Abstract
The Taibai stream salamander (Batrachuperus taibaiensis) is a recently described species of the genus Batrachuperus that occurs in the Zhouzhi Heihe River and is endangered in its native range. Here, we have established a method for water environmental DNA (eDNA) analysis of Batrachuperus using a series of optimizations. We have designed a specific set of primers for the genus Batrachuperus to amplify a 160 bp fragment of Cytb. The sequences were obtained from nested PCR on eDNA extracted from water samples, after which DNA barcoding was performed according to sequence analysis to determine the presence of the target species in the water. The method was validated using water from the Zhouzhi Heihe River with known B. taibaiensis populations and found that B. taibaiensis eDNA can move at least 150 m downstream from its point of origin. This study is the first to establish an optimal method for obtaining the eDNA of Batrachuperus from water samples, which provides a theoretical basis for resource investigation and the protection of B. taibaiensis in future research. It is also an example of the eDNA extraction of other species that live in similar waters and are less genetically diverse between species.