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DNA metabarcoding has proven to be an accessible, cost-effective, and non-invasive tool for dietary analysis of predators in situ. Although DNA metabarcoding provides numerous benefits in characterizing diet—such as detecting prey animals that are difficult to visually identify—this method has seen limited application in amphibian species. Here, we used DNA metabarcoding to characterize the diet of fire salamanders (Salamandra salamandra) (Linnaeus, 1758) in three distinct regions across the northwestern Iberian Peninsula. To test the efficiency of COI-based metabarcoding in determining salamanders’ diet diversity, we compared our COI-based results with results from traditional diet studies from neighboring and distant populations, as well as with recent findings obtained in a DNA metabarcoding study using 18S. Two COI primers were used in combination to investigate the potential impact of primer bias in prey detection. Our COI metabarcoding approach increased taxonomic resolution and supported a generalist diet in S. salamandra. Between primers, there were no significant differences in the diversity and richness of prey detected. We observed differences in the prevalence of prey identified between sampling regions both in our study and in other studies of S. salamandra diet. This COI metabarcoding study provides recommendations and resources for subsequent research using DNA metabarcoding to study amphibian diets.

COI Metabarcoding Provides Insights into the Highly Diverse Diet of a Generalist Salamander, Salamandra salamandra (Caudata: Salamandridae)