Safe and genotyped. A non-invasive method for extraction of amphibian DNA from water baths and its application on Northern spectacled salamanders, Salamandrina perspicillata (Savi 1821)

The global decline of amphibian populations, with 40.7% of species classified as threatened, calls for innovative and ethical approaches in conservation genetics. Molecular biology advancements have introduced environmental DNA (eDNA) analysis, primarily focusing on aquatic environments. However, the present study explores a novel non-invasive protocol using water samples to extract DNA from terrestrial and semi-terrestrial amphibians, specifically the endangered Italian endemic salamander, Salamandrina perspicillata (Savi, 1821). Unlike traditional invasive methods involving tissue sampling, this protocol immerses animals briefly, eliminating the need for digit or tail amputations or manipulation for buccal swabs. The study validated the protocol through DNA extraction, amplification, and sequencing, yielding results comparable to traditional methods. The non-invasive nature of the protocol aligns with the 3Rs principles (Replace, Reduce, Refine) and offers a streamlined, stress-minimizing alternative for studying protected and endangered species. Future experiments should also explore further refinements, including reduced soaking times and additional applications, such as skin microbiota analysis. This protocol represents a significant step towards ethical and effective research practices in amphibian conservation genetics, encouraging a paradigm shift in wildlife research ethics. Continued innovation in non-invasive methodologies is essential for comprehensive understanding and robust conservation strategies amid the ongoing biodiversity crisis.