挥着重要作用，但温度对底栖有孔虫的影响机制尚不明确。本研究通过采集胶州湾陆架区（水深3-26米）的表层沉积物样品，在实验室不同温度条件下（6℃、10℃、12℃、18℃和24℃）对携带活体底栖有孔虫的沉积物样品进行未投喂饵料的培养，培养实验持续10周，尝试探究底栖有孔虫群落对不同温度的响应。实验一共挑选鉴定有孔虫2331枚，其中活体有孔虫476枚。活体有孔虫丰度在6.80 ind/g-13.25 ind/g之间。实验结果表明：活体有孔虫丰度与温度显著负相关；随着温度升高，有孔虫物Margalef指数、Shannon-Wiener指数先升高后下降，均匀度指数表现平稳，并且它们与温度没有显著相关性；此外，不同温度下有孔虫群落的优势种不同。研究结果揭示了在本实验的温度范围和培养条件下，外界环境温度的变化会影响有孔虫的群落参数和多样性，有孔虫群落优势种的改变表征着有孔虫群落的演替。

2.  温度培养实验中有孔虫壳体常用染色剂的安全性使用研究

有孔虫壳体Mg/Ca是最常见的古温度指标之一，实验室培养的方法被广泛应用于构建有孔虫不同种类Mg/Ca与温度关系的研究中。在培养实验中，钙黄绿素是标记有孔虫新形成碳酸钙壳体结构最常用的染色剂。钙黄绿素是否对有孔虫的生物特征（如丰度、虫体大小）有影响尚不清楚。本研究通过采集青岛湾表层沉积物样品，将携带活体底栖有孔虫的沉积物样品在4个钙黄绿素浓度（0 mg/L、1 mg/L，5 mg/L和10 mg/L）与3个培养时间（2周、4周和6周）下进行实验室培养，研究不同浓度与培养时间下钙黄绿素对活体底栖有孔虫群落的影响。

实验一共检获活体底栖有孔虫10393枚，其中不同处理下有孔虫丰度介于122.72 ± 13.69-458.65 ± 234.29 ind./g样品干重之间。丰度最高值出现在第2周的5 mg/L浓度组，而最低值出现在培养时间最长（6周）浓度最高（10 mg/L）的处理中。统计分析结果表明钙黄绿素浓度对群落参数（有孔虫丰度、Margalef指数、Shannon-Wiener指数）影响显著，培养时间只对有孔虫丰度影响显著。对有孔虫的壳质类型进行分析，发现实验组（1 mg/L、5 mg/L和10 mg/L）的瓷质有孔虫所占比例显著高于对照组（0 mg/L），而玻璃质有孔虫比例低于对照组。此外，实验中得到3种优势种：Ammonia aomoriensis、Cribrononion gnythosuturatum和Quinqueloculina seminula。不同优势种的丰度对钙黄绿素浓度与培养时间的响应不同。随着钙黄绿素浓度增加，C. gnythosuturatum和Q. seminula的丰度显著下降，A. aomoriensis的丰度变化不显著，随着培养时间增长，只有Q. seminula的丰度显著下降。不同实验处理下优势种虫体大小发育不同。对照组优势种虫体平均大小在第2周达到最大值，而实验组优势种虫体平均大小在第4或第6周才达到最大值。本研究结果显示：在实验设置的浓度与时间范围内，钙黄绿素可能通过增强瓷质有孔虫类群而对有孔虫群落产生有利影响。此外，考虑到高钙黄素浓度和长时间的培养对有孔虫有负面影响，我们建议采用比较安全的钙黄绿素使用方法：在培养实验开始时，加入钙黄绿素（≈5 mg/L）作为初始标记，在实验中途用天然海水替代。大约在培养实验结束前4周，可以再次将钙黄绿素加入海水培养液中。

3.  利用形态学与分子生物学方法揭示温度对底栖有孔虫的影响及内在机制

随着全球气候变暖，海水温度对海洋生物的影响越来越大。有孔虫作为海洋学研究中最重要的环境指示生物之一，建立有孔虫与温度的关系至关重要，但目前温度对有孔虫影响的机制尚不清楚。本实验通过采集胶州湾陆架区A5（水深9.2m）、C4（水深6m）、D6（水深26m）站位的表层沉积物样品，将携带活体底栖有孔虫的沉积物样品在实验室不同温度（6ºC、12ºC、18ºC、24ºC和30ºC）条件下进行培养，整个培养实验一共持续80天，结合传统形态鉴定与高通量测序技术研究不同温度对底栖有孔虫群落的影响与内在机制。

