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

<p>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 <i>Batrachochytrium salamandrivorans</i> (<i>Bsal</i>) is infecting natural fire salamander populations (<i>Salamandra salamandra</i>) and causes chytridiomycosis resulting in infrequent regional extinctions of populations across Central Europe. With regard to the expanding distribution of <i>Bsal</i> 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 <i>S. salamandra</i> 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 <i>S. salamandra</i> 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 <i>S. salamandra</i> larvae, but also confirms the limits to this eDNA approach in that it requires optimized laboratory protocols.</p>