海洋生态与环境科学重点实验室知识库

海洋生物的谱系地理格局与群体遗传结构受其分布范围内地质历史、地貌、水文环境、物种自身生物学特性等多重因素的影响。西北太平洋具有丰富的边缘海结构、独特的地质历史以及复杂的水文环境，其沿岸的潮间带复杂多变，既有广阔的泥质浅滩，也有丰富多样的岩相环境；西北太平洋纬度跨度大，不同气候类型海域间存在明显的环境异质性；以上因素对西北太平洋广布海洋生物的种下水平的演化历史和群体间的基因交流，以及不同地理种群对本地环境适应性的产生均具有重要影响。此外，潮间带是人类活动和全球气候变化影响下的敏感区域，部分潮间带海洋生物的分布范围在人类活动和全球气候变化得影响下发生了改变。因此，西北太平洋为海洋生物谱系地理学和群体遗传学研究提供了理想的天然实验室。短滨螺（Littorina brevicula, Philippi, 1844）是西北太平洋沿岸岩相潮间带生境的广布种，其分布范围内存在明显的环境异质性，既包括不同纬度条件下的温度差异，也包括不同沿岸栖息地的盐度差异，因此是研究西北太平洋海洋生物谱系地理格局、群体遗传结构及潮间带生物本地适应性进化机制的理想物种。

本研究以西北太平洋环境梯度下分布的30个短滨螺地理群体为研究对象，基于线粒体ND6（mtND6）、微卫星标记（SSR）以及全基因组水平的单核苷酸多态性（SNP）三种分子标记技术，查明了短滨螺分布范围内自然群体的谱系地理格局以及群体遗传结构；通过长江三角洲短滨螺新形成群体的溯源分析深刻了我们对冬季长江口与苏北水域间的生态连通性的认知；探讨了环境梯度下不同地理群体本地适应性分化的机制及相关的环境驱动因子；主要研究结果如下：

1、查明了西北太平洋短滨螺群体谱系地理格局及群体遗传结构，推测了短滨螺群体的演化历史。

完成了我国沿岸短滨螺自然群体mtND6全长的扩增测序，并结合已发表的日本沿岸短滨螺群体数据开展了谱系地理分析，结果显示短滨螺分布范围内存在两个以长江三角洲为分界的具有显著分化的谱系，其中谱系Ⅰ主要分布在长江三角洲以北的中国和日本沿岸的自然群体，谱系Ⅱ分布在长江口以南的中国自然群体，提示末次冰期海平面上升后长江三角洲不适宜的生境导致长江三角洲南北的自然群体间基因流中断，产生谱系分化。群体遗传结构的分析结果显示：西北太平洋沿岸短滨螺群体可分为四个遗传组群，其中日本和韩国沿岸的群体为一个组群（日本组群）；除青岛、日照和连云港之外的中国北方群体为一个组群（中国北方组群）；青岛、日照和连云港三个群体为一个组群（青岛组群）；长江口以南的中国南方群体为一个组群（中国南方组群）。长江三角洲浅滩导致的栖息地不连续性；边缘海之间交流，沿岸流、涡旋等洋流系统对群体间基因流的限制等是影响西北太平洋短滨螺群体遗传结构的主要因素。群体演化历史分析显示中国北方组群最为古老，中国南方组群和日本组群先后分化出来，最后青岛组群从中国北方组群分化出来。此外，群体历史动态分析结果显示短滨螺群体大约从20,000年前开始出现了明显的群体扩张，可能与末次冰期之后海平面上升形成大量新的适宜岩相潮间带生物的生境、短滨螺群体对新生境的快速拓殖导致种群的分布范围及数量的快速增长相关。

