Single-locus species delimitation of Pronophilina butterflies (Nymphalidae: Satyrini) in the Colombian Andes: congruence between morphology and MOTUs

The subtribe Pronophilina (Nymphalidae: Satyrinae: Satyrini) is one of the most diverse subtribes within the Lepidoptera. In Colombia, these butterflies are distributed throughout the Andes, where they play a key role in montane ecosystems and exhibit high levels of endemism. Despite their complex genital morphology, species within the same genus often exhibit highly similar wing patterns, complicating taxonomic identification. In this study, we analyzed the concordance between DNA barcode-based species delimitation and morphological identifications using 334 cytochrome c oxidase subunit I barcodes from 94 a priori identified morphospecies in Colombia. We applied four molecular species delimitation methods (Automatic Barcode Gap Discovery, Refined Single Linkage, Poisson Tree Processes, and Assemble Species by Automatic Partitioning), delimitating between 98 and 108 molecular operational taxonomic units. Overall, the methods showed high consistency, with high congruence between morphological and molecular delimitations (81%). Additionally, 96.8% of the morphospecies exhibited a barcode gap, indicating clear genetic differentiation. However, we found some inconsistencies, including 6 cases of species merging and 12 cases of species splitting. Our findings underscore the utility of DNA barcoding for species delimitation in Pronophilina, while highlighting the need for integrative approaches to resolve taxonomic uncertainties in this diverse group.