Small swabs, small frogs: buccal swabbing for minimally-invasive genetic sampling of Leiopelma species

Genomics can provide conservation-relevant insights into population size, genetic diversity, and connectivity with relatively little sampling effort. However, for frogs and other small vertebrates, invasive genetic sampling (e.g. toe-clipping) can impact animals’ long-term survival and welfare. Minimally-invasive genetic sampling methods may be required to facilitate robust conservation genomic studies of small vertebrates. In Aotearoa | New Zealand, previous genetic studies of the three native Leiopelma spp. frogs have relied almost entirely on toe-clips or whole specimens, creating an ethical barrier to genomic studies. Here, we trialled duplicate buccal swabbing as a minimally-invasive genetic sampling method on the small-bodied pepeketua, Hamilton’s frog (Leiopelma hamiltoni) and Hochstetter’s frog (Leiopelma hochstetteri). DNA yield was primarily influenced by our duplicate swabbing method rather than by frog body size, condition, or species. The second swab had a significantly higher DNA yield (721 ± 104 ng), more than twice that of the first swab (317 ± 49 ng). We compared the utility of buccal swabs and toe clips for genotyping-by-sequencing (GBS) in Hamilton’s frogs and assessed the suitability of buccal swabs for long-range PCR in Hochstetter’s frogs. Buccal swabs were as effective as toe-clips for GBS, with the proportion of missing SNPs (single-nucleotide polymorphisms) not significantly different between sampling methods. However, only 7/20 buccal swabs amplified successfully with long-range PCR. Our results indicate that genetic sampling of Leiopelma spp. could move towards buccal swabbing as a standard method. Toe-clipping and euthanasia of individuals should be limited to situations where high molecular weight DNA is crucial for analysis (e.g. genome assembly) or whole voucher specimens are needed for taxonomy.