2、对长江三角洲沿岸短滨螺新形成群体进行了溯源分析，发现长江口与苏北水域在冬季存在很强的连通性。

本部分选取了长江三角洲沿岸6个新形成群体以及长江三角洲以北和长江口以南各3个自然群体进行分析。单倍型网络图构建结果显示12个群体可被分为以北纬33.5度为界线的两个具有显著分化的谱系，其中谱系Ⅰ主要分布在北纬33.5度以北的五个群体，谱系Ⅱ主要分布在北纬33.5度以南的7个群体。对新形成群体的个体进行溯源分析发现，北纬33.5度以北的两个群体中有94%（45）的个体来源于北方自然群体，北纬33.5度以南的4个群体中有98%（96）的个体来源于南方自然群体。这暗示了冬季西南黄海存在北向的沿岸流，且影响范围向北可达北纬33.5度左右的水域。遗传结构分析呈现出和谱系分析一致的结果，即以北纬33.5度为界，两侧的群体分别成组群，两个组群间存在显著的遗传分化。本研究结果首次为冬季西南黄海存在北向的沿岸流提供了遗传学证据，并查明了该沿岸流的影响范围向北可达北纬33.5度左右的水域。这也暗示了冬季长江口会作为西南黄海生态系统重要的物质和能量输入来源，为黄海生态系统的管理和保护提供了可参考的科学依据。

3、解析了短滨螺群体间的精细遗传结构，初步探讨了西北太平洋不同海域的短滨螺地理群体对本地环境的适应性进化的遗传机制。

基于重测序技术，获得了短滨螺8个地理群体的群体基因组学数据。群体遗传结构分析显示8个自然群体可分为7个遗传组群，其中我国的舟山和厦门群体为一个组群，其它群体均单独成组群。适应性分析结果显示：多个与盐度适应（如SLC16a13等）、渗透压调节（如AQP等）及温度适应（如HSP70等）等相关的功能基因被注释到，同时GO注释分析也发现多个与能量代谢、渗透压调节、温度适应等重要生理过程相关的基因集，提示西北太平洋短滨螺的不同地理群体对温度及盐度等环境因子的本地适应性进化。

综上，本论文采用三种分子标记技术，查明了西北太平洋短滨螺群体的谱系地理格局、群体遗传结构，并探讨了其本地适应性机制。相关研究结果丰富了我们对西北太平洋海洋生物演化历史、群体遗传结构及相关影响因素的认知，为西北太平洋潮间带生物谱系地理格局和群体遗传结构的研究提供了可参考资料；从分子生态学角度提示了长江口是冬季西南黄海水域重要的物质和能量来源，为黄海生态系统的管理和保护提供科学依据；同时对深入理解海洋生物群体对本地环境适应性分化的遗传机制具有重要的科学意义。

The phylogeographic history and population genetic structure of the marine species are influenced by multiple factors including geological history, geomorphology, hydrological environment, and the biological characteristics etc. The northwestern Pacific is characterized by a series of marginal seas, unique geographical history and complex hydrological environments. The intertidal habitats of northwestern Pacific are diverse, including wide mudflat and variety of rocky intertidal zone. In addition, there are latitudinal environment heterogeneity among marginal seas of the northwestern Pacific. All factors might have played important roles in intraspecific evolutionary history, gene flow, and adaptation of geographical population to local environments for species widely distributed in the northwestern Pacific. Furthermore, the intertidal habitats of northwestern Pacific have been influenced by athropogenic activities and global change, and the distribution of some intertidal species have been changed. Hence, the northwestern Pacific provides a natural laboratory for studying phylogeography and population genetics of marine species. The gastropod, Littorina brevicula (Philippi, 1844) is a common periwinkle snail widely distributed in the rocky littoral fringe of the temperate coast of the Northwestern Pacific. There are obvious environmental heterogeneity in the distribution range of L. brevicula, such as temperature regime at different latitudes and salinity in different ocean areas. Considering its wide distribution range in the intertidal rocky habitat of northwestern Pacific, L. brevicula is an ideal species for the study of phylogeographic history, population genetic structure, and genetic mechanism of local adaptation.

Here we collected samples of L.brevicula from 30 localities in the northwestern Pacific, by using mitochondrial ND6 gene sequence, microsatellite and genome-wide SNP markers, we clarified the phylogeographic pattern and population genetic structure of L.brevicula across its range of distribution; the connectivity between the Yangtze River Estuary (YRE) and the Subei coastal water was explored by the genetic assignment/exclusion test of newly colonized population on the Yangtze River Delta (YRD); we also investigated the genetic mechanisms of local adaptation of geographical populations and discussed possible environment factors driving adaptation for different populations. Major findings of the present study are shown below:

1) The phylogeographic pattern and population genetic structure of L. brevicula were clarified, and population demographic history of L. brevicula were investigated.

