Integrative taxonomy of cryptic Pachypus chafers using museomics, morphometrics, barcoding, and genomic DNA analysis (Coleoptera: Scarabaeidae: Pachypodinae).

The taxonomy of the endemic Mediterranean genus Pachypus Dejean, 1821 has long been complicated by cryptic diversity and limited morphological differentiation. Based on extensive results from previous studies and new genomic data analyses, we perform here an integrative taxonomic revision of the genus. Using the morphological examination of the type material of most species and de novo sequencing of 978 metazoan-level universal single-copy orthologous genes (mzl-USCOs) of the almost 200-year-old lectotype of Pachypus impressus Erichson, 1840, we proposed refined species hypotheses within the genus. Resulting species entities from a recent study using mzl-USCOs were aligned within the framework of existing nomenclatorially available names inferred from Museomics and comparative morphological analyses. We additionally removed two existing taxon names from synonymy with Pachypus candidae and raised them to valid species: Pachypus cornutus (Olivier, 1789) and P. impressus Erichson, 1840. Pachypus excavatus Fabricius, 1792 is confirmed here as a valid species. This study also revealed one new synonymy: Pachypus impressus Erichson, 1840 (= P. melonii Sparacio, 2008, syn. n.). Furthermore, we described seven new species which had been already delineated in our earlier study: Pachypus baroniensis sp. n., P. franginii sp. n., P. gallurensis sp. n., P. matzaccara sp. n., P. occidentalis sp. n., P. pelegrinus sp. n., and P. sulcis sp. n. Extensive morphometric analyses of five datasets (one based on distance measurements and four on body outlines), including about 1,900 specimens, confirmed the cryptic character of most Pachypus species, as morphospace plots of all traits largely overlapped among most of the species. This made the assignment of analyzed type specimens to retrieved morphometric clusters a poor predictor of the true identity of mzl-USCO-defined species. We designated a lectotype of Pachypus impressus Erichson, 1840 and a neotype of Pachypus cornutus (Olivier, 1789). Finally, for the purpose of enhancing applied DNA-based studies and metabarcoding, we extracted the 5' COI DNA sequences from the raw DNA reads of our target enrichment nucleotide data as well as from the newly sequenced lectotype of P. impressus. The phylogenetic tree inferred from these data was compared with that obtained from the previously produced 3' COI data generated by Sanger sequencing. The habitus and male genitalia of all species are illustrated, an identification key is provided, and the distribution of the species is shown. As a result of our long-term studies of the genus, we also provide new insights into the ecology and distribution of the group, which may be relevant to conservation across the entire circum-Tyrrhenian region. Zoobank link: urn:lsid:zoobank.org:pub:34F3F1E1-2A40-4450-95B3-0336E6E2CF52.