Exploration of an eDNA procedure for surveying Chinese giant salamanders: a comparison with conventional field methods

Comprehensive field surveys are vital steps in gathering specific information on the population status and genetic diversity of threatened species, which is essential for formulating effective conservation and management plans. The Chinese giant salamanders (Andrias, CGS), the largest extant amphibian species, has undergone a significant population decline over the past century, making it one of the most globally threatened species. The nocturnal and cave-preferred habits of CGS, along with its typically small population size, present significant challenges for field detection. This study explores an environmental DNA (eDNA) procedure for conducting CGS surveys and compares it with conventional techniques. Thirty-nine species-specific primers were developed in silico to amplify mitochondrial gene regions of CGS. Three optimal primer pairs were validated in both laboratory and field settings, with one pair selected for the eDNA survey across seven sites in Hunan Zhangjiajie Giant Salamander National Nature Reserve. The eDNA method successfully detected CGS in all seven sampling sites, outperforming the conventional survey, which failed to find CGS in five sites during the same period. Haplotypes from eDNA samples provided comparable information on the phylogenetic lineages of CGS compared to the three mitochondrial gene markers from conventional survey samples. This study highlights the effectiveness of eDNA in monitoring CGS presence and identifying phylogenetic lineages. It offers a valuable method for conducting field surveys of CGS in different regions. The presence of two lineages in most sampling sites from eDNA haplotype analyses cautions against potential mixed lineage scenarios, possibly resulting from previous reintroduction conservation practices.