Molecular Identification of a <i>Hyla </i><i>annectans</i> Population Distributed in Fenggang County, Guizhou Province in China

华西雨蛙(Hyla annectans)首次发现于印度，在中国广泛分布，因此无论是物种还是亚种都一直是两栖动物多样性的研究热点，理清相关分类单元的分类地位有赖于更丰富的种群样本和研究手段。本研究于2019年5月在贵州省遵义市凤岗县进化镇采集到2个蛙类标本，经初步形态鉴定为华西雨蛙，但之前并没有该物种在凤岗县分布的文献记载。因此对标本进行DNA提取，并PCR扩增其线粒体16S rRNA基因片段，使用分子系统发育学方法对其进行分子鉴定。结果表明，凤岗所采蛙类样本16S rRNA基因序列与7条华西雨蛙同源序列聚为一个支持率较高的单系，支系内序列间平均遗传距离为0.0109，且对应序列单倍型均在同一个单倍型网络内。自测序列与华西雨蛙兴义种群标本的16S rRNA基因序列遗传距离较小。因此，初步判断该标本属华西雨蛙(Hyla annectans)。种群中性检验分析，没有检测到华西雨蛙在较近的历史上有种群扩张的证据。另外，本文探讨了相距较远的印度种群同源序列对相关系统发育分析结果的影响。Hyla annectans was first discovered in India and is widely distributed in China. Therefore, both the species and its subspecies have always been research hotspots for amphibian diversity. Clarifying the taxonomic status of related taxonomic units relies on richer population samples and research methods. In May 2019, two frog specimens were collected in Jinhua Town, Fenggang County, Zunyi City, Guizhou Province. Preliminary morphological identification indicated that they were Hyla annectans, but there was no previous literature record of this species distributed in Fenggang County. Therefore, DNA extraction was performed on the specimens, and their mitochondrial 16S rRNA gene fragments were amplified by PCR. Molecular phylogenetic methods were used for molecular identification. The results showed that the 16S rRNA gene sequence of the specimens collected in Fenggang clustered with seven homologous sequences of Hyla annectans into a highly supported monophyletic clade. The average genetic distance between sequences within the clade was 0.0109, and the corresponding haplotypes were all within the same haplotype network. The genetic distance between the self-tested sequence and the 16S rRNA gene sequence of the Xingyi population of Hyla annectans was relatively small. Therefore, it is preliminarily determined that the specimen belongs to Hyla annectans. Population neutrality test analysis did not detect evidence of population expansion in Hyla annectans in recent history. In addition, this article discussed the impact of homologous sequences from distant Indian populations on the results of related phylogenetic analysis.