Multilocus re-evaluation of species boundaries in the Central European Psilopteryx psorosa species group (Trichoptera, Limnephilidae) with shallow morphological differentiation.

The caddisfly genus Psilopteryx Stein, 1874 (Limnephilidae) represents a taxonomically challenging group in which species boundaries have traditionally been defined solely by adult morphology, as larval stages lack reliable diagnostic characters. This difficulty is particularly evident within the Psilopteryx psorosa species group in Central Europe, where species delimitation has relied primarily on subtle and often variable differences in male genital morphology, while reliable diagnostic characters for larvae and females remain unavailable. In this study, we applied a multilocus molecular approach to evaluate species boundaries within this complex. We analysed 58 specimens collected across eight montane regions in the Czech Republic, Slovakia, and Germany, including material from the type locality of P. psorosa. Based on male genital morphology and following the taxonomic concept of Mey and Botosaneanu (1985), the analysed specimens were initially assigned to two subspecies: P. psorosa psorosa and P. psorosa bohemosaxonica. Two mitochondrial genes (COI, cytB) and one nuclear marker (wingless) were analysed using haplotype-based analysis, genetic distances, and phylogenetic inference. Across all analysed markers, genetic divergence was low, with median uncorrected p-distances not exceeding 0.3% and maximum divergence below 2%. Mitochondrial haplotypes were widely shared among regions and taxa, and neutrality tests performed on the complete mtDNA dataset yielded significantly negative values. Haploweb analysis of the nuclear marker recovered a single field for recombination (FFR), while both single-gene phylogenetic reconstructions and the concatenated multilocus analysis failed to recover reciprocally monophyletic groups corresponding to the traditionally recognised taxa. Our results indicate that the analysed forms represent a single evolutionary lineage and that the observed morphological differentiation reflects geographically structured intraspecific variation rather than distinct species.