Teran-Valdez, A. | Search bioRxiv

bioRxiv2026

Dorsal skin biopsies: A non-lethal sampling method for studying amphibians, including the highly endangered Harlequin frogs (Bufonidae: Atelopus)

Non-lethal sampling methods are increasingly essential for amphibian research as global declines intensify and many species persist in small, vulnerable populations. Skin biopsies offer a promising alternative to whole-animal collection and other minimally invasive approaches; however, systematic evaluations of recovery and impacts on body condition remain limited. Here, we assess the effects of small (2-mm) dorsal skin biopsies in four frog species, including three highly endangered Harlequin frogs (Atelopus bomolochos, A. balios, A. longirostris) and the Gualataco marsupial frog (Gastrotheca riobambae). Under controlled laboratory conditions and in semi-natural enclosures, we monitored wound healing, survival, and body mass trajectories in biopsied and control individuals over a one-month period. Across all species, biopsy sites fully healed within approximately three weeks, following consistent stages of re-epithelialization and subsequent repigmentation. No biopsy-related mortality was observed, and body mass did not differ between biopsied and control individuals, indicating no detectable effects of skin biopsies on body condition during the wound-healing period. Occasional minor post-biopsy reactions resolved without intervention within the observation period. We additionally report anecdotal field recovery observations for three other species (A. coynei, A. laetissimus, and A. sp. aff. longirostris), indicating survival and visible wound closure following release. Together, these results indicate that small dorsal skin biopsies represent a safe, non-lethal sampling method for amphibians, including highly endangered taxa. By providing sufficient tissue for diverse downstream applications--such as chemical analyses, genomics, transcriptomics, microbiome characterization, and disease detection--this approach expands the range of questions that can be addressed while minimizing harm to threatened species.

zoology↗

bioRxiv2025

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.

zoology↗