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题名	西太平洋沿岸大弹涂鱼群体遗传学及中国背眼虾虎鱼亚科系统进化研究; Population genetics of Boleophthalmus pectinirostris in the coast of western Pacific and phylogeny of Oxudercinae species in China
作者	陈维
答辩日期	2013 ; 2012
导师	洪万树
关键词	大弹涂鱼 Boleophthalmus pectinirsotris 线粒体基因组 mitochondrial genome 分子系统地理学 molecular phylogeography 群体遗传学 population genetics AFLP AFLP 背眼虾虎鱼亚科 Oxudercinae
英文摘要	大弹涂鱼（Boleophthalmus pectinirostris）为广温广盐的两栖鱼类，喜栖息于港湾和河口潮间带淤泥滩涂，营穴居生活，主要分布于中国、日本、韩国、越南和马来西亚。我国产于江苏、浙江、福建、台湾、广东和广西等省沿海滩涂，为我国东南沿海地区的名特优养殖鱼类。本研究以西太平洋沿岸9个地理群体的大弹涂鱼为研究对象，采用线粒体DNA（mtDNA）序列分析和AFLP指纹分析，分别检测了核外基因和核基因的遗传变异，对线粒体基因组全序列和遗传多样性进行了研究，阐明大弹涂鱼的起源与进化、群体历史动态、群体遗传结构及其谱系关系，为大弹涂鱼的资源管理和保护提供理论依据。另外还通过mtDNA序列分析，对我国现存的背眼虾虎鱼亚科中8个种的系统进化进行初步研究。主要研究结果如下： 1. 依据虾虎鱼科鱼类mtDNA各基因保守区段设计引物，测定了大弹涂鱼线粒体基因组全序列，其全序列长度为17118bp，包括13个蛋白编码基因、2个核糖体RNA、22个tRNA及非编码区，在碱基组成上出现明显的反G偏倚。除了COⅠ基因的起始密码子为GTG以外，其余12个蛋白基因的起始密码子均为标准起始密码子ATG。ND1，ND2，ATP8，ND4L和ND6基因的终止密码子为TAA，COⅠ的终止密码子为AGA，ND5为TAG，其余6个基因则为不完全终止密码子T。大弹涂鱼的控制区全长1454bp，从中识别出了1个终止相关序列区、 3个保守序列区以及一段长串联重复区，长串联重复区中包括5个130bp的完整重复单元和1个53bp的不完全重复单元。 2. 对采自西太平洋沿岸4个国家9个地理群体大弹涂鱼的mtDNA细胞色素b全序列进行分析，研究大弹涂鱼的系统发育地理格局和群体历史动态。在248尾样本中，检测到119个单倍型。9个地理群体整体体现出了很高的单倍型多样度（0.9645±0.006）和相对较为中等的核苷酸多态性（0.00636±0.00021）。根据邻接关系树将大弹涂鱼分为了两大谱系：中越谱系和日韩谱系，中越谱系又可分为2个支系，支系1和支系2，单倍型类群的分布体现出了明显的地理差异。日韩谱系的遗传多样性低于中越谱系。两两群体间的FST值均体现出了显著的差异（P<0.05）。AMOVA分析结果显示大部分的遗传变异来自两个单倍群谱系间。中性检验和核苷酸不配对分布分析显示，大弹涂鱼的两个单倍型谱系都经历了晚更新世的群体扩张，中越谱系的扩张时间在17万年前，日韩谱系的扩张时间在8万年前。谱系的形成可能源于更新世冰期的隔离。 3. 应用AFLP标记分析了西太平洋沿岸的不同地理群体大弹涂鱼的遗传多样性和群体结构。利用6对选择性引物对9个群体132尾大弹涂鱼进行扩增，在15-500bp之间得到186条条带，其中多态性片段160条，多态性比例为86.02%。NJ系统树显示大弹涂鱼的9个群体被分为了两支，日本、韩国及台湾的群体聚为岛屿支系，江苏、浙江、福建、广东、广西和越南群体聚为大陆支系。群体间的遗传距离较大，群体间遗传分化指数FST值统计检测到显著的遗传结构（P=0.000）。AMOVA分析显示，大部分的变异组分来自于各群体之间。群体间遗传分化系数Gst为0.3857，群体间的基因流Nm为0.7964，群体间基因交流较弱。遗传分化分析结果支持mtDNA 检测到的两个完全分化且异域分布的系统发育类群。 4. 利用线粒体COⅠ基因全序列对分布于中国的银线弹涂鱼、大鳍弹涂鱼、大弹涂鱼、青弹涂鱼、大青弹涂鱼、蜥形副平牙虾虎鱼、长身拟平牙虾虎鱼和犬齿背眼虾虎鱼这8种背眼虾虎鱼亚科鱼类的系统进化关系进行了研究。8个种的COⅠ基因全长在1558-1560bp之间，存在9个插入/缺失，保守位点有1082个，此外有474个变异位点，转换/颠换比Ts/Tv为1.53。在碱基组成上，均体现出了明显的反G偏倚。种间平均核苷酸差异数K在166.984-248.5之间，采用Kimura2-parameter模型计算的遗传距离D在0.1191-0.1836之间。转换和颠换的数目与遗传距离呈良好的线性关系，表明序列进化未达到饱和。在构建的四种系统树（NJ、MP、ML和Bayes树）中，弹涂鱼属与其他6种鱼类出现了明显的分支，并得出了拟平牙虾虎鱼最先分化的结论。结果与新的分类系统相一致。背眼虾虎鱼的平均种内遗传距离为0.33%，平均种间遗传距离为16.10%，种间遗传距离是种内遗传距离的48.6倍，对于背眼虾虎鱼亚科而言，COI基因是一个有效的DNA条形码，可以用于背眼虾虎鱼类的物种鉴定。; The mudskipper (Boleophthalmus pectinirostris) is an amphibious fish, adapting to a wide range of temperature and salinity, and inhabiting the mudflats of intertidal zone and the mangrove forest. The fish lives mainly on benthic diatoms and organic detritus. It is distributed throughout China, Korea, Vietnam, and Japan, and in China, it is mainly found in coastal mudflats of Jiangsu, Zhejiang, Fujian, Taiwan, Guangdong and Guangxi provinces. Due to its economic value, this species is widely cultured in the southeast coastline of China. In this study, the sequence of mitochondrial DNA (mtDNA) and AFLP markers were employed to analyze the complete mitochondrial genomic sequences and genetic diversity of the 9 B. pectinirostris populations from Western Pacific coast, in an attemp to illustrate the origin and