DNA metabarcoding analysis of stomach flushing contents reveals the exceptionally diverse diet of the golden alpine salamander.

Amphibians, particularly salamanders, are facing dramatic population declines worldwide; therefore, investigating their role as predators through diet analysis could inform targeted conservation programs. DNA metabarcoding significantly outperforms morphology-based identification of prey taxa in other vertebrates, but has rarely been applied to amphibians, and then only for faecal samples. Here, we chose the golden Alpine salamander Salamandra atra aurorae, an endangered endemism of the Italian Alps, to (i) develop a new metabarcoding protocol for amphibian stomach flushing samples; (ii) investigate the diet of this subspecies, including differences in the Frequency of Occurrence (%FO) of prey taxa between sexes; and (iii) compare these results with a recently published morphology-based identification of diet for the same samples. From 53 golden Alpine salamanders from the Province of Trento (Italy) population, targeting two COI fragments with DNA metabarcoding, we detected an extraordinary 177 prey taxa (103 to species), obtaining a much higher proportion of positive samples and taxonomic resolution compared to the morphological study. The FO of these taxa showed a strong positive correlation between sexes. In amphibian diet studies, the inclusion of metabarcoding protocols for stomach flushes could accelerate our understanding of their trophic ecology and improve future conservation actions.