Linda Hagberg, Enrique Celemin, Iker Irisarri, Oliver Hawlitschek, Jose L. Bella, Tami Mott, Ricardo J. Pereira

• The process of species formation is characterized by the accumulation of multiple reproductive barriers. The evolution of hybrid male sterility, or Haldane's rule, typically characterizes later stages of species formation, when reproductive isolation is strongest. Yet, understanding how quickly reproductive barriers evolve and their consequences for maintaining genetic boundaries between emerging species remains a challenging task because it requires studying taxa that hybridize in nature. Here, we address these questions using the meadow grasshopper Pseudochorthippus parallelus, where populations that show multiple reproductive barriers, including hybrid male sterility, hybridize in two natural hybrid zones. Using mitochondrial data, we infer that such populations diverged some 100,000 years ago, at the beginning of the last glacial cycle in Europe. Nuclear data show that contractions at multiple glacial refugia, and post-glacial expansions have facilitated genetic differentiation between lineages that today interact inThe process of species formation is characterized by the accumulation of multiple reproductive barriers. The evolution of hybrid male sterility, or Haldane's rule, typically characterizes later stages of species formation, when reproductive isolation is strongest. Yet, understanding how quickly reproductive barriers evolve and their consequences for maintaining genetic boundaries between emerging species remains a challenging task because it requires studying taxa that hybridize in nature. Here, we address these questions using the meadow grasshopper Pseudochorthippus parallelus, where populations that show multiple reproductive barriers, including hybrid male sterility, hybridize in two natural hybrid zones. Using mitochondrial data, we infer that such