中国科学院海洋研究所机构知识库

蛰龙介亚目（Terebelliformia）隶属于环节动物门（Annelida），多毛纲 （Polychaeta），蛰龙介目（Terebellida），是一类营管栖生活的以有机碎屑为食的沉积食性底栖蠕虫。蛰龙介亚目物种丰富，迄今已发现的物种超过1100种。它们分布广泛，从浅海潮间带到深海均有记录，其中阿尔文虫科（Alvinellidae）和双栉虫科（Ampharetidae）的一些物种可以生活在鲸落和深海热液等极端环境中。

蛰龙介亚目物种身体柔软且形态多变，在分类鉴定中存在很多困难，例如一些口触手和鳃丝等鉴别特征容易被破坏，双栉虫科的鉴定缺少明显的量化特征，早期的物种记录缺少详细描述，有的种模式标本丢失，造成了分类鉴定的困难甚至混肴。蛰龙介亚目目前共有6个科，但是科之间的系统发育关系尚存在很多争议。例如，米列虫科（Melinnidae）曾经是双栉虫科的亚科，毛鳃虫科（Trichobranchidae）与蛰龙介科（Terebellidae）和双栉虫科的亲疏关系以及蛰龙介科各亚科的划分，都存在问题。

本研究利用传统形态分类学和分子生物学相结合的方法，对中国海蛰龙介科扁蛰虫属（Loimia）和双栉虫科扇栉虫属（Amphicteis）物种进行了修订；利用高通量测序得到蛰龙介亚目3科共8个物种的线粒体基因组,分别分析了其结构组成并利用比较基因组学方法对其差异进行了分析；结合GenBank上蛰龙介亚目物种的线粒体基因数据，使用贝叶斯法和最大似然法分析不同线粒体基因组的不同数据集，重建了蛰龙介亚目内部的系统发育关系。主要结论如下：

1. 鉴定出中国沿海分布的扁蛰虫属物种5种，包括2个新物种：巨鳃扁蛰虫Loimia macrobranchia Wang, Sui, Kou & Li, 2020和北方扁蛰虫Loimia borealis Wang, Sui, Kou & Li, 2020。其中北方扁蛰虫鉴别特征是其独特的腹腺垫结构，在分类上首次提出腹腺垫特征是扁蛰虫属重要的鉴别特征；巨鳃扁蛰虫区别于该属其它物种的鉴别特征主要是巨大的第1对鳃。通过比较，确认以前中国海记录的扁蛰虫L. medusa（Savigny，1822）为错误鉴定，该物种在中国海没有分布。带扁蛰虫L. bandera Hutchings，1990和树扁蛰虫L. arborea Moore，1903以前仅在中国海生态学文献中被记录过，没有鉴别特征的描述，本研究依据采集到的新鲜标本，为两个物种提供了详尽的形态学描述。本研究还结合GenBank上该属物种序列和本研究获得的物种序列（COI、16SrRNA、18SrRNA）对该属物种进行了分子系统学分析，遗传距离和分子系统学结果支持了在形态学上将中国海采集到的样品分为5个物种的结论，即：树扁蛰虫、精巧扁蛰虫Loimia ingens（Grube，1878）、带扁蛰虫、北方扁蛰虫和巨鳃扁蛰虫，也否定了以前文献中认为中国海域的精巧扁蛰虫为复合种的假设。

2. 鉴定出中国沿海分布的扇栉虫属物种6种，包括1个新物种：黄海扇栉虫Amphicteis hwanghaiensis Wang, Sui, Li, Hutchings & Nogueria, 2020。前人仅对该属的扇栉虫Amphicteis gunneri （Sars，1835）和蔽扇栉虫A. obscurior Chamberlin，1919进行了详细的描述，本文作为补充，基于采集的新鲜标本，对中国海其他物种（无毛扇栉虫A. glabra Moore，1905、扁鳃扇栉虫A. scaphobranchiata Moore，1906、中华扇栉虫A. chinensis Sui & Li，2017）作了详细描述。首次发现中国沿海该属的物种可以根据稃刚毛的形态分为两组，即稃刚毛末端钝圆型和末端尖细型。

