Cryptic diversity within the genus Tiroloscia Verhoeff, 1926 (Oniscidea, Philosciidae): a preliminary investigation

The genus Tiroloscia Verhoeff, 1926 currently includes eight epigean species, mainly distributed in Italy, south-central France, and northern Spain. However, the taxonomic status of the genus is controversial, and a thorough revision is needed. In this study, we provide a preliminary evaluation of the genetic diversity within the genus Tiroloscia, with particular attention to the alpine and pre-alpine species Tiroloscia exigua (Budde-Lund, 1885). The phylogenetic relationships between 25 distinct populations of this species, sampled across most of its distribution range, are reconstructed based on two mtDNA (COI and 16S rRNA) and one nuDNA (28s rRNA) gene fragments. Sequences of two other species belonging to this genus, Tiroloscia corsica (Dollfus, 1888) and Tiroloscia macchiae (Verhoeff, 1931), along with Philoscia affinis Verhoeff, 1908 were also included. Phylogenetic analyses of both single and combined datasets were performed using Bayesian inference and Maximum Likelihood, and a molecular clock was used to define a time window for diversification of the different clades and to explore aspects of their evolutionary history. We then applied three molecular species delimitation methods (mPTP, GMYC and ABGD) to delineate species boundaries in our T. exigua dataset and to assess whether its current taxonomy reflects the observed genetic diversity. Finally, the delimited putative species were compared with the groups resulting from a morphological analysis, in order to test the hypothesis of T. exigua as a complex of pseudo-cryptic species distinguishable on the basis of a few morphological characters. Our results showed a relatively high genetic diversity within T. exigua and identified five lineages grouped into two main clades, which probably originated between the Middle-Upper Miocene and the Lower Pliocene (between ~11 and ~5 Mya). Molecular species delimitation analyses identified the five lineages as distinct putative species, in agreement with the morphological evidence. The limited sampling of other species does not allow a complete reconstruction of phylogenetic relationships within the genus, but some valuable considerations can be made. The high genetic divergence found between the two vicariant subspecies of T. corsica (i.e. T. corsica corsica from Corsica and Sardinia and T. corsica giustii from the island of Capraia) suggests that they probably represent two distinct species, as already hypothesised on a morphological basis. Finally, the genetic distance between the three Tiroloscia species, which is comparable to that between some of them and P. affins, together with some important morphological differences, suggests that they may refer to different genera, stressing the need for further studies using an integrative approach in the future.