Differentiation in the marbled white butterfly species complex driven by multiple evolutionary forces

[Aim] Genetic and phenotypic data may show convergent or contrasting spatial patterns. Discrepancies between markers may develop in response to different evolutionary forces. In this study we analyse inter- and intraspecific differentiation of closely related taxa in the marbled white butterfly species group. Based on genetic and phenotypic characters we test for potential evolutionary drivers and propose a taxonomic revision.[Location] Western Palaearctic (including north-western Africa).[Methods] We compared distributions of mitochondrial cytochrome c oxidase subunit I gene (COI) sequences, of several allozyme loci, and of the shape of wings and genitalia obtained by applying landmark-based techniques for the three butterfly species Melanargia galathea (central and eastern Europe), M. lachesis (Iberia) and M. lucasi (North Africa).[Results] All studied markers showed a strong spatial structure, although discordance among their patterns was detected. COI sequences, wing shape and genitalia indicated a main split between M. galathea and M. lucasi. A lower differentiation between M. galathea and M. lachesis was found in wing shape and reflected in two mutations of the COI gene, while allozymes indicated a strong divergence. Within M. galathea, allozyme data and COI, but not morphology, revealed the existence of a slightly differentiated lineage in the Italian Peninsula, France and Switzerland. Based on COI, Melanargia lucasi was split into two subgroups, a western and an eastern Maghreb lineage.[Main conclusions] Long-term isolation of Melanargia populations between North Africa and Europe led to divergence between M. galathea and M. lucasi. This was followed by a recent differentiation among populations isolated during the cold periods of the Pleistocene, such as M. lachesis in Iberia. These lineages are characterized by a tendency not to overlap in secondary sympatry. The different patterns of the four markers may arise from divergent evolutionary processes and pressures: wings may be mainly affected by natural selection, genital structures by sexual selection, whereas long-term isolation and drift may have driven divergence of mitochondrial DNA and allozymes.Funding for this research was provided by the project ‘Barcoding Italian Butterflies’, by the European Union's Seventh Framework programme for research and innovation under the Marie Skłodowska-Curie grant agreement No 609402-2020 researchers: Train to Move (T2M) postdoctoral fellowship to R. Vodă, by European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant (project no. 658844 Eco-PhyloGeo) to L. Dapporto and by the Spanish Ministerio de Economía y Competitividad CGL2013-48277-P and PRX15/00305 to R. Vila.N