Vences Miguel, Nagy Zoltán T, Sonet Gontran, Verheyen Erik
Division of Evolutionary Biology Zoological Institute, Technical University of Braunschweig, Braunschweig, Germany.
Methods Mol Biol. 2012;858:79-107. doi: 10.1007/978-1-61779-591-6_5.
Only a few major research programs are currently targeting COI barcoding of amphibians and reptiles (including chelonians and crocodiles), two major groups of tetrapods. Amphibian and reptile species are typically old, strongly divergent, and contain deep conspecific lineages which might lead to problems in species assignment with incomplete reference databases. As far as known, there is no single pair of COI primers that will guarantee a sufficient rate of success across all amphibian and reptile taxa, or within major subclades of amphibians and reptiles, which means that the PCR amplification strategy needs to be adjusted depending on the specific research question. In general, many more amphibian and reptile taxa have been sequenced for 16S rDNA, which for some purposes may be a suitable complementary marker, at least until a more comprehensive COI reference database becomes available. DNA barcoding has successfully been used to identify amphibian larval stages (tadpoles) in species-rich tropical assemblages. Tissue sampling, DNA extraction, and amplification of COI is straightforward in amphibians and reptiles. Single primer pairs are likely to have a failure rate between 5 and 50% if taxa of a wide taxonomic range are targeted; in such cases the use of primer cocktails or subsequent hierarchical usage of different primer pairs is necessary. If the target group is taxonomically limited, many studies have followed a strategy of designing specific primers which then allow an easy and reliable amplification of all samples.
目前只有少数几个主要研究项目针对两栖动物和爬行动物（包括龟鳖目和鳄目）这两类主要的四足动物进行细胞色素氧化酶亚基I（COI）条形码研究。两栖动物和爬行动物物种通常较为古老，分化程度高，且包含深度的同种谱系，这可能会在参考数据库不完整的情况下导致物种鉴定出现问题。据了解，没有一对COI引物能保证在所有两栖动物和爬行动物分类群中，或在两栖动物和爬行动物的主要亚分支内都有足够高的成功率，这意味着需要根据具体研究问题调整聚合酶链式反应（PCR）扩增策略。一般来说，已有更多的两栖动物和爬行动物分类群对16S核糖体DNA（rDNA）进行了测序，至少在更全面的COI参考数据库可用之前，16S rDNA在某些情况下可能是一个合适的补充标记。DNA条形码技术已成功用于识别物种丰富的热带群落中的两栖动物幼体阶段（蝌蚪）。在两栖动物和爬行动物中，组织采样、DNA提取和COI扩增都很简单。如果针对的是广泛分类范围的分类群，单引物对的失败率可能在5%至50%之间；在这种情况下，需要使用引物混合物或随后分层使用不同的引物对。如果目标群体在分类学上有限，许多研究采用了设计特异性引物的策略，这样就能轻松可靠地扩增所有样本。