3. 分别获得了蛰龙介科4个物种（带扁蛰虫，吻蛰虫Artacama proboscidea Malmgren，1866；树扁蛰虫；似蛰虫Amaeana occidentalis（Hartman，1944））、双栉虫科3个物种（中华副栉虫Paramphicteis sinensis（Sui & Li，2014）；细沟栉虫Anobothrus gracilis（Malmgren，1866）；短须双栉虫Ampharete lindstroemi Malmgren，1867）和米列虫科1个物种（泥米列虫Melinna elisabethae McIntosh, 1914）的线粒体基因组。分析结果表明，（1）蛰龙介科4个物种线粒体基因组和GenBank中毛鳃虫科线粒体基因组的基因排列顺序完全一致，包括13个蛋白编码基因（PCGs）、2个rRNA基因（rmL和rmS）和23个tRNA基因，都存在一个trnM基因复制，暗示这两科的关系较近。4个物种基因组总长度为15797-16304bp，蛋白编码基因长度保守，都具有较高的A+T含量，在密码子使用上对于NNU和NNA这类密码子具有强烈的偏好性。转运RNA结构为典型的三叶草型，trnS1基因的DHU臂都发生了缩短或缺失现象。控制区位置都位于trnM和trnH基因之间，但是各物种控制区的长度和结构有所不同。（2）双栉虫科3个物种的长度为10980bp-11022bp，细沟栉虫没有得到全线粒体基因组，没有得到trnM和trnH基因的序列，另外两个物种都包含37个基因（13个PCGs+2个rRNA+22个tRNA）。蛋白编码基因长度保守，都具有较高的A+T含量，在密码子使用上对于NNU和NNA这类密码子具有强烈的偏好性，转运RNA结构为典型的三叶草型。（3）米列虫科物种的线粒体基因组为首次报道，也具有37个基因（13个PCGs+2个rRNA+22个tRNA），全长为16309bp，具有较高的A+T含量。rrnL位于trnV和trnL1基因之间，rrnS基因位于trnM和trnV基因之间，与蛰龙介科物种rRNA的位置相同。

4. 结合本研究测序获得的蛰龙介科、双栉虫科和米列虫科线粒体基因组以及GenBank中已公布的10种蛰龙介亚目线粒体基因组（蛰龙介科、双栉虫科、阿尔文虫科、笔帽虫科（Pectinariidae）和毛鳃虫科），对蛰龙介亚目各个科的系统发育关系进行了分析。结果显示蛰龙介亚目内部6个科分为三支，笔帽虫科单独一支位于基部，米列虫科和毛鳃虫科为姐妹类群，先聚为一支再与蛰龙介科聚为一支。阿尔文虫与双栉虫科关系较近聚为一支，但有的数据集重建的系统发育树不支持双栉虫科为单系群，其系统学地位有待进一步研究。

The suborder Terebelliformia, belonging to the order Terebellida of Polychaeta in Annelida is a group of tube-dwelling sedentary worms that are deposite feeders. Up to date, more than 1100 species have been recorded in this suborder. Terebelliforms are widely distributed, ranging from the intertidal zone to deep sea. Species of Alvinellidae and Ampharetidae were found in extreme environments such as whale falls and deep-sea hydrothermal vents.

There are a lot of difficulties in identification and classification of these diverse groups with soft body. For example, buccal tentacles and branchiae as important features are easy to damage; Species in Ampharetidae lack obvious quantitative traits for identification.

Early species records lacked detailed description and type materials were not well-preserved, which caused the existence of numerous monotypic genera. Currently, there are 6 families in Terebelliformia with many controversies about the phylogenetic relationship between the families. For example, the subordinate relationship between Melinnidae and Ampharetidae, the affinities of Trichobranchidae, Terebellidae and Ampharetidae, and validity of the subfamilies in Terebellidae has not been sufficiently investigated.

