Abstract：
Environmental DNA (eDNA) metabarcoding has emerged as a powerful, non-invasive tool for biodiversity assessments. However, the accuracy and limitations of these assessment techniques are highly dependent on the choice of primer pairs being used. Although several primer sets have been used in eDNA metabarcoding studies of amphibians, there are few comparisons of their reliability and efficiency. Here, we employed lab- and field-tested sets of publicly available and de novo-designed primers in amplifying 83 species of amphibian from all three orders (Anura, Caudata, and Gymnophiona) and 13 families present in China to evaluate the versatility and specificity of these primers sets in amphibian eDNA metabarcoding studies. Three pairs of primers were highly effective, as they could successfully amplify all the major clades of Chinese amphibians in our study. A few non-amphibian taxa were also amplified by these primers, which implies that further optimization of amphibian-specific primers is still needed. The simultaneous use of three primer sets can completely cover all the species obtained by conventional survey methods and has even effectively distinguished quite a number of species (n = 20) in the Wenshan National Nature Reserve. No single primer set could individually detect all of the species from the studied region, indicating that multiple primers might be necessary for a comprehensive survey of Chinese amphibians. Besides, seasonal variations in amphibian species composition were also revealed by eDNA metabarcoding, which was consistent with traditional survey methods. These results indicate that eDNA metabarcoding has the potential to be a powerful tool for studying spatial and temporal community changes in amphibian species richness.
摘要：
环境DNA(eDNA)元编码已经成为一种强大的，生物多样性评估的非侵入性工具。然而,这些评估技术的准确性和局限性在很大程度上取决于所使用的引物对的选择。尽管几种引物组已经被用于两栖动物的eDNA元编码研究，它们的可靠性和效率几乎没有比较。这里,我们使用实验室和现场测试的公开可用和从头设计的引物集来扩增来自所有三个订单的83种两栖动物（Anura，Caudata,和Gymnophiona）和中国存在的13个家族，以评估这些引物在两栖动物eDNAmetabarcoding研究中的多功能性和特异性。三对引物是非常有效的，因为它们可以成功地放大我们研究中中国两栖动物的所有主要分支。一些非两栖动物类群也被这些引物扩增，这意味着仍需要进一步优化两栖动物特异性引物。同时使用三个引物组可以完全覆盖通过常规调查方法获得的所有物种，甚至有效地区分了文山国家级自然保护区中的许多物种（n=20）。没有一个引物组能单独检测到所研究区域的所有物种，这表明对中国两栖动物进行全面调查可能需要多种引物。此外,eDNA元编码也揭示了两栖动物物种组成的季节性变化，这与传统的调查方法是一致的。这些结果表明，eDNA元编码有可能成为研究两栖动物物种丰富度的时空群落变化的有力工具。
