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宋超,于亚男,张涛,杨刚,章龙珍.2014.基于线粒体COⅠ基因部分序列的长江口舌鳎科鱼类系统分类研究.动物学杂志,49(5):716-726.
基于线粒体COⅠ基因部分序列的长江口舌鳎科鱼类系统分类研究
Phylogenetic Analysis of Cynoglossidae in the Yangtze Estuary on Partial Sequence of Mitochondrial COⅠ
投稿时间:2013-11-20  修订日期:2014-06-27
DOI:10.13859/j.cjz.201405011
中文关键词:  舌鳎科  COⅠ  DNA条形码  分子系统分类
英文关键词:Cynoglossidae  COⅠ  DNA barcodes  Molecular systematic
基金项目:长江水环境教育部重点实验室开放课题(YRWEE201106), 公益性行业(农业)科研专项(201203065), 长江口中华鲟自然保护区专项(2012), 青草沙水库邻近水域生态修复专项
作者单位E-mail
宋超 中国水产科学研究院东海水产研究所农业部东海与长江口渔业资源环境科学观测实验站 上海 200090 songchao_2006@163.com 
于亚男 ①中国水产科学研究院东海水产研究所农业部东海与长江口渔业资源环境科学观测实验站 上海 200090②南京农业大学渔业学院 江苏南京 20095  
张涛 中国水产科学研究院东海水产研究所农业部东海与长江口渔业资源环境科学观测实验站 上海 200090  
杨刚 中国水产科学研究院东海水产研究所农业部东海与长江口渔业资源环境科学观测实验站 上海 200090  
章龙珍* 中国水产科学研究院东海水产研究所农业部东海与长江口渔业资源环境科学观测实验站 上海 200090 longzhen2885@hotmail.com 
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中文摘要:
      为了确定线粒体COⅠ基因在长江口舌鳎科鱼类系统分类及物种鉴定中的作用,本实验采用线粒体COⅠ基因特异扩增测序及GenBank已有序列联合配对分析的方法,对长江口舌鳎科2属9种鱼类39个COⅠ基因片段的序列进行比较和系统进化研究。采用MEGA 5.0软件进行统计分析,舌鳎科鱼类该片段的平均AT含量高于GC含量,第1密码子位点含量最高(51.8%~57.3%,平均53.8%),第2密码子的含量稳定,均为42.0%,第3密码子变化范围最大(28.1%~37.8%,平均32.4%)。依据Kimura-2-parameter模型,9种舌鳎科鱼类种间遗传距离平均值为0.191,种内为0.003,种间遗传距离是种内的63.7倍。采用最大简约法(MP) 和邻接法(NJ)构建系统发育树,显示长江口舌鳎科鱼类为明显的单系群,但舌鳎科鱼类内部的系统发育关系与形态分类划分的亚属并不完全一致,其中须鳎属的日本须鳎(Paraplagusis japonica)与拟舌鳎亚属的宽体舌鳎(Cynoglossus robustus)聚为一支。虽然三线舌鳎亚属的种类均可聚为独立的分支,但短吻三线舌鳎(C. abbreviatus)与紫斑舌鳎(C. purpureomaculatus)、长吻红舌鳎(C. lighti)与短吻红舌鳎(C. joyneri),两组间种间遗传距离分别为0.002和0.007,两组物种间均存在同种异名现象。本研究表明,线粒体COⅠ基因作为分子标记除了能筛选出同种异名种类外,还能够对舌鳎科鱼类进行有效的物种鉴定和系统进化分析,线粒体COⅠ基因作为分类条形码是可行的。
英文摘要:
      Objective: To determine the role of mitochondrial COⅠ gene on classification and identification of species, a total of 39 single individuals of 9 species pertaining to 2 genera of Cynoglossidae in the Yangtze Estuary were barcoded by COⅠ, sequenced and compared with that of other Cynoglossidae species recorded in GenBank (Table 1). Methods: Total genomic DNA was extracted from each scale sample using the classic phenol / chloroform extraction method. ~650 bp COⅠ fragments were amplified using the primers, i.e., F1: 5’-TCA ACC AAC CAC AAA GAC ATT GGC AC-3’, R1: 5’-TAG ACT TCT GGG TGG CCA AAG AAT CA-3’. Each PCR amplification was performed in a total volume of 50μl of PCR mixture. PCR products were purified and then sequenced in both forward and reverse directions using an ABI PRISMTM 3730 XL Automated Sequencer. DNA sequences were aligned with ClustalW using default parameters. Base composition, variable and parsimony informative sites were determined using MEGA 5.0. Neighbor-joining (NJ) and Maximum parsimony (MP) phylogenetic trees were constructed for COI haplotypes (Kimura 2 Parameter substitution model, K2P; 1000 bootstraps pseudoreplications) using MEGA 5.0. Results: Using the MEGA 5.0 software for statistical analysis, the averaged AT content is higher than the GC content (Table 2). The GC content of codon position 1 averaged 53.8% (51.8%-57.3%), that of position 2 for 42.0%, and that of position 3 ranged from 28.1% to 37.8% in average of 32.4% (Table 4). The transitionsal pairs (si) was slightly more than the transversional pairs (sv), with the ratio (R=si/sv) was 1.45 (Table 3). Analysis of the frequency of amino acids in COⅠ gene encoding protein, which showed that the highest frequency of amino acid is leucine, and the lowest frequency of amino acid is tryptophan (Table 5). The average K2P distances pairwise-species and within-species were 0.191 and 0.003, respectively (Table 6). The K2P distance pairwise-species was 63.7 times of that within-species. According to the MP and NJ trees for all 39 sequences, it demonstrated that Cynoglossinae in the Yangtze Estuary is a monophyletic group (Fig. 1). However, the phylogenetic relationships revealed by the COⅠ sequence analyses were not consistent with those inferred from morphological classification. In contrast with morphological classification, Paraplagusis japonica of Rhinoplagusia and Cynoglossus robustus of Cynoglossoides were placed in a same clade with support of a high bootstrap value (Fig. 1). The species in the sub-genera of Areliscus clustered on an independent branch. But, synonymic phenomena existed in the following two groups of species: C. abbreviatus and C. purpureomaculatus, C. lighti and C. joyner, with the pairwise-species of 0.002 and 0.007, respectively (Table 6). Conclusions: Our results highlight that the information from COⅠ sequences not only can filter out the synonym of the same species, but also be able to carry out effective identification for Cynoglossidae species, which further showed that mitochondrial COⅠ is feasible as the classification barcode.
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