Hidden species’ diversity and phylogenetic relationships within the terrestrial isopod genus Tiroloscia (Crustacea: Isopoda: Oniscidea)

Defining species boundaries may result challenging and has been a widely debated issue in the last decades. In cases of cryptic or “pseudocryptic” species, molecular approaches can be a valuable tool to provide taxonomic hypotheses and significantly complement morphological taxonomy. Here, two mitochondrial and one nuclear markers were used to study the phylogenetic relationships within the genus Tiroloscia, paying particular attention to Tiroloscia exigua. Moreover, we used a molecular clock to define a time window for the diversification of the main lineages within the species and explore aspects of its evolutionary history. Finally, four species delimitation methods were applied to clarify taxonomy and validate species boundaries. We found strong evidence against the monophyly of Tiroloscia and a surprisingly high level of genetic diversity within Tiroloscia exigua, supported by morphology. Notably, five evolutionary lineages were identified within T. exigua, suggesting the presence of distinct taxonomic entities. Divergence time estimation places the onset of T. exigua diversification around the middle Miocene (~12.2 Mya). Based on phylogenetic and morphological results, we propose the resurrection of Tiroloscia squamuligera as a valid species. Our results underscore the importance of molecular approaches to uncover hidden diversity, particularly in terrestrial isopods which may hide still underestimated biodiversity.