Detection of elusive fire salamander larvae ( Salamandra salamandra ) in streams via environmental DNA

In the face of the global biodiversity crisis, the monitoring of species richness and diversity is experiencing an increased demand entailing a raise in cost and time investment. The analysis of species-specific DNA fragments in environmental samples (eDNA) such as from water or soil, facilitate the molecular detection of species without the specific sampling of individuals. The invasive chytrid fungus Batrachochytrium salamandrivorans ( Bsal ) is infecting natural fire salamander populations ( Salamandra salamandra ) and causes chytridiomycosis resulting in infrequent regional extinctions of populations across Central Europe. With regard to the expanding distribution of Bsal over the last years, cost-effective monitoring of fire salamanders is important for the conservation of this species. Based on a real-time quantitative PCR (qPCR) assay, we developed a new protocol to detect S. salamandra larvae in streams via eDNA, using species-specific primers of the mitochondrial control region (D-loop). We tested the efficiency of qPCR primer sets for six combinations of DNA extraction kits coupled with subsequent PCR inhibitor removal kits for obtaining qPCR-detectable S. salamandra eDNA from water filters, that were taken both from natural streams and artificial water tanks in the laboratory as positive controls. We found that the DNeasy Blood & Tissue Kit in combination with the DNeasy PowerClean CleanUp Kit performed best for detecting salamander larvae from natural streams. Our experimental protocol paves the way for resource-saving approaches to monitor S. salamandra larvae, but also confirms the limits to this eDNA approach in that it requires optimized laboratory protocols.