Metabarcoding and microtomography reveal new insights into the dietary niche of near-extinct amphibians

Harlequin toads ( Atelopus ) are among the most threatened vertebrates, yet their trophic ecology remains poorly understood due to the virtual disappearance of most populations and non-invasive sampling constraints. Here, we combine DNA metabarcoding of faecal samples from six surviving harlequin toad species from Ecuador with synchrotron-based microtomography of historic, fluid-preserved material of seven species across all major clades of the genus to assess dietary composition. Metabarcoding revealed a diverse invertebrate diet with marked ecological segregation between habitats, suggesting specialization on Hymenoptera in species inhabiting low to mid elevation forests and broader prey spectra in high-Andean taxa. Synchrotron scanning for the first time allowed non-destructive recovery of 3D-images of arthropod exoskeletons from the intestinal system of amphibians, confirming hymenopterans as key prey for forest-associated Atelopus in historical specimens. This dual approach overcomes the limitations of traditional and single-method studies, offering a scalable, non-invasive framework for dietary analyses. By providing curated step-by-step commands for sequence processing, we further aim to make dietary metabarcoding more accessible to zoologists. Our study substantially expands dietary data for near-extinct harlequin toads and supports conservation efforts with urgently needed ecological insights as a baseline for adapted conservation breeding and integrative conservation of trophic webs.