21种大眼蟹线粒体COI基因比较及系统进化分析

Comparison of 21 mitochondrial COI gene sequences of Macrophthalmidae species and phylogenetic analysis

【目的】比较大眼蟹科物种线粒体COI基因序列差异,探究COI基因作为大眼蟹科物种鉴定分子标记的可行性;同时基于COI基因序列构建目前最全面的大眼蟹科系统发育树,为大眼蟹科系统发育研究提供理论依据。【方法】测定拉氏大眼蟹、日本大眼蟹和太平大眼蟹3种大眼蟹的线粒体COI基因全序列,结合GenBank已公布的18种大眼蟹科COI基因部分序列,采用MatGAT 2.02进行多序列比对分析,并以三疣梭子蟹和红星梭子蟹为外类群,通过MEGA X中的最大似然法(ML)和最大简约法(MP)分别构建系统发育进化树。【结果】拉氏大眼蟹、日本大眼蟹和太平大眼蟹线粒体COI基因全序列长均为1534 bp,编码511个氨基酸残基,且均以ATG作为起始密码子及T作为不完全终止密码子。用于多序列比对的序列长度为657 bp,连续编码219个氨基酸残基,不存在碱基插入或缺失现象,碱基含量为28.9%~35.9%T、18.9%~27.2%C、25.3%~29.7%A及16.6%~19.0%G。核苷酸序列和氨基酸序列比对分别发现267和20个变异位点,且绝大多数变异位点出现在第3位密码子,而第2位密码子非常保守,即均表现出遗传密码的简并性.遗传距离、序列相似性及系统发育进化分析结果均表明,日本大眼蟹与万岁大眼蟹的亲缘关系最近、太平大眼蟹与绒毛大眼蟹的亲缘关系最近,而拉氏大眼蟹的进化地位及大眼蟹属是否为单系群还需进一步研究。【结论】21种大眼蟹线粒体COI基因序列均有区别于其他种类的特异位点,可考虑作为物种鉴定的分子标记;但用于多序列比对的COI基因部分序列包含有效信息位点有限,在解析某些物种间的亲缘关系上仍显不足,部分种类间的亲缘关系可信度较低,因此后续研究应基于更多基因序列及更多物种数以解决这一问题。

Mitochondrial COI gene can provide important information for species identification and phylogenetic studies within family.【Objective】To explore the feasibility of COI gene as a molecular marker for species identification of Macrophthalmidae by comparing the differences in mitochondrial COI gene sequences of Macrophthalmidae species. At the same time,comprehensive phylogenetic tree of Macrophthalmidae was constructed based on COI gene sequence,which provided a theoretical basis for the phylogenetic study of Macrophthalmidae.【Method】In this study,the complete COI sequence of 3 Macrophthalmus species,Macrophthalmus latreillei,M. japonicas and M. pacificus were determined and other 18 partial COI sequences of Macrophthalmidae species were downloaded from GenBank database. Multiple sequence alignment analysis was performed using MatGAT 2.02,and phylogenetic trees were constructed using the maximum likelihood(ML)and maximum parsimony(MP)methods in MEGA X with Portunus trituberculatus and P. sanguinolentus as outgroups.【Result】COI gene full-length sequence of M. latreillei,M. japonicas and M. pacificus was 1534bp,encoding 511 amino acid residue,and ATG was the start codon and T was the incomplete stop codon. Sequences for multiple sequence alignment was 657 bp,encoding 219 amino acids continuously,with 28.9%-35.9% T,18.9%-27.2%C,25.3%-29.7% A,and 16.6%-19.0% G;respectively. Sequence alignment of nucleotide and amino acid sequences revealed 267 and 20 variable sites,with most of the variation sites appeared in the third codon,and the second codon was very conservative,which showed the degeneracy of genetic code. The genetic distance,sequence similarity and phylogenetic analysis showed M. japonicas and M. pacificus had the closest relationships with M. banzai and M. tomentosus;respectively. However,further studies were needed to evaluate the evolutionary status of M. latreillei and the monophyletic origin of Macrophthalmus.【Conclusion】The mitochondrial COI gene sequences of 21 Macrophthalmidae species are different from each other,which can be considered as a molecular marker for species identification. However,it is still insufficient to analyze the phylogenetic relationships between certain species because it contains too few effective genetic sites and the genetic relationship between some species is less reliable,so subsequent studies should be based on more gene sequences and more species to solve this problem.