海洋生物分类与系统演化实验室知识库

广义上的帘蛤科贝类（帘蛤科sensu lato，以下简称帘蛤科s.l.）是海洋双壳贝类中经济价值较大、多样化水平较高的类群。由于其分布范围极广、生境类型多样、形态变异较大，导致依据传统形态分类学建立的帘蛤科s.l.分类系统和系统演化关系存在较大争议，这阻碍了其他相关研究的进展。本研究利用现代分子生物学技术，通过测定我国近海常见帘蛤科s.l.部分核基因及线粒体基因组的序列，同开放数据库（NCBI等）中已有帘蛤科s.l.分子序列进行比对，构建分子系统树，查明其各亚科间的系统演化关系，并对我国近海分布的部分帘蛤科s.l.疑难属种的分类学地位进行修订和完善，以构建更为准确的帘蛤科s.l.贝类分类体系。主要结论如下：

1. 帘蛤科s.l.的线粒体基因组特点。线粒体基因组序列长度为16,856 bp（皱纹蛤Periglypta puerpera）至22,676 bp（菲律宾蛤仔Ruditapes philippinarum），具有37~39个功能基因，包括13个蛋白编码基因、2个rRNA和22 ~ 24个tRNA；所有功能基因均编码在正链，并表现出明显的AT富集，AT含量为62.5%~74.9%；蛋白编码基因和tRNA均表现出明显的T偏好（AT-skew＜0），全部基因表现出明显的G偏好（GC-skew＞0）。帘蛤科贝类线粒体基因组基因排列顺序的保守程度与系统发育关系的远近相关：单系发生的属中物种的基因排列顺序几乎完全一致，tRNA的数量差异仅在伊萨伯雪蛤Placamen isabelline、墨氏巴非蛤Paphia motsei、短文蛤Meretrix petchialis和琴文蛤Meretrix lyrata 等4个物种中发现；单系发生的亚科（除缀锦蛤亚科）中物种倾向于共享相同的蛋白编码基因排列顺序。发现cox1-trnl1-nad1-nad2-nad4l-trnI-cox2-trnP-cytb-rrnl-atp8-nad4-trnH-trnE-trnS2-atp6-nad3-nad5……nad6……cox3是最接近帘蛤科祖先的基因排列顺序。本研究首次发现杂色蛤仔Ruditapes aspera线粒体双单亲遗传现象存在。

2. 帘蛤科s.l.系统演化关系及分类修订。基于多基因短片段（CB）和线粒体全基因组序列（MT）分别构建的帘蛤科贝类系统演化树显示：广义上的帘蛤科贝类分成了两个支持度较高的主要支系，即支系A和支系B；支系A共包括四个亚科，即雪蛤亚科Chioninae、镜蛤亚科Dosiniinae、缀锦蛤亚科Tapetinae和帘蛤亚科Venerinae，其中，雪蛤亚科表现为单系发生，而镜蛤亚科、缀锦蛤亚科和帘蛤亚科均在MT树中为支持度较高的单系群，但在CB树中分成了两个或多个支系；支系B共包括10个亚科，即仙女蛤亚科Callistinae、和平蛤亚科Clementiinae、青蛤亚科Cyclininae、芽蛤亚科Gemminae、美女蛤亚科Gouldiinae、文蛤亚科Meretricinae、住石蛤亚科Petricolinae、卵蛤亚科Pitarinae、楔形蛤亚科Sunettinae和蚤蛤亚科Turtoninae，其中，仙女蛤亚科在CB树和MT树中均为多系群，美女蛤亚科在CB树和MT树中均为单系群，文蛤亚科在MT树中表现为单系群，但在CB树中表现为多系群。其它亚科没有线粒体基因组数据，只能探讨其在CB树种的演化关系。和平蛤亚科和卵蛤亚科在CB树中表现为多系群，芽蛤亚科、住石蛤亚科和楔形蛤亚科在CB树中表现为单系群。

