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 Based on Partial Sequence of Mitochondrial COⅠ

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。

Objective： To determine the role of mitochondrial COⅠ gene in classification and identification of species,a total of 39 single individuals from 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 inthe 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 50 μl of PCR mixture. PCR products were purified and then sequenced in both forward and reverse directions using an ABI PRISMTM3730 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; 1 000 bootstraps pseudoreplications） using MEGA 5. 0. Results： Using the MEGA 5. 0 software for statistical analysis,the averaged AT content was 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 3ranged from 28. 1% to 37. 8% in average of 32. 4%（ Table 4）. The transitional pairs（ si） was slightly more than the transversional pairs（ sv）,and the ratio（ R = si /sv） was 1. 45（ Table 3）. Analysis of the frequency of amino acids in COⅠ gene encoding protein showed that the highest frequency of amino acid was leucine,and the lowest frequency of amino acid was tryptophan（ Table 5）. The average K2 P distances pairwise-species and within-species were 0. 191 and 0. 003,respectively（ Table 6）. The K2 P distance pairwise-species was 63. 7times of that within-species. According to the MP and NJ trees for all 39 sequences,it was demonstrated that Cynoglossinae in the Yangtze Estuary was 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 used to carry out effective identification for Cynoglossidae species,which further shows that mitochondrial COⅠ is feasible as the classification barcode.