Integrative characterization of genetic and phenotypic differentiation in an ant species complex with strong hierarchical population structure and low dispersal abilities.

Low dispersal, occurrence of asexual reproduction and geographic discontinuity increase genetic differentiation between populations, which ultimately can lead to speciation. In this work, we used a multidisciplinary framework to characterize the genetic and phenotypic differentiation between and within two cryptic ant species with restricted dispersal, Cataglyphis cursor and C. piliscapa and used behavioral experiments to test for reproductive isolation. Their distribution is segregated by the Rhône River and they have been traditionally distinguished only by hair numbers, although a statistical assessment is still lacking. We found strong genetic (microsatellites, nuclear and mitochondrial sequences), morphological (number of hairs, tibia length, male genitalia) and chemical (cuticular hydrocarbons) differentiation not only between species but also among localities within species. However, inter-specific differentiation was slightly higher than intra-specific differentiation for most markers. Overall, this pattern could either reflect reproductive isolation or could result from a longer period of geographic isolation between species than among localities within species without necessarily involving reproductive isolation. Interestingly, our behavioral experiments showed an absence of mating between species associated to a higher aggressiveness of workers towards heterospecific males. This suggests that sexual selection may, at least partially, fuel reproductive isolation. We also showed that cuticular hydrocarbons, mtDNA sequences and number of hairs provide reliable criteria allowing species discrimination. Overall, this species complex offers a case study to further investigate varying stages of a speciation continuum by estimating reproductive isolation between pairs of localities varying by their level of genetic differentiation.