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 owing to the virtual disappearance of most populations and constraints on noninvasive sampling. 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 from 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 potential specialisation on Hymenoptera in species inhabiting low- to mid-elevation forests and broader prey spectra in high-Andean taxa. Synchrotron scanning, for the first time, enabled 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.