基于线粒体COI序列的DNA条形码在中国海口足类物种鉴定中的应用分析

Application of mitochondrial COI-based DNA barcoding for the identification of stomatopod species(Crustacea, Stomatopoda) in the China seas

中国海口足类动物区系具有丰富的物种多样性,是我国海洋底栖生物中的重要经济类群。口足类的属内种间鉴别特征有的极为相似,仅依靠传统的形态分类方法很难对近缘种和疑难种进行准确的鉴定。DNA条形码技术可以弥补传统形态学鉴定的某些局限,为物种鉴定提供了有效的工具。该研究探讨了利用线粒体COI序列对中国海口足类进行物种鉴定的可行性,共获得口足目4总科6科24属38个种的204条线粒体COI序列,与Gen Bank收录的14种42条口足类同源序列进行比对,结果显示口足类COI基因不存在碱基插入缺失现象,碱基组成偏倚明显,A+T含量(63.5%)显著高于G+C含量(36.5%)。基于Kimura双参数模型计算遗传距离,结果显示遗传距离随着分类阶元的增高而增大。同物种种内个体间的遗传距离变化范围在0%~3.91%,平均值为0.76%。同属内各物种间的遗传距离变化范围为6.55%~18.99%,平均值为12.91%。同科内不同属间的遗传距离变化范围为9.16%~23.32%,平均值为16.89%。不同科间的遗传距离变化范围为16.52%~26.6%,平均值为21.31%。由此可见,口足类COI基因的种间和种内遗传距离存在明显的间隙。基于COI序列构建的口足类邻接关系树显示所有包含大于1个个体的物种均可形成单系群,且节点支持率均为100%。本研究证明了COI序列作为DNA条形码标准基因在口足类物种鉴定中的有效性。此外,研究发现中国沿海分布的口虾蛄可能至少存在两个隐存种,实证了基于COI序列的DNA条形码技术能够用于口虾蛄隐存多样性的探究。

Commonly known as mantis shrimps,stomatopods are commercially valuable and taxonomically diverse.In spite of high species diversity,stomatopod species exhibit subtle morphological differences,which make species identification a difficult task based on taxonomic keys alone.DNA barcoding has been proposed as a promising tool for species identification and discovery in a wide range of animal taxa.In this study,the performance of DNA barcoding in delineating stomatopod species in the China seas was assessed.The nucleotide sequences of mitochondrial cytochrome c oxidase subunit-I(COI)gene were obtained from 204 individuals of 38 stomatopod species in 24 genera in a wide phylogenetic range.These 204 sequences were compared with the other 42 homologous sequences from 14 species of Stomatopoda retrieved from GenBank.Sequence comparisons revealed 223 polymorphic sites,217 of which were parsimony informative,and no InDels were found.The COI sequences showed a strong AT bias(63.5%)in nucleotide composition.As expected,the mean Kimuara-2-parameter(K2P)genetic divergence increased with an increase in the taxonomic rank.The pairwise genetic divergences among conspecific individuals ranged from 0%to 3.91%,with a mean of 0.76%.The mean K2P pairwise divergence between individuals of congeneric species was 12.91%,ranging from 6.55%to 18.99%.The mean K2P pairwise divergence between individuals of different genera that belong to the same family was 16.89%(range 9.16%–23.32%),and mean K2P pairwise divergence between individuals of different stomatopod families was 21.31%(range 16.52%–26.6%).Thus,an obvious“barcoding gap”was found between intraspecific and interspecific divergences of COI sequences within Stomatopoda.The neighbor-joining phylogenetic tree reconstructed by COI sequences recovered most species as monophyletic with high bootstrap support,except for the species represented by a single individual.In addition,two reciprocally monophyletic,highly supported lineages together with large inter-lineage relative to intra-lineage divergences recovered from COI data concur in suggesting the existence of two possible cryptic species in Oratosquilla oratoria(De Haan,1844).These results demonstrate the effectiveness of mitochondrial COI-based DNA barcoding for species identification and cryptic discovery in stomatopods.