Integrative analysis of DNA phylogeography and morphology of the European rose chafer (Cetonia aurata) to infer species taxonomy and patterns of postglacial colonisation in Europe.

Integrative taxonomy has been proposed as a framework to unify new conceptual and methodological developments in quantitative assessment of trait variation used in species delimitation, but empirical studies in this young branch of systematics are rare. Here we use standard phylogenetic and parsimony network analyses on nuclear and mitochondrial DNA (Cox1, ITS1) of 230 individuals from 65 European sampling sites in order to deduce population structure of Cetonia beetles from geno- and haplotypes. Statistical measures of population differentiation are inferred on genealogical and geographical scales to test hypotheses about species limits and population history. By combining results of phylogenetic structure with features of morphology, including genital shape morphometrics and discrete external body characters, as well as with measures of population genetics, we attempt to integrate the results as a test of the validity of species limits, in particular of currently recognised subspecies. Despite high Cox1 divergence between some haplotype lineages, even some sympatric lineages (9%, e.g. N2 vs. N4), nDNA and morphology, as well as pattern of geographical and genealogical divergence measured by AMOVA analysis did not support the hypothesis of separate species. Highest divergence in nuclear markers was found among Italian populations of C. aurata pisana and C. a. sicula, and moderately high fixation indices along measurable morphological divergence suggest the correctness of their status as 'subspecies'. Divergence time estimates of the lineages suggest a glacial divergence in different refugia between the major haplogroups, while population differentiation in mtDNA among these was primarily attributable to restricted gene flow caused by geographic isolation.