基于线粒体COI基因序列的壮真蝎与普洱真蝎的分子鉴定

Molecular Identification of Euscorpiops validus and E. puerensis Based on Mitochondrial COI Gene Sequences

对蝎类物种的传统分类主要依靠形态和行为特征,但由于该类群种间形态特征极为相似,物种的划分和鉴定困难。为弥补传统分类方法的不足,本研究以线粒体细胞色素氧化酶亚基Ⅰ(COI)基因作为分子标记,对形态相似的壮真蝎Euscorpiops validus和普洱真蝎E.puerensis进行分子水平的物种鉴定。采用PCR扩增测序获得壮真蝎与普洱真蝎共24个样本的COI基因部分片段序列(660 bp),进行了遗传距离、系统发育及单倍型网络图分析。结果显示:壮真蝎15个样本中共检测到4个单倍型,单倍型之间的相似度为99.3%~99.8%;普洱真蝎9个样本中共检测到4个单倍型,单倍型之间的相似度为99.6%~99.8%;2种蝎的种间序列相似度为90.1%~90.6%,单倍型间的稳定差异核苷酸位点数为61个。壮真蝎与普洱真蝎种内平均遗传距离分别为0.004 0、0.002 3,种间平均遗传距离为0.103 9,且种间遗传距离为种内的34.6倍。此外,分子系统发育树显示壮真蝎与普洱真蝎的单倍型序列各自聚为2个单系枝,且具有很高的分枝自举值(100%)。单倍型网络图结果也显示壮真蝎与普洱真蝎8个单倍型明显分为2大类群,且壮真蝎的单倍型HAP2与普洱真蝎的单倍型HAP7之间的突变步数高达62步。上述结果不仅进一步确认壮真蝎与普洱真蝎为2个不同的物种,且表明线粒体COI基因可用于开展真蝎属Euscorpiops物种的分子鉴定。

The traditional classification and taxonomy of scorpiones are mainly based on morphological and behavioral characteristics. However,due to the similar morphological characteristics among species,it is difficult to classify and identify distinct species among scorpions. To compensate for the insufficiency of traditional taxonomic methodology,this study used mitochondrial cytochrome oxidase subunit Ⅰ（ COI） gene as molecular marker to conduct molecular identification of 2morphologically similar scorpion species Euscorpiops validus and E. puerensis. The partial COI gene sequences（ 660 bp）from 24 samples of E. validus and E. puerensis were amplified by PCR followed by gene sequencing. The genetic distances,phylogenetic and haplotype network analyses were then carried out. The results showed that： 4 haplotypes with similarity of99. 3%-99. 8% were detected in 15 individuals of E. validus,and 4 haplotypes with similarity of 99. 6%-99. 8% were found in 9 individuals of E. puerensis; the interspecific similarity was 90. 1%-90. 6% and the number of stable differentiated nucleotide sites between the haplotypes of 2 species was 61. The intraspecific average genetic distances among E. validus haplotypes and among E. puerensis haplotypes were 0. 004 0 and 0. 002 3,respectively,while the interspecific average genetic distance between E. validus and E. puerensis was 0. 103 9,being 34. 6 times higher than that of intraspecific values. In addition,molecular phylogenetic tree clearly showed that the haplotype sequences of E. validus and E. puerensis clustered as 2 reciprocally monophyletic clades with high bootstrap values（ 100%）. The haplotype network also showed that8 haplotypes of E. validus and E. puerensis can be clearly divided into 2 clades,and the mutation steps between HAP2 haplotype of E. validus and HAP7 haplotype of E. puerensis reached 62. These results further confirmed that E. validus and E. puerensis were 2 distinct species,and indicated that mitochondrial COI gene was suitable for molecular species identification of Euscorpiops species.