基于系统演化研究结果，对帘蛤科贝类分类系统进行修订，建议将广义上的帘蛤科分成两个科：支系A为狭义上的帘蛤科（下称帘蛤科s.s.），典型特征为壳表通常具有形态变化较大的同心肋；支系B为文蛤科Meretricidae，典型特征为壳表通常光滑或有较弱的同心刻纹。对常见帘蛤科贝类的分类地位进行了修订和完善，主要包括：雪蛤属Placamen应归为镜蛤亚科；皱纹蛤属Periglypta应归为帘蛤亚科；凸卵蛤属Pelecyora应归为卵蛤亚科；将光壳蛤属Lioconcha和齿纹卵蛤属Hyphantosoma归入美女蛤亚科；认为日本闭壳蛤Claudiconcha japonica不属于住石蛤亚科，其亚科归属需要进一步研究；将裂纹格特蛤和日本格特蛤归为缀锦蛤属中，并更名为裂纹缀锦蛤Tapes hiantina和日本缀锦蛤Tapes japonica；将鳞杓拿蛤归为帝汶蛤属Timoclea，拉丁名更改为Timoclea squamosus，同时将杓拿蛤属Anomalodiscus降为帝汶蛤属的亚属；将曲波提加芒蛤（曲波皱纹蛤）归为对角蛤属，中文名修改为曲波对角蛤Antigona chemnitzi，同时认为提加芒蛤亚属应归为对角蛤属；将短管仙女蛤Ezocallista brevisiphonata归为紫石房蛤属，并更名为短管石房蛤Saxidomus brevisiphonata，同时将虾夷仙女蛤属Ezocallista降为石房蛤属的亚属。同时，本研究认为雪蛤亚科、帘蛤亚科、仙女蛤亚科和卵蛤亚科均为独立有效的亚科，皱纹蛤属为独立有效的属，杂色蛤仔和菲律宾蛤仔应隶属于2个不同属，加夫蛤Gafrarium pectinatum和凸加夫蛤Gafrarium tumidum为两个独立有效的物种。此外，本研究还记录了翘鳞蛤属1新记录种（石桥翘鳞蛤Irus ishibashianus）和1未定种（Irus sp.）。

3. 帘蛤科sl.贝类分化及生物地理历史分析。利用分子钟工具推算帘蛤科不同支系分化时间，结果显示，帘蛤科Veneridae s.s.起源于约287.24 Mya（Million years ago, 百万年前）的二叠纪时期，于254.47 Mya开始分化形成两个主要支系，其中Clade A（帘蛤科s.s.）大约于218.36 Mya开始分化，Clade B（文蛤科）大约于234.77 Mya开始分化。化石数据分析结果显示，在160~110 Mya（侏罗纪晚期到白垩纪早期）、80~70 Mya（白垩纪中晚期）和60~20 Mya（古近纪早期到新近纪早期）三个时间段，帘蛤科中属级类群的多样性明显增加。其中，和平蛤亚科、楔形蛤亚科和芽蛤亚科的化石记录均在古新世（66 Mya）之后出现；在古新世和始新世（66-33.9 Mya）时期，广义上的卵蛤亚科的属级类群数量最多；渐新世之后（23.03-2.5 Mya）帘蛤亚科和雪蛤亚科的属级类群多样性明显增多。

新生代时期，在生物多样性热点区，属级类群多样性水平明显提升，并随生物多样性热点区的迁移而发生地域性改变。本研究推测，在中新世时期，特提斯海区域属级类群多样性的变化与特提斯海峡的阶段性关闭有关；渐新世之后大西洋-东太平洋领域两侧属级阶元多样性的结构变化可能与洋流方向变化有关。

The family Veneridae sensu lato (Veneridae s.l.) is one of the most economical and diverse groups in marine bivalves. Since Veneridae s.l. species have a wide distribution, various habitats and highly varied morphological characters, which hindered the development of related research. The taxonomic infrastructure and phylogenetic relationships within Veneridae s.l. have long been controversial. Here, modern molecular biology techniques were used in this study to sequence the nuclear fragments and complete mitochondrial genomes in common venus clams from coastal China. The sequences were then analyzed and compared with that published in the open database (like NCBI) before phylogenetic trees construction. This study aims to find out the relationships between subfamilies of Veneridae s.l. The classifications of some dubious genera and species of venus clams from China were confirmed and modified. We hope to build a more accurate and convincing taxonomic infrastructure for the Veneridae s.l. The main findings of this study were listed as follows.

