DNA barcoding of large oak-living cerambycids: diagnostic tool, phylogenetic insights and natural hybridization between Cerambyx cerdo and Cerambyx welensii (Coleoptera: Cerambycidae).

Three large saproxylic cerambycids with different pest/legal status co-occur in the Iberian oak woodlands, Cerambyx welensii (Cw), Cerambyx cerdo (Cc) and Prinobius myardi (Pm): Cw is an emerging pest, Cc is a protected but sometimes harmful species and Pm is a secondary/minor pest. A precise taxonomic diagnosis is necessary for research, management or protection purposes, but may be problematic mainly because Cw and Cc larvae are morphologically indistinguishable. To resolve this constraint, we genotyped adults, larvae and eggs collected over a wide geographical range using the mitochondrial barcoding of the cytochrome c oxidase subunit I (COI). A Neighbour-Joining tree phylogram revealed three distinct clusters corresponding to Cw, Cc and Pm. We further first sequenced for Cw and Cc two mitochondrial (12S rRNA and 16S rRNA) and one nuclear (28S rRNA) gene fragments. For the first two genes, interspecific divergence was lower than in COI, and for the 28S (lower mutation rate), the two species shared identical haplotypes. Two approaches for species delimitation (General Mixed Yule Coalescent (GMYC), Barcode Index Number (BIN)) confirmed the species distinctiveness of Cc and Cw. The Bayesian COI gene tree showed a remarkable genetic divergence between Cc populations from Iberia and the rest of Europe. Such divergence has relevant taxonomic connotations and stresses the importance of a wide geographical scale sampling for accurate DNA barcoding species identification. Incongruities between morphology/lineage and COI barcodes in some individuals revealed natural hybridization between Cw and Cc. Natural hybridization is important from a phylogenetic/evolutionary perspective in these cerambycids, but the prevalence of (and the behavioural/ecological factors involved in) interspecific cross-breeding remain to be investigated.