The complete mtND6 sequence was amplified and sequenced for natural population along coast of China, and phylogeographic analyses were conducted by combining previously published mtND6 data of natural population around Japan. Clear phylogeographic and genetic differentiation were detected between natural rocky populations south and north of the YRE, which resulted from the lack of hard substrate for rocky invertebrates in the large YRD coast after the Last Glacial Maximum (LGM). The result of genetic structure analysis based on microsatellites demonstrated that the populations of L. brevicula in the Northwestern Pacific could be divided into four genetic clusters, populations from the coast of Japan and Korea (hereafter Japan cluster), populations from the northern China but excluding Qingdao, Rizhao and Lianyungang (hereafter northern cluster of China), populations of Qingdao, Rizhao and Lianyungang (hereafter Qingdao cluster), and populations south of YRE (hereafter southern cluster of China). Habitat fragmentation the lack of hard substrate for rocky invertebrates in the large YRD and the restriction of gene flow caused by ocean current system are crucial factors shaping genetic structure of L. brevicula. Evolution history analysis suggested that the northern cluster of China was the ancestry cluster, the southern cluster of China and the Japan cluster derived from the Japan cluster successively, and the Qingdao cluster derived from the northern cluster of China at last. Population demographic analysis showed that populations of L. brevicula experienced expansion from about 20,000 years ago, probably due to the rapid growth of population sizes and the expansion of their distribution range accompanied by availability of new habitats due to the rise of sea level after the LGM.

2) The source of new colonized populations on the coast of YRD were explored by genetic assignment/exclusion tests, and results demonstrated strong ecological connectivity between the YRE and the Subei coastal water in winter time.

We investigated ecological connectivity between the YRE and inner southwestern Yellow Sea in wintertime by precisely pinpointing the source of six newly colonized populations of the winter-spawning rocky intertidal invertebrate, L. brevicula, on artificial structures along the coast of the Yangtze River Delta (YRD) using mitochondrial ND6 sequences and microsatellite data. Most individuals (98%) to the south of ~33.5°N were from natural rocky populations to the south of the YRE and most of those (94%) to the north of ~33.5°N were from the northern natural rocky populations, which demonstrated strong ecological connectivity between the inner southwestern Yellow Sea and the YRE in winter time. We presented the first genetic evidence that demonstrated a northward wintertime coastal current in the inner southwestern Yellow Sea, and precisely illustrated the boundary of the coastal current recently proposed by numerical experiment. These results indicated that the YRE serves as an important source of materials and energy for the inner southwestern Yellow Sea in winter, which can be crucial for the function of the Yellow Sea ecosystem.

3) By using population genomic approaches, the population genetic structure and genetic mechanisms underlying adaptive evolution to local environments of L. brevicula populations were clarified.

Genome-wide population genomic dataset of eight L.brevicula geographic populations were obtained based on whole genome sequencing. Population genetic structure analyses showed that eight natural populations should be divided into seven genetic clusters. One cluster was formed by Zhoushan and Xiamen populations, and each of the rest populations formed a separate cluster respectively. The analysis of local adaptation found multiple functional genes related to salinity adaptation (e.g. SLC16a13), osmotic pressure regulation (e.g. AQP) and thermal adaptation (e.g. HSP70). Meanwhile, GO annotation analysis also revealed multiple GO terms that involved in important physiological processes such as energy metabolism, osmotic pressure regulation, and temperature adaptation, which demonstrated local adaptation to salinity and thermal regime of different populations in the northwestern Pacific.

In summary, by using different molecular approaches, we investigated the phylogeographic history, population genetic structure, and the mechanisms of local adaptation of L. brevicula in the northwestern Pacific. These results will broaden our understanding of the intraspecific evolutionary history, population genetic structure, and the underlying driving factors of marine species. Our results indicated that the YRE serves as an important source of materials and energy for the inner southwestern Yellow Sea in winter, which will be useful for the management and protection of the Yellow Sea ecosystem. At last, our understanding of the genetic mechanism of adaptation of L. brevicula to local environments could provide insights into the way marine organism coping with global climate change in the future.