‘Bad’ species? RADsequencing, DNA barcoding, and a role for Wolbachia in the evolutionary history of the Erebia tyndarus species complex

The Erebia tyndarus species complex is a cluster of closely related alpine butterflies. Members of this group have been characterized so far by morphological and ecological characters, cross-breeding experiments, karyology, and molecular data from allozymes and mitochondrial DNA (mtDNA). However, the delimitation of species, their relationships, as well as the origin of their peculiar geographic distribution, are still largely unclear. We employed restriction sites associated DNA (RAD) sequencing in order to clarify the systematic and genetic relationships among taxa of the E. tyndarus “Alpine” clade. Our results showed, for the first time, that the four species traditionally recognized in this clade (E. tyndarus, E. calcaria, E. nivalis, E. cassioides) indeed correspond to very well defined genetic units, which can be easily identified even by analysing small subsets of 15-20 of polymorphic markers. A species tree analysis based on a subset of 400 SNPs indicate that the four species gradually diverged from each other within the last 150,000 years. In contrast to the neat delimitation provided by nuclear RAD data, we did not find any species-specific synapomorphies at 2100 bp of mtDNA (Figure 1b), despite a clear intraspecific geographic structure. Moreover, our RADsequencing approach allowed to reveal the presence ofWolbachia endosymbionts in all of the species and geographic regions. We thus used coalescent simulations to compare the probability of the observed mtDNA data under a ‘neutral’ scenario of divergence fitted to our RAD-SNPs data and under a non-neutral scenario involving mtDNA replacement driven by Wolbachia infection. Results clearly indicate that a neutral scenario is extremely unlikely, and that the lack of interspecific mitochondrial differentiation in the E. tyndarus ‘Alpine’ clade is most probably due to a Wolbachia-driven mitochondrial replacement occurred ca. 15,000 – 40,000 years BP.