1. The characters of mitogenomes of Veneridae s.l. The length of the complete mitochondrial genomes arranges from 16,856 bp to 22,676 bp, with the shortest one appearing in Periglypta puerpera and the longest one in Ruditapes philippinarum. The mitochondrial genomes consist of 13 protein-coding genes (cox1-3, nad1-6, nad4l, cytb, atp6, and atp8) and 2 rRNAs (rrnl and rrns). The quantities of tRNA in the mitochondrial genomes vary from 37 to 39. All the functional genes are encoded in the forward strand and AT-biased, with AT content ranging from 62.5% to 74.9%. All the protein-coding genes and tRNAs are T-biased (AT-skew<0), while all the functional genes are G-biased (GC-skew>0).

The conservation in gene orders of mitochondrial genomes in Veneridae s.l. corresponds to the phylogenetic relationship. For the species in the monophyletic genus, the gene orders are almost identical, except for variation of quantities of tRNAs in 4 species (Placamen isabelline, Paphia motsei, Meretrix petchialis, and Meretrix lyrata); species of monophyletic subfamilies tend to share the same protein-coding gene order. Besides, the gene order, which is thought to be the most related to ancestors, is proposed as cox1-trnl1-nad1-nad2-nad4l-trnI-cox2-trnP-cytb-rrnl-atp8-nad4-trnH-trnE-trnS2-atp6-nad3-nad5……nad6……cox3. In this study, we find the Doubly Uniparental Inheritance in Ruditapes aspera.

2. The phylogenetic relationships and the classification revision in Veneridae s.l. Two phylogenetic trees are built based on multi-gene fragments (CB tree) and mitochondrial genomes (MT tree). The result shows that species of Veneridae s.l. are divided into two clades, Clade A and Clade B, as described in this study. There are four subfamilies in Clade A, Chioninae, Dosiniinae, Tapetinae, and Venerinae. Subfamily Chioninae is monophyletic in both trees, and the other three subfamilies are monophyletic with high supports in the MT tree but polyphyletic in the CB tree. There are ten subfamilies in Clade B: Callistinae, Clementiinae, Cyclininae, Gemminae, Gouldiinae, Meretricinae, Petricolinae, Pitarinae, Sunettinae, and Turtoninae. The Callistinae is polyphyletic in both trees, while Gouldiinae is monophyletic. Meretricinae is monophyletic in the MT tree, but polyphyletic in the CB tree. There is a lack of mitochondrial genomes in the other subfamilies, and the phyletic status can only be observed in the CB tree. Clementiinae and Pitarinae are polyphyletic, while Gemminae, Petricolinae, and Sunettinae are monophyletic groups.

The taxonomic system of Veneridae s.l. is modified based on the phylogenetic results. We propose to divide Veneridae s.l. into two families: the Clade A is named Veneridae sensu stricto (Veneridae s.s.), with the typical morphological character being highly varied concentric sculpture; the Clade B is named Meretricidae, with the typical morphological being that the surface is smooth or with weak sculpture. Some classifications of venus clams are modified as follows. Firstly, Placamen is placed into Dosiniinae, Periglypta is placed into Venerinae, and Pelecyora is put into Pitarinae. Secondly, Lioconcha and Hyphantosoma are placed into Gouldiinae and we synonymize Lioconchinae by Gouldiinae. Thirdly, Claudiconcha japonica should not be included in Petricolinae, but the accurate position should be further studied. Fourthly, two Macira species, Macira hiantina and Macira japonica are actually Tapes species and they are renamed as Tapes hiantina and Tapes japonica. Fifthly, the genera of Anomalodiscus squamosus and Tigammona chemnitzi are changed to Timoclea and Antigona, and Anomalodiscus and Tigammona are considered as a subgenus of Timoclea and Antigona, respectively. Sixthly, Ezocallista is adopted as a subgenus of Saxidomus, and thus, Ezocallista brevisiphonata is renamed Saxidomus brevisiphonata. Meanwhile, we confirm that Chioninae, Venerinae, Callistinae and Pitarinae are independent and available subfamilies, Periglyta is an independent and available genus, Ruditapes aspera and Ruditapes philippinarum are actually not congeneric species and Gafrarium tumidum is not supported as the synonym of Gafrarium pectinatum. Besides, we described a new record species (Irus ishibashianus) and an undefined species (Irus sp.) in this study.

