11种鲈形目鱼类ITS2特征及系统应用

ITS2 (Internal transcribed spacer 2)是位于核糖体5.8S和28S基因之间的非编码序列。为了探讨该片段的多样性特征以及进化模式,本研究选取了鲈形目(Perciformes) 5科11种鱼类为研究对象,共获得了444条ITS2克隆序列,其长度范围为332～515 bp。比较种内不同序列的长度发现,金带细鲹(Selaroides leptolepis)在种内存在24 bp的差异,剑鱼(Xiphias gladius)在种内存在32 bp的差异,这2种鱼类的差异较为明显;其余9种鱼类的长度相对比较保守,长度差异小于14 bp。依据11种鱼类的保守位点数、变异位点数、简约信息位点数、单倍型数、保守位点比例、单倍型多样性指数、核苷酸多样性等特征分析发现,种内存在着不同程度差异,特别是金带细鲹的ITS2序列存在着Type A、Type B和Type C 3种类型,各类型间差异较大。根据序列的多样性特征推断,金带细鲹和剑鱼的进化方式为非协同进化;蓝圆鲹(Decapterus maruadsi)、大甲鲹(Megalaspis cordyla)、吉打副叶鲹(Alepes djedaba)和日本竹筴鱼(Trachurus japonicas)的长度和变异位点均存在着一定程度的差异,但差异并不明显,视为不严格的协同进化;泰拉鰆鲹(Scomberoides tala)、布氏鲳鲹(Trachinotus blochii)、尖吻鲈(Lates calcarifer)、射水鱼(Toxotes chatareus)和军曹鱼(Rachycentron canadum) 5种鱼类为协同进化;另外,协同和非协同进化状态与分类系统没有相关性。序列比对发现,大甲鲹种内存在着由协同进化方式演变为非严格的协同进化方式的过度序列;在金带细鲹的3个不同个体中,序列间存在着从协同进化、非严格的协同进化演变为非协同进化的3种进化方式。基于ITS2序列构建的11种鱼类的邻接系统树显示,每种鱼类的克隆都分别按种单独聚为一支,鲹科7属鱼类各属也是单独聚支,表明ITS2不仅可以用在种类的分子鉴定,同时也可以作为分子标记应用于鲹科和属级水平的系统关系研究。

Study on feature of ITS2 in 11 Perciformes species and the application on phylogenetic relationship

The nuclear ribosomal RNA (nrRNA) genes of eukaryotes are organized in clusters of tandem repeat units, including three genes (18S, 5.8S, and 28S) and two internal transcribed spacers (ITS1 and ITS2). The ITS2 is located between the 5.8S and 28S genes. Because multiple copies of ITS2 have different intra-and inter-species evolution rates, ITS2 is often used as the molecular marker to identify species or to infer the phylogenetic relationship at the rank of species or genera. In this study, 11 species from five families of Perciformes were selected as the representatives to investigate the characteristics and the evolutionary pattern of the ITS2 in fishes. A total of 444 cloning sequences of ITS2 were obtained from 24 samples through PCR and cloning methods. The length of these sequences ranged between 332-515 bp. A comparison of sequences within species found that the length of Selaroides leptolepis (24 bp) and Xiphias gladius (32 bp) were extremely varied, and that of others, less than 14 bp, were relatively conservative. The evolutionary pattern of ITS2 of 11 species within species were conjectured based on the difference in several polymorphism characteristics, including the difference in length, conserved and variable sites, parsimony-informative site, the number of haploid type, proportion of conserved and variable sites, haploid type diversity index, nucleotide diversity and genetic diversity. The two out of 11 species (S. leptolepis and X. gladius) had obvious differences, especially the three different sequence types (Type A, Type B and Type C) detected in three individuals, suggesting a non-concerted evolution. Although, to a certain extent, length and variable sites were observed in four other species (Decapterus maruadsi, Megalaspis cordyla, Alepes djedaba, and Trachurus japonicas), those differences were not as obvious as those in S. leptolepis and X. gladius, therefore, the four species were not subjected to strict concerted evolution (no-strict concerted evolution). The five other species (Scomberoides tala, Trachinotus blochii, Lates calcarifer, Toxotes chatareus, and Rachycentron canadum) almost had no intraspecific variations, suggesting a concerted evolution process. Meanwhile, there was no correlation between the evolution pattern of 11 species and the taxonomic system. In addition, sequence comparison revealed that transitive sequences between concerted evolution and non-concerted evolution were observed in M. cordyla. In S. leptolepis, all three evolutionary patterns (concerted evolution, no-strict concerted evolution and non-concerted evolution) were detected in three individuals. Based on 444 cloning sequences of ITS2 from 11 species with Epinephelus adscensionis as the outgroup, two phylogenetic trees were constructed using the neighborjoining and maximum likelihood methods. The results showed that both trees were largely congruent with each other. The topology showed that sequences from the same species clustered together and sequences from each genus of seven genera in Carangidae claded together. These results supported that the ITS2 is applicable as a molecular marker for species identification, but also useful for phylogenetic relation analysis at the rank of genus in Carangidae.