DNA taxonomy, phylogeny and Pleistocene diversification of the Cicindela hybrida species group (Coleoptera: Cicindelidae).

Species delimitation is complicated where morphological variation is continuous or poorly subdivided, but for taxonomic convenience it is common practice to separate and name geographical groups to capture this variation. DNA-based approaches may be used to test if these groups in fact represent historically divided, discrete species entities. The Cicindela hybrida complex (Coleoptera: Cicindelidae) is an assemblage of up to seven morphologically recognized species and 15 subspecies with wide distribution in the Palaearctic region. We sequenced a discontinuous segment of 1899 bp of mtDNA including three regions (coxI, rrnL+trnL2+nad1, cob) for a total of 99 specimens from 36 sampling localities across Europe, revealing 48 haplotypes. Four major clades could be identified corresponding to geographical groups from central Iberia, Ukraine, central Europe, and a band from the Atlantic Iberian coast to northern Europe. Taking into account further subdivisions within these clades, four of the six named species included in the analysis were recognizable by applying various procedures for species delimitation. Age estimates from calibrated molecular clocks date the diversification of the hybrida group within the past 2 million years (Myr), and the separation of the northern clade within 0.4 Myr. Nested clade analysis revealed the rapid range expansion of the northern group consistent with postglacial dispersal, but we did not find support for specific source population(s) in the postulated southern refugia. The evolutionary framework based on mtDNA sequences is shown to identify species entities as discrete clusters of closely related sequences and provides an objective system for delineating and recognizing hierarchically structured groups. In the case of the C. hybrida complex, these groups largely coincided with those established from morphology. The study adds further support to the utility of mtDNA-based sequence profiles (the 'DNA taxonomy') as a rapid and objective synthesis of evolutionary diversity and as reference system for communication.