基于线粒体COⅠ基因序列的DNA条形码在中国南海绯鲤属鱼类鉴定中的应用

Application of Mitochondrial Cytochrome Oxidase Subunit Ⅰ sequence in DNA barcoding Upeneus in the South China Sea

为探讨COⅠ基因作为DNA条形码对绯鲤属（Upeneus）鱼类鉴定的有效性,明确物种的分类地位,通过COⅠ基因5＇端652 bp序列研究中国南海绯鲤属的黑斑绯鲤（Upeneus tragula）、马六甲绯鲤（Upeneus moluccensis）、纵带绯鲤（Upeneus subvittatus）、黄带绯鲤（Upeneus sulphureus）、吕宋绯鲤（Upeneus luzonius）以及条尾绯鲤（Upeneus bensasi）6个种56 ind标本的标准DNA条形码序列的种内种间遗传距离,并构建分子系统树。研究表明：所测样品的碱基组成为T：29.2%,C：29.6%,A：21.4%,G：19.8%,A＋T含量（50.6%）略高于G＋C含量（49.4%）,转换/颠换率为3.08。6种绯鲤属鱼类组成5个自展支持率为100%的分支,其中黑斑绯鲤和马六甲绯鲤混杂分布于同一分支上,其余4种绯鲤独自成支;分支间平均遗传距离10.10%（8.60%~12.40%）,是分支内平均遗传距离0.18%（0.00%~0.30%）的56倍。纵带绯鲤、黄带绯鲤、吕宋绯鲤、条尾绯鲤种间平均遗传距离为10.70%（8.80%~12.40%）,约为这4个种种内平均遗传距离0.125%（0.00%~0.20%）的85倍,种间遗传距离大于种内遗传距离的10倍以上,确定了它们的物种有效性。黑斑绯鲤和马六甲绯鲤种内遗传距离分别为0.40%和0.20%,与其它4种绯鲤为同一水平;但种间遗传距离仅为0.30%,属于一般物种的种内遗传距离范围,因此推测二者或为同一物种,但还需从形态学和基因组序列分析等方面加以进一步确证,其是否受到种间杂交和近期辐射进化的影响也需要今后更多的研究。标准的DNA条形码虽然能够有效地区分中国南海绯鲤属鱼类中的纵带绯鲤、黄带绯鲤、吕宋绯鲤和条尾绯鲤,但DNA条形码对黑斑绯鲤和马六甲绯鲤的鉴定与形态学的鉴定结果不一致,表明在物种鉴定时,母系遗传的线粒体COⅠ基因有时需要结合其它核基因分子标记或生态调查资料加以辅助。

To test the role of 652 bp segments of mt DNA cytochrome oxidase subunit I gene as DNA barcoding in identifying Upeneus species,reliably determining species status and constructing a reference sequence library,specimens collected in the South China Sea were morphologically identified,the intra-species and inter-species genetic distances of the genus Upeneus were calculated, and phylogenetic trees were reconstructed. DNA barcoding from 56 specimens representing 6 species of genus Upeneus revealed that the nucleotide compositions were calculated as T = 29. 2%,C = 29. 6%,A = 21. 4%,G = 19. 8%,A ＋ T（ 50. 6%） was slightly higher than C ＋ G（ 49. 4%）,the average value of transition /transversion ratios was3. 08. In the NJ tree 6 species exhibited 5 discrete clades with 100% bootstrap supports,of which Upeneus tragula and Upeneus moluccensis were intertwined in the same clade,and the remaining 4 species formed 4independent clades,the average genetic distances among clades [10. 1%（ 8. 6%- 12. 4%） ] were 56 times that within clades [0. 18%（ 0. 0%- 0. 3%） ]. Inter-species distances among Upeneus subvittatus,Upeneus sulphureus,Upeneus luzonius,and Upeneus bensasi[10. 7（ 8. 8%- 12. 4%） ] were 85 times that of the intraspecies [0. 125%（ 0. 0%- 0. 2%） ],the inter-species distances were 10 times greater than that of intraspecies divergences（ the ＂ 10 x rule＂）,which confirmed their species status. Although intra-species genetic distances of U. tragula and U. moluccensis were 0. 4% and 0. 2% respectively,which were similar to the other 4 congeneric species,the genetic distances between U. tragula and U. moluccensis were only 0. 3%,which fell within the scope of most intra-species distances,suggesting that they might be the same species,but it needs to be confirmed by morphological and genomic sequence analysis,and interspecific hybridization or recent evolutionary radiation also should be excluded in further studies. Although DNA barcoding permitted the discrimination of 66. 7%（ 4 /6） Upeneus species,demonstrating the utility of barcoding for regional species identification,the inconsistence of DNA barcoding and morphological identification of U. tragula and U.moluccensis necessitates the worth of combination of more detailed morphological examination and ecological investigation,and sequence information from nuclear genes and / or fast-evolving mitochondrial DNA genes.