DNA Barcode Library of Megadiverse Lepidoptera in an Alpine Nature Park (Italy) Reveals Unexpected Species Diversity

Species inventories are a prerequisite for biodiversity monitoring and conservation, particularly in protected areas. However, the possibilities of a standardized survey of species diversity using DNA barcoding have so far hardly been implemented, especially in species-rich groups. A first-time molecular-based and nearly complete inventory of the megadiverse insect order Lepidoptera in a protected area in the Alps (Cottian Alps, Italy) was intended to test the possibilities and reliability of DNA-based identifications. From voucher material collected between 2019 and 2022, we successfully sequenced 1213 morphospecies that grouped into 1204 BINs (barcode index numbers), whereas DNA barcoding failed for another 18 species. A total of 35 species shared a BIN with one or more taxa, but a majority of 19 species could still be discriminated by divergent sequences. A total of 12 morphospecies split into two BINs. These species and a further 22 taxa with unique BINs and barcode divergences >2% to the nearest neighbor require taxonomic re-assessment. Two additional cryptic species from the study area were described recently. Finally, 16 species are newly recorded for Italy. Our study, therefore, demonstrates the importance of DNA barcoding for both faunistics and the discovery of cryptic diversity, even in apparently well-studied protected areas