Pinpointing the level of isolation between two cryptic species sharing the same microhabitat : a case study with a scarabaeid species complex

Understanding the processes of speciation is an important challenge in improving knowledge of the origin of biodiversity. One crucial point is to assess the causes of reproductive isolation, especially in the case of co-occurring species. Differences in microscale spatial distribution in small organisms may blur the issue. We explored biological processes underlying speciation within dung beetles belonging to the vacca species complex (Scarabaeidae: Onthophagus). The two taxa of this complex, O.vacca and O.medius, not only are known to have a large overlapping Palearctic distribution range but also share the same cowpat with no physical barriers and no observed specific aggregated patterns in the local distribution. The present study aimed at determining the level of isolation between the two taxa and discusses the most likely scenario of the speciation (sympatry vs. allopatry) based on the Coyne & Orr's (2004) four criteria. We conducted a full study on populations sampled within the Mediterranean region integrating morphological analysis (digital image analysis of the elytral melanism pattern), two-gene phylogenies, population genetic analyses on populations sampled from an area where both species occur and another one with O.vacca only, as well as intra- and interspecific mating and crossing bioassays. The variation in the elytral melanism pattern clearly followed a bimodal distribution, with O.medius being more melanic than O.vacca, with a very limited overlapping area. The two taxa are reproductively isolated, with a strong postzygotic incompatibility despite the absence of sexual isolation. Sequence analysis of both nuclear and mitochondrial markers revealed a deep divergence between the two taxa dating back to 8.7Mya. All findings concurred with some phenological observations and the conclusion that the most likely scenario for speciation in the vacca complex was an allopatric speciation followed by secondary contact