实验中通过形态挑选鉴定活体有孔虫4162枚，分子测序获得Effective Tags 3871615条。研究结果显示：随着培养温度升高，A5、C4、D6站位的活体有孔虫丰度均呈下降趋势，Spearmen相关性分析表明活体有孔虫丰度与温度呈显著负相关，分子数据的有孔虫reads与温度的相关性不显著；形态与分子数据得到的A5、C4、D6站位的有孔虫Margalef指数均随温度升高而升高，相关性分析表明形态上活体有孔虫Margalef指数与温度呈显著正相关，但形态与分子数据得到的有孔虫Shannon-Wiener指数与温度均没有显著相关性；玻璃质有孔虫以及胶结质有孔虫的丰度随着温度升高而显著降低，玻璃质有孔虫以及胶结质有孔虫的reads随温度升高而升高，但相关性检验并不显著，不同站位的瓷质有孔虫和单房室有孔虫的reads随温度升高变化不同；形态学上得到的优势种有Ammonia aomoriensis、A. beccarii、Buccella frigida、Cribrononion subincertum和Trochammina inflata，分子得到的优势种与形态不一致，并且不同站位的不同优势种对温度的响应不同。该研究揭示了温度显著影响有孔虫群落的群落丰度、多样性、壳质类型以及优势种组成；通过相关性与函数拟合分析，建立了有孔虫丰度、Margalef指数、不同壳质类型以及部分优势种的丰度与温度的线性函数关系，为古温度与古海洋的重建提供第一手数据资料和函数参考；此外，分子生物学能捕获形态学无法检获的小个体隐存种以及单房室有孔虫（多数为无壳类），导致分子生物学所得到的有孔虫物种数、多样性提高，并且两者有孔虫物种组成的差异，引发了有孔虫在形态与分子上对温度响应的不同。

4.  利用高通量测序技术探究有孔虫对低纬度冷泉（低温低氧环境）的响应

冷泉是深海中一种罕见的生境，它通常与海洋中碳氢化合物资源（如天然气、石油）的分布密切相关。底栖有孔虫作为冷泉渗漏活动的潜在指示而受到广泛关注，但目前冷泉区有孔虫的研究绝多大数基于传统形态分类。本实验中，通过搭载“深海勇士”号下潜器在海马冷泉区采集了SY186-1、SY186-3、SY186-5和SY186-7站位（平均水深超过1380m）0-28cm以浅的沉积物样品。在实验室提取了样品中的总基因组DNA，利用有孔虫特异性引物扩增其中的有孔虫DNA，应用高通量测序技术对获得的有孔虫DNA片段进行测序，分析了海马冷泉沉积物中有孔虫的分子多样性与群落组成。

原始数据经过序列拼接和严格的质量控制，一共得到3109553条Effective tags和432个OTUs。研究结果发现SY186-1和SY186-3站位（距离冷泉活动中心更近）的reads、OTUs、Margalef指数以及Shannon-Wiener指数等群落参数比SY186-5和SY186-7站位（距离冷泉活动中心更远）更高。此外，发现冷泉区有孔虫绝大多数为玻璃质有孔虫（>85%），其次为少量的胶结质有孔虫和单房室有孔虫，未在有孔虫数据库中比对到瓷质有孔虫（平均Identity值为97.7%）。另外发现冷泉有孔虫群落主要分为三种生态类群：底栖有孔虫冷水类群、底栖有孔虫反硝化类群以及浮游有孔虫类群。我们的研究揭示了冷泉区活动中心低温低氧的条件造成冷泉活动中心的有孔虫reads、分子多样性高于非活动中心；冷泉区有孔虫分子多样性高于一般海区而低于一些高营养海区；冷泉区有孔虫以高比例的玻璃质有孔虫为特征；反硝化有孔虫是冷泉区重要的特征类群，Uvigerina属是其中最常见的有孔虫属。因此有孔虫可以作为环境指示的有效生物。

5.  利用高通量测序技术探究有孔虫对高纬度亚北极（低温环境）的响应

北极地区是全球海洋气候系统的重要组成部分，对于研究全球气候变化（如温室效应）等问题具有重要意义。然而我们对这片海洋过去和现在的了解仍然非常有限。钙质有孔虫被认为是重建北极地区海洋古环境的最有用的替代指标之一。为了探究亚北极沉积物中有孔虫的分子多样性与群落组成特征，通过采集亚北极11个站位的表层沉积物，提取了样品中的总基因组DNA，利用有孔虫特异性引物扩增其中的有孔虫DNA。