3. Divergence and biogeographic history analysis of Veneridae s.l. The divergence time of lineages within Veneridae is estimated by means of the molecular clock. The result shows that Veneridae s. novo originated in Permian, about 287.24 Mya (Million years ago) and the two main clades were formed in about 254.47 Mya, and diverged in 218.36 Mya and 234.77 Mya, respectively. Fossil records further indicate that the genus-level quantity increased during the late Jurassic to Cretaceous (160~110 Mya), middle and late Cretaceous (80~70 Mya), and from early Paleogene to early Neogene (60~20 Mya). The fossils of Clementiinae, Sunettinae and Gemminae were recorded after Paleocene (66 Mya), and Pitarinae s.l. had the most abundant genera during Paleocene and Eocene (66~33.9 Mya). The genera quantities of fossils from Chioninae and Venerinae increased after Oligocene (23.03~2.5 Mya).

In the Cenozoic era, the distribution variation of genera in Veneridae indicated the migratory route of biodiversity hot spots. We found that the variation of genera in the Tethys Sea was related to the periodic closure of the Tethys Strait during the Miocene period. And the variation of genera in both sides of the Atlantic-Eastern Pacific realm after the Oligocene was likely to be related to the varied direction of the ocean currents.

第1章  研究背景... 1

1.1  帘蛤科贝类概况... 1

1.1.1  帘蛤科贝类形态特征... 2

1.1.2  帘蛤科贝类生态习性与分布... 5

1.2  帘蛤科贝类分类学研究... 6

1.2.1  帘蛤科贝类分类学研究国际概况... 6

1.2.2  帘蛤科贝类分类系统国内研究概况... 13

1.2.3  帘蛤科贝类分类系统争议... 16

1.3  帘蛤科贝类系统演化研究... 17

1.3.1  帘蛤科贝类系统演化国际研究概况... 17

1.3.2  帘蛤科贝类系统演化国内研究概况... 23

1.4  线粒体基因组在双壳贝类系统演化中的应用... 23

1.4.1 双壳贝类线粒体基因组结构特征... 23

1.4.2 双壳贝类线粒体基因组遗传和演化特征... 24

1.5  本研究的目的和意义... 26

第2章  材料与方法... 27

2.1  实验材料... 27

2.2  基因组DNA提取... 28

2.3  基因片段扩增与测序... 28

2.4  线粒体基因组测序、组装及注释... 38

2.4.1  高通量测序... 38

2.4.2  线粒体基因组组装... 38

2.4.3  线粒体基因组注释及分析... 40

2.5  系统发育分析... 41

2.6  帘蛤科分化时间和物种多样性推断... 42

第3章  结果... 44

3.1  帘蛤科贝类短片段及线粒体基因组特征... 44

3.1.1  帘蛤科贝类短片段特征... 44

3.1.2  帘蛤科线粒体基因组结构特征... 45

3.1.3  蛋白编码基因选择压力分析... 48

3.1.4  线粒体基因排列顺序... 49

3.2  帘蛤科贝类系统发育分析... 52

3.2.1  支系A的系统发育分析... 53

3.2.2  支系B的系统发育分析... 55

3.3  帘蛤科贝类分化时间及多样性研究... 58

3.3.1  帘蛤科贝类分化时间... 58

3.3.2  帘蛤科贝类多样性历史变化... 61

3.4  帘蛤科s.l.部分疑难属种的分类学研究结果... 63

3.4.1  缀锦蛤亚科Tapetinae部分疑难属种分类学研究结果... 63

3.4.2  帘蛤亚科Venerinae部分疑难属种分类学研究结果... 71

3.4.3  闭壳蛤属Claudiconcha分类学研究结果... 72

3.4.4  仙女蛤亚科Callistinae疑难属种分类学研究结果... 73

3.4.5  美女蛤亚科Gouldiinae疑难属种分类学研究结果... 74

第4章  讨论... 78

4.1  帘蛤科贝类线粒体基因组研究... 78

4.1.1  蛋白编码基因选择压力分析... 78

4.1.2  线粒体基因排列顺序... 79

4.2  帘蛤科s.l.贝类系统发育研究... 80

4.3  帘蛤科s.l.物种多样性历史变化研究... 82

4.4  帘蛤科s.l.的分类修订... 83

4.4.1  帘蛤科Veneridae s.s的分类修订... 84

4.4.2  文蛤科Meretricidae的分类修订... 93

第5章  结论与展望... 97

参考文献... 105

附  录... 120

致谢... 142

作者简历及攻读学位期间发表的学术论文与研究成果... 143