Multi-locus DNA sequence variation in a complex of four leaf beetle species with parapatric distributions: mitochondrial and nuclear introgressions reveal recent hybridization.

While the importance for including multiple independent loci in phylogeographic studies is largely acknowledged, a majority of these still focus on a single species. We combine the study of multilocus DNA sequence variation (one mitochondrial and four unlinked nuclear fragments) at both the inter- and intraspecific levels to explore the evolutionary history of four closely related specialized herbivorous insects (Chrysomelidae, genus Gonioctena). Analyzing the sequences for samples collected across their entire European range allows us to (1) characterize the genetic boundaries among species, i.e. the degree of lineage sorting, (2) infer their phylogenetic relationships and (3) explore reproductive barriers among them in regions where their ranges overlap. For two sister species, we identify multiple independent cases of unidirectional transfer of genetic material (introgression) at both mitochondrial and nuclear loci, demonstrating recent hybridization between both species in the overlapping regions of their range. The highlighted pattern of genetic variation suggests that Gonioctena intermedia expanded its range into that of G. quinquepunctata, and that both species may experience mutual exclusion. Overall, this study illustrates that interpreting intraspecific genetic variation for the purpose of evolutionary inference without the broader context of the other closely related species could lead to erroneous conclusions.