DNA barcoding and species delimitation of crickets, katydids, and grasshoppers (Orthoptera) from Central and Southern Europe, with focus on the Mediterranean Basin

The Mediterranean Basin, recognized as a global biodiversity hotspot, harbors a remarkable diversity of grasshoppers, katydids, and crickets, many of which are endemic and potentially contain cryptic lineages. In this study, we generated a comprehensive dataset comprising 1,441 barcodes from 270 identified species within the Ensifera and Caelifera suborders. These were combined with existing data to form a dataset of 2,606 barcodes representing 351 species. We employed Maximum Likelihood (ML) topology reconstruction and applied five species delimitation methods (BIN, ABGD, ASAP, GMYC, and PTP) to detect potential incongruences between Operational Taxonomic Units (OTUs) and existing taxonomic classifications. Our analysis revealed that OTUs delimited by these methods corresponded to 71.39% of the evaluated species, with a notably higher congruence in Ensifera (88.53%) compared to Caelifera (52.15%). Across the dataset, we identified 54 lineages comprising cryptic species, indicating significant unrecognized diversity within these groups. Additionally, 21 instances of species being merged into consensus OTUs were observed, suggesting either the need for taxonomic revision or highlighting the limitations of current genetic markers. Among the methods tested, ABGD, particularly with the Kimura two-parameter model, was the most consistent with traditional taxonomy, yielding the highest consensus rates. In contrast, the PTP method exhibited the lowest consensus, often leading to an oversplitting of lineages. These findings underscore the complexity of species delimitation in recently radiated taxa and emphasize the importance of using multiple methodologies to accurately capture biodiversity, especially in regions characterized by a high prevalence of cryptic species.