evolution, phylogeographic patterns, demographic history and genetic diversity. Our findings will provide insights into the future fishery management and conservation for B. pectinirostris. In addition, based on the COⅠ mitochondrial gene sequences we performed phylogenetic analyses for Oxudercinae species living in China coast. The main results and conclusions are as follows: 1. The primers were designed based on the conserved regions in mitochondrial DNA of Gobiidae species, and genomic sequence of the mitochondrial DNA of B. pectinirostris was determined. The complete mitogenomic sequence of B. pectinirostris was 17118 bp, including 13 protein-coding genes, 2 rRNA, 22 tRNA, and noncoding control regions. The base composition of B. pectinirostris showed an obvious antiguanine bias commonly observed in fishes. All e genes were encoded on the H-strand, except ND6 subunit gene and eight tRNA genes (tRNAGln, tRNAAla, tRNAAsn, tRNACys, tRNATyr, tRNASer, tRNAGlu, tRNAPro), which were encoded on the L-strand. Twelve genes required ATG as the start codon, while COI used GTG instead. Three types of complete stop codons were detected in open reading frames of B. pectinirostris : TAA for ND1, ND2, ATP8, ND4L and ND6; AGA for COI; and TAG for ND5, respectively; the rest 6 genes ended with incomplete terminate codons T. The control region of B. pectinirostris mitogenome was indentified between tRNAPro and tRNAPhe and found to be 1454 bp. Within this sequence, we identified one termination-associated sequence (TAS), 3 conserved block (CSB-1,CSB-1 and CSB-3) and a long tandem repeat area that included 5 complete repeat units (130bp) and one incomplete repeat units (53bp). 2. The genetic diversity and population structure of 9 populations of B. pectinirostris from the coast of Western Pacific were used to investigate the phylogeographic patterns and demographic history using the complete cytochrome b sequences of mitochondrial DNA. Among 248 individuals of B. pectinirostri 119 haplotypes were detected. Generally, high haplotepe diversity (h=0.9645±0.006) and moderate nucleotide diversity (π=0.00636±0.00021) were observed in each population. The topology of the neighbor-joining tree of B. pectinirostris haplotypes was assigned into the two closely related lineages (A and B) that appeared to have geographic structure. The A lineage included the populations from Jiangsu, Zhejiang, Fujian, Taiwan, Guangdong, Guangxi and Vietnam; the B lineage included those from Japan and Korea. All the pairwise comparisons of FST were statistically significant (P<0.05). The analyses of molecular variance (AMOVA) showed that most of the variances were found between 2 lineages. The neutrality tests and mismatch distribution analysis revealed that B. pectinirostris had undergone population expansion in the late Pleistocene. And population expansion occurred approximately 170,000 years ago for the lineage A, while 80,000 years ago for the lineage B. The sea level changes of the late Pleistocene may be responsible for the formation and expansion of the two lineages . 