经过严格的质量控制流程和数据处理，实验一共获得了2170762条Effective tags和1046个OTUs。其中，有孔虫reads最高的站位为BR10，有孔虫Margalef指数以及Shannon-Wiener指数在BR06站位取得最大值。Spearman相关性检验结果显示，有孔虫群落参数（reads、OTU、Margalef指数和Shannon-Wiener指数）与环境因子（温度、盐度和水深）均没有显著相关性。有孔虫群落中玻璃质有孔虫占比最高，其次为胶结质和单房室有孔虫，瓷质有孔虫占比最低。研究揭示了亚北极地区有孔虫分子多样性水平低于其他研究海区；高比例的玻璃质有孔虫和低比例的瓷质有孔虫是亚北极有孔虫的重要特征；Nonionella labradorica是亚北极有孔虫的特征优势种，Reophax属、Globobulimina属以及Elphidium属为特征类群。

Foraminifer is a kind of single-celled protozoa which is living mostly in the marine environment. They constitute the most diverse shelled microorganism group in the modern ocean. Most of them secrete a calcium-carbonate shell while a few have agglutinated or organic shell. Benthic foraminifera are the main group of the modern foraminifera. Moreover, only about 40 to 50 species are planktonic foraminifera. Benthic foraminifera are not only more diverse than planktonic foraminifera, they also have a longer geological record. The oldest fossils of benthic foraminifera appeared in the Cambrian period, while those of planktonic foraminifera appeared in the Jurassic period. The establishment of the quantitative relationship between benthic foraminiferal shell elements and marine environmental factors (temperature, salinity, pH, etc.) has become one of the most important methods to reconstruct the palaeoenvironment of bottom seawater. With the global warming, more and more attention has been paid to the influence of temperature on organisms. Foraminifera is one of the most important biological indicators for the study of the palaeotemperature of ocean. It is of great scientific significance to explore the response of benthic foraminifera to temperature for the reconstruction of palaeoclimate and palaeoocean.

In this study, benthic foraminifera were used as the research object. The sediment samples of the continental shelf in Jiaozhou bay (2017, 2018), the intertidal zone of Qingdao bay (2018), the “Haima” cold seep in the south China sea (2019), and the subarctic region (2019) were collected. Combining the traditional morphological method with the next-generation sequencing technology (high-throughput sequencing technology), we used the field investigation and laboratory culture experiment to analyze the response of the abundance, diversity, shell type, and dominant species of benthic foraminifera to temperature in different marine areas. This study tries to reveal the internal mechanism of the effect of temperature on the benthic foraminifera and establish the relationship between the biological indicators of benthic foraminifera and temperature. The specific research contents are divided into the following five parts:

1. A preliminary experiment to study the response of the benthic foraminifera community from the continental shelf to temperature in laboratory culture

As one of the most important marine environmental indicators, benthic foraminifera play an important role in the reconstruction of the paleotemperature of the bottom seawater. However, the mechanism of the effect of temperature on the benthic foraminifera is not clear. The surface sediment samples from the Jiaozhou bay (3-26 m) of the continental shelf were collected. Sediment samples with living benthic foraminifera communities were cultured without extra food under different temperatures (6°C, 10°C, 12°C, 18°C, and 24°C) in the laboratory. The whole culture experiment lasted for 8 weeks. This study tried to explore the response of the benthic foraminifera communities to different temperatures.

A total of 2,331 benthic foraminifera were identified in the experiment, including 476 living foraminifera. The abundance of living foraminifera ranged from 6.80 ind/g to 13.25 ind/g dry weight of the sample. The results showed that the abundance of living foraminifera was negatively correlated with temperature. With the increase of temperature, the Margalef index and Shannon-Wiener index of foraminifera increased first and then decreased while the evenness index was stable. Moreover, all the three diversity index had no significant correlation with temperature. The response of dominant species of foraminiferal communities were specific to different temperatures. The study indicated that the change of ambient temperature would affect the foraminiferal community parameters, diversity, and community structure under the temperature range and culture conditions of this experiment. The difference of dominant species in the different temperatures suggested a succession of the foraminiferal community.

