Non-Invasive Sampling Reveals Genetic Differentiation and Lineage Structure in the Rare Butterfly Luehdorfia chinensis Leech (Lepidoptera Papilionidae).

Conservation genetics research on rare and legally protected butterflies is often constrained by restrictions on destructive sampling, resulting in longstanding knowledge gaps regarding population structure and evolutionary differentiation. Non-invasive sampling (NIS) offers a potential solution, yet its reliability for generating conservation-relevant genetic data in endangered Lepidoptera remains insufficiently evaluated. The feasibility of NIS was assessed in the Chinese endemic butterfly Luehdorfia chinensis by comparing DNA yield and quality from naturally shed materials, including larval and pupal exuviae and frass, across developmental stages and preservation methods. All material types yielded amplifiable COI sequences, with fifth-instar larval exuviae preserved at -20°C or in anhydrous ethanol consistently providing the highest DNA quality, indicating strong suitability for COI-based population-level analyses and long-term monitoring. By integrating NIS-derived sequences with publicly available COI data from additional populations, two geographically structured genetic lineages were identified, corresponding to low-altitude populations in the Yangtze River basin (L. c. chinensis) and high-altitude populations in the Qinling Mountains (L. c. huashanensis). These lineages were separated by five mutational steps and accounted for 82.9% of among-group genetic variance (AMOVA), consistent with recognized subspecific boundaries and reflecting the role of mountain-basin topography in limiting gene flow. Lower genetic diversity in populations broadly consistent with L. c. huashanensis may indicate potential conservation concern under environmental change and habitat fragmentation. Overall, the results demonstrate that non-invasive materials can yield robust genetic data for resolving population structure in protected butterflies, supporting their broader application in conservation genetics and management.