3. The population genetic structure and genetic diversity of B. pectinirostris in the coast of Western Pacific were analyzed using amplified fragment length polymorphism (AFLP). Among 132 individuals from 9 populations, 186 loci between 15bp and 500bp were detected by 6 selective amplification primers, and 160 of which were polymorphic (86.02%). Nei’s analysis showed that genetic distances were higher between populations. NJ tree constructed on the base of genetic distance between populations showed that there were 2 groups among 9 populations. 0ne group included Japan, Korea and Taiwan populations; the other included the rest of the populations. All the pairwise comparisons of FST were statistically significant (P=0). The analyses of molecular variance (AMOVA) showed that most of the variances were found among populations. The coefficient of population differentiations (Gst) was 0.3857 and the estimate of geneflow from Gst (Nm) was 0.7964, which was comparatively lower than those of other species. Phylogenetic patterns by AFLP analysis were consistent with those by cyt b analysis. 4. Based on the mitochondrial COⅠgene sequences we performed phylogenetic analyses for 8 species (Periophthalmus argentilineatus, Periophthalmus magnuspinnatus, Boleophthalmus pectinirsotris, Scartelaos viridis, Scartelaos gigas, Parapocryptes serperaster, Pseudapocrytes elongates and Oxuderces dentatus.) in the family Oxudercinae living in China coast. The complete COⅠgenes of these 8 species were between 1558-1560bp. There existed 9 gaps/missing, 1082 invariable (monomorphic) sites and 474 polymorphic sites (including 5 singleton variable sites and 469 parsimony informative sites). The transitionsal/ transversional ratio was 1.53. Base composition bias was obvious in the sequences, and the average G level was significantly lower than other three nueleotides. The average number of nucleotide differences between populations (K) was between 166.984 and 248.5, and the Kimura2-Parameter genetic distance (D) ranged from 0.1191 to 0.1826. The number of both transitions and transversions showed an approximately linear relationship with genetic distances, indicating that the sequences haven’t saturated. According to the results of four phylogenetic trees (NJ、MP、ML and Bayes trees), Periophthalmus was clustered first and other 6 species were clustered, then the 2 clades met at last. The results support the existing taxonomy. The average interspecies genetic distance was16.10% ,while the average intraspecies genetic distance was 0.33%, the former was 48.6 folds of the later.It is suggested that the mitoehondrial COI gene should be a good DNA bareoding marker for Oxudercinae species identification.; 学位：理学博士; 院系专业：海洋与环境学院海洋学系_海洋生物学; 学号：22420090153257
语种	zh_CN
出处	http://210.34.4.13:8080/lunwen/detail.asp?serial=38175
内容类型	学位论文
源URL	[http://dspace.xmu.edu.cn/handle/2288/53318]
专题	海洋地球－学位论文
推荐引用方式 GB/T 7714	陈维. 西太平洋沿岸大弹涂鱼群体遗传学及中国背眼虾虎鱼亚科系统进化研究, Population genetics of Boleophthalmus pectinirostris in the coast of western Pacific and phylogeny of Oxudercinae species in China[D]. 2013, 2012.

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