2. A study on the safe use of commonly used marker for foraminiferal shell in temperature culture experiments

The foraminiferal shell Mg/Ca is one of the most common paleotemperature indicators. The method of laboratory culture has been widely used to establish the relationship between Mg/Ca and temperature of different foraminiferal species. Calcein has been usually used as an effective marker for tagging newly formed biogenic calcite in shell structure of foraminifera in culture experiments. Whether calcein has any effect on the biological characteristics of foraminifera, such as abundance and size, is unclear. The surface sediment samples from Qingdao bay were collected. The sediment samples with living benthic foraminiferal communities were cultured under four calcein concentration (0 mg/L, 1 mg/L, 5 mg/L, and 10 mg/L) and three incubation time (2 weeks, 4 weeks, and 6 weeks) in the laboratory experiment. This study aimed to explore the impact of calcein on living benthic foraminifera community under different calcein concentration and incubation time.

A total of 10,393 living benthic foraminifera were collected. The abundance of the living foraminifera under different treatments ranged from 122.72 ± 13.69 to 458.65 ± 234.29 ind./g dry weight of sample. The highest abundance was found in the 5 mg/L concentration group at 2 weeks, and the lowest value was found in the treatment with the longest incubation time (6 weeks) and the highest concentration (10 mg/L). The results of statistical analysis showed that the concentration of calcein significantly affected the community parameters (foraminiferal abundance, Margalef index, and Shannon-Wiener index) while the incubation time only had a significant effect on the abundance of foraminifera. As for the shell type of foraminifera, the proportion of the porcelanous foraminifera in the experimental groups (1 mg/L, 5 mg/L, and 10 mg/L) was significantly higher than that in the control group (0 mg/L) while the proportion of hyaline foraminifera was lower than that in the control group. In addition, three dominant species were obtained: Ammonia aomoriensis, Cribrononion gnythosuturatum, and Quinqueloculina seminula. The abundance of different dominant species had different responses to calcein concentration and incubation time. The abundance of C. gnythosuturatum and Q. seminula decreased significantly with the increase of calcein concentration. However, the abundance of A. aomoriensis did not change significantly. Moreover, only the abundance of Q. seminula decreased significantly with the increase of incubation time. The shell size and development of dominant species were different under different experimental treatments. The average size of the dominant species in the control group reached the maximum value in the second week while the average size of the dominant species in the experimental group did not reach the maximum value until the fourth or sixth week. The results of this study suggested that calcein might have a positive effect on foraminifera community by enhancing the porcelanous foraminifera at the set range of the calcein concentration and incubation time in the experiment. In addition, considering the negative effects of high calcein concentration and long incubation time on foraminifera, we suggested a relatively safe method of calcein use: Calcein (≈ 5 mg/L) was added as an initial marker at the beginning of the culture experiment, and natural seawater was substituted halfway through the experiment. About 4 weeks before the end of the culture experiment, calcein can be added to the seawater culture medium again.

3. A study to reveal the effect of temperature on benthic foraminifera and its internal mechanism based on morphological and molecular methods

With the global warming, more and more attention has been paid to the influence of temperature on organisms. Foraminifer is one of the most important environmental indicators in oceanography. Thus, it is very important to establish the relationship between temperature and foraminifera. However, the mechanism of the effect of temperature on foraminifera is still unclear. The surface sediment samples from the stations (A5, 9.2m; C4, 6m; D6, 26m) of Jiaozhou bay continental shelf were collected. The sediment samples with living benthic foraminifera were cultured under different temperatures (6ºC, 12ºC, 18ºC, 24ºC, and 30ºC) in the laboratory. The whole culture experiment lasted for 80 days. The effects of different temperatures on benthic foraminiferal communities and its internal mechanisms were studied by combining traditional morphological identification and next-generation sequencing technology.

In the experiment, 4,162 living foraminifera were identified by morphological identification, and 3,871,615 effective tags were obtained by molecular sequencing technology. The results showed that the abundance of living foraminifera at A5, C4, and D6 stations decreased with the increase of culture temperature, and Spearmen correlation analysis showed that the abundance of living foraminifera was significantly negatively correlated with temperature. However, the correlation between foraminiferal reads and temperature was not significant. The Margalef index of foraminiferal community at A5, C4, and D6 stations obtained from morphological and molecular data all increased with the increase of temperature. Correlation analysis showed that the Margalef index of foraminiferal community was significantly positively correlated with temperature. However, the Shannon-Wiener index of foraminifera obtained from morphological and molecular data had no significant correlation with temperature. The abundance of hyaline foraminifera and agglutinated foraminifera decreased significantly with increasing temperature. The reads of hyaline foraminifera and agglutinated foraminifera increased with increase of temperature, but the correlation test was not significant. The reads of porcelanous foraminifera and single-chambered foraminifera at different stations varied with temperature. The dominant species obtained based on the morphology were as follows: Ammonia aomoriensis, A. beccarii, Buccella frigida, Cribrononion subincertum, and Trochammina inflata. The dominant species obtained from the molecular method were not consistent with those from the morphology, and different dominant species at different stations showed diverse responses to temperature. This study revealed that temperature significantly affected the abundance, diversity, shell type, and dominant species of the foraminiferal community. After correlation analysis and function fitting, the linear relationships between foraminiferal abundance, Margalef index, abundance of different shell types, and some dominant species and temperature were established. It will provide first-hand data and function reference for the reconstruction of palaeotemperature and palaeo-ocean. In addition, the presence of small cryptic species and single-chambered foraminifera (most without shell) could be detected by molecular methods, which resulted in the increase of species and diversity of foraminifera obtained based on the molecular methods. Moreover, the differences in the species composition resulted in different responses of foraminifera to temperature between morphological and molecular methods.