In this study, the Loimia species of Terebellidae from China were reviewed by combining traditional taxonomic and molecular biological methods. Species of the genus Amphicteis of Ampharetidae from China were reviewed and by traditional taxonomic methods. The mitochondrial genomes of 8 species from 3 families of Terebelliformia were obtained by next-generation sequencing. The structural variation of mitochondrial genomes among species of the 3 families were analyzed. Combined with mitochondrial genomes data from GenBank, Bayesian inference and Maximum likelihood methods were used to reconstruct phylogenetic relationship of the families depending on the different datasets.

The main conclusions are divided into the following four parts:

1. Five species of the genus Loimia including two new species Loimia macrobranchia Wang, Sui, Kou & Li, 2020, and Loimia borealis Wang, Sui, Kou & Li, 2020, have been identified from coastal areas of China. Among them, the unique structure of the ventral shields is the distinguishing feature of L. borealis. It was first proposed that the characteristic of the ventral shields is an important identification feature of the genus Loimia. Loimia macrobranchia differs from congeners by the shape of its large first pair of branchiae. The species L. medusa (Savigny, 1822), previously recorded in China sea, was proved did not exist in China sea and previous record was misidentification. L. bandera Hutchings, 1990, and L. arborea Moore, 1903, have only been recorded in ecological literature in China. Based on collected specimens, this study provides a detailed description of both species. We also combine all the sequences (COI, 16 SrRNA, 18 SrRNA) from GenBank and this study to analyze systematic relationship of the genera. Genetic distance and molecular systematics results support the samples collected in China sea can be divided into five species: L. macrobranchia, L. borealis, L. bandera, L. arborea and L. ingens(Grube, 1878), repudiating former hypothesis that L. ingens in Chinese waters was a species complex.

2. Six species of the genus Amphicteis have been identified in coastal China, including one new species: A. hwanghaiensis Wang, Sui, Li, Hutchings & Nogueira, 2020. Only A. gunneri(Sars, 1835)and A. obscurior Chamberlin, 1919 have been described in detail in this genus. In this study, other species (A. glabra Moore, 1905, A. scaphobranchiata Moore, 1906 and A. chinensis Sui & Li, 2017) from the China Sea have been described in detail based on collected specimens. We found for the first time that species of the genus from coastal China can be divided into two groups (blunt terminal and tapering terminal) according to the paleae.

3. Mitochondrial genomes of four species from the family Terebellidae (L. bandera; Artacama proboscidea Malmgren, 1866; L. arborea; Amaeana occidentalis (Hartman, 1944)), three species from the family Ampharetidae (Paramphicteis sinensis (Sui & Li, 2014); Anobothrus gracilis (Malmgren, 1866); Ampharete lindstroemi Malmgren, 1867) and one species from the family Melinnidae (Melinna elisabethae McIntosh, 1914) were obtained, respectively. Gene arrangement of the four species in the family Terebellidae were identical to those of the family Trichobranchidae in GenBank, including 13 protein-coding genes (PCGs), 2 rRNA genes (rrnL and rrnS), and 23 tRNA genes, all of which had one trnM gene copy, suggesting that the two families were closely related. The total length of the genomes of the four species was 15797-16304bp, and the length of the protein-coding genes was conserved. They all had a high A+T content, and had a strong preference for NNU and NNA codons in the use of codon. The structure of the transport RNA was a typical cloverleaf type, and the DHU arms of trnS1 gene were shortened or deleted. All the control regions were located between trnM and trnH genes, but the length and structure of the control regions were different in different species. The length of the three species of the Ampharetidae ranged from 10980 bp to 11022bp. The mitochondrial genome of Anobothrus gracilis was incomplete, without trnM and trnH genes. The other two species both contained 37 genes (13 PCGs+2 rRNA+22 tRNA). The length of the protein coding genes is conservative, and they all have high A+T content. In terms of the use of codon, there is a strong preference for NNU and NNA codon, and the structure of the transport RNA is typical cloverleaf type. Mitochondrial genome of Melinnidae species was sequenced for the first time also contained 37 genes (13 PCGs+2 rRNA+22 tRNA), with a total length of 16,309bp and a high A+T content. rrnL gene is located between the trnV and trnL1 genes, and the rrnS gene is located between the trnM and trnV genes, which are the same positions as the rRNAs in the species of Terebellidae.

