Disentangling Chemical Defence and Prey Composition of Oophaga sylvatica

Abstract Poison frogs acquire alkaloids from arthropod prey, yet linking diet to chemical defence remains challenging due to methodological and ecological complexity. We investigated this relationship in 23 adult Oophaga sylvatica from an early-stage cacao regeneration plot in the Ecuadorian Chocó. Using non-destructive alkaloid sampling and GC-MS profiling, combined with stomach flushing, morphological prey identification, and DNA metabarcoding, we characterised individual alkaloid richness and abundance (total alkaloid quantity), described prey diversity, and tested whether diet similarity correlated with alkaloid similarity. We also compared metabarcoding and morphological identification for stomach content analysis. We detected 130 alkaloids spanning 16 structural classes and found substantial inter-individual variation in richness and abundance. Most alkaloids occurred in low quantities, whereas a few classes dominated chemical profiles. Total alkaloid quantity increased with alkaloid richness. Diet analysis recovered 280 prey taxa across 22 orders, with ants, flies, beetles, and mites contributing most to richness. Metabarcoding recovered more diversity, improved taxonomic resolution, and detected highly digested and soft-bodied prey. Morphological identification recovered fewer taxa overall but performed better for small, sclerotized groups, particularly mites. Prey composition could not be correlated with alkaloid profiles, likely reflecting temporal mismatches between short-term diet data and long-term toxin accumulation, limited knowledge of alkaloid sources, and methodological constraints. Using available literature, only 15% of detected alkaloids could be linked to known arthropod sources among the recovered prey. We demonstrate that combining morphological prey identification with DNA metabarcoding provides a more comprehensive understanding of poison frog diets while highlighting existing knowledge gaps regarding alkaloid sources and the need for more complete DNA barcode reference databases.