4. A study to explore the response of foraminifera to cold seep (low temperature and low oxygen environment) using high-throughput sequencing technology

Cold seep is a kind of rare deep-sea system, which is closely related to the distribution of hydrocarbon resources (such as natural gas and oil) in the ocean. Benthic foraminifera have attracted much attention as a potential indicator of cold seep activity. However, almost all the studies on foraminifera in cold seep area are limited to traditional morphological classification. In this experiment, we collected sediment samples from the 4 stations (SY186-1, SY186-3, SY186-5, and SY186-7; depth > 1380m) in the “Haima” cold seep area. The total DNA was extracted and a region of the small subunit (SSU) rRNA gene was amplified with the foraminiferal specific primers. Then we analyzed the molecular diversity and community composition of foraminifera in the cold seep.

In total, 3,109,553 Effective tags and 432 OTUs were gained after strict quality control. The results showed that the foraminiferal community parameters (reads, OTUs, Margalef index, and Shannon-Wiener index) at SY186-1 and SY186-3 (closer to the cold seep activity center) were higher than those at SY186-5 and SY186-7 (farther away from the center). In addition, we found that the majority of foraminifera were Rotaliida (>85%), followed by a small amount of Textulariida and Monothalamida, without Miliolida (Identity value > 97.7%). Moreover, three ecological foraminiferal groups were found: cold-water benthic foraminifera, denitrifying benthic foraminifera, and planktonic foraminifera. Our study revealed that low temperature and low oxygen conditions in the cold seep center caused foraminiferal reads and diversity higher than that in the non-seep sites; The diversity of foraminifera in the cold seep was higher than that in the general sea area but lower than that in some high nutrient zone; The cold seep foraminifera are characterized by high proportion of Rotaliida; Denitrifying foraminifera is a characteristic group in the cold seep, among which Uvigerina is the most common foraminifera. Thus, foraminifera can be used as an effective environmental indicator of cold seep.

5. A study to explore the response of foraminifera to subarctic area (low temperature environment) using high-throughput sequencing technology

The arctic ocean is an important part of the global marine climate system, which is of great significance for the study of global climate change (e.g. global warming). However, our understanding of the ocean's past and present is still very limited. Calcareous foraminifera are considered to be one of the most useful proxies to reconstruct the marine paleoenvironment of the arctic ocean. In order to investigate the molecular diversity and community composition characteristics of foraminifera in subarctic sediments, total genomic DNA was extracted from surface sediments collected from 11 stations in the subarctic. Then the foraminiferal DNA was amplified using foraminiferal specific primers.

After strict quality control process and data processing, a total of 2,170,762 effective tags and 1,046 OTUs were obtained in the experiment. The result showed BR10 had the highest reads of foraminifera. The highest values of foraminiferal Margarlef index and Shannon-Wiener index were obtained at BR06. The spearman correlation test showed that the foraminiferal community parameters (reads, OTU, Margarlef index, and Shannon-Wiener index) had no significant correlation with environmental factors (temperature, salinity, and water depth). In the foraminiferal community, hyaline foraminifera accounted for the highest proportion, followed by agglutinated and single-chambered foraminifera, then porcelanous foraminifera accounted for the lowest proportion. The results indicated that the molecular diversity of foraminifera in the subarctic was lower than those in other areas. High proportion of hyaline foraminifera and low proportion of porcelanous foraminifera were the important characteristics of subarctic foraminifera. Nonionella labradorica was the characteristic dominant species of the subarctic foraminifera, with the genera Reophax, Globobulimina, and Elphidium as the characteristic groups.