4. Combined with the mitochondrial genomes obtained in this study and 10 Terebelliformia species published in GenBank, the phylogenetic relationships of each family of Terebelliformia were analyzed. Results showed that the six families of the suborder were divided into three clades. Pectinariidae was located at the base of the suborder, and Melinnidae and Trichobranchidae were sister groups, which clustered first into one clade and then into one clade with Terebellidae. Alvinellidae is close to Ampharetidae, but the phylogenetic trees reconstructed in some data sets do not support Ampharetidae as a monophyletic group.

第1章  引言... 1

1.1  蛰龙介亚目分类学及系统发育现状... 1

1.1.1  蛰龙介亚目概述... 1

1.1.2  蛰龙介亚目的生物学特性... 2

1.1.3  蛰龙介目分类学研究进展... 3

1.1.4  蛰龙介亚目系统发育研究进展... 5

1.1.5  蛰龙介亚目国内研究现状... 7

1.2  蛰龙介科概况... 8

1.3  双栉虫科概况... 12

1.4  米列虫科概况... 14

1.5  分子系统发育研究方法概述... 14

1.5.1  多毛类分子系统学中的常用分子标记... 15

1.5.2  线粒体基因组的测序方法... 15

1.5.3  多毛类线粒体基因组的主要特征... 16

1.5.4  多毛类线粒体基因组的研究进展... 17

1.5.5  蛰龙介亚目线粒体基因组的研究进展... 18

1.6  本研究的目的和意义... 19

第2章  扁蛰虫属分类及分子系统学研究... 21

2.1  扁蛰虫属概况... 21

2.2  常用的分类学性状... 22

2.3  研究材料... 22

2.4  研究方法... 24

2.4.1  形态学部分... 24

2.4.2  分子系统学部分... 25

2.5  实验结果... 27

2.5.1  扁蛰虫属分类学研究结果... 27

2.5.2  扁蛰虫属分子系统学研究结果... 46

2.6  小结... 51

第3章  扇栉虫属分类学研究... 53

3.1  扇栉虫属概况... 53

3.2  常用的分类学性状... 54

3.3  研究材料... 54

3.4  研究方法... 54

3.5  扇栉虫属的分类学研究结果... 55

3.6  小结... 65

第4章  蛰龙介亚目几个科线粒体基因组结构分析... 66

4.1  实验材料与方法... 66

4.1.1  样品采集、鉴定、提取DNA.. 66

4.1.2  二代Illumina测序... 66

4.1.3  基因组组装... 66

4.1.4  基因组注释分析... 67

4.2  蛰龙介科线粒体基因组组成结构与成分分析... 68

4.2.1  蛰龙介科线粒体基因组的结构组成和碱基成分分析... 68

4.2.2  蛋白编码基因分析... 77

4.2.3  RNA.. 80

4.2.4  控制区（Control Region）... 81

4.3 双栉虫科线粒体基因组组成结构与成分分析... 83

4.3.1  双栉虫科线粒体基因组的结构组成和碱基成分分析... 85

4.3.2  蛋白编码基因分析... 90

4.3.3  RNA.. 92

4.3.4  控制区（Control Region）... 92

4.4  米列虫科线粒体基因组分析... 93

4.4.1  基因组组成... 93

4.4.2  蛋白质编码基因和密码子使用偏好性... 96

4.4.3  RNA.. 98

4.4.4  控制区... 98

4.5  总结... 98

第5章  基于线粒体基因组的蛰龙介亚目系统发育研究... 100

5.1  研究背景... 100

5.2  研究材料... 100

5.3  研究方法... 100

5.3.1  全线粒体基因组数据的获取... 100

5.3.2  数据集的选取... 101

5.3.3  系统发育分析... 102

5.4  结果... 102

5.5  基因排列顺序... 105

5.6  讨论... 108

第6章  结论及展望... 110

参考文献... 113

附  录... 127

致  谢... 141

作者简历及攻读学位期间发表的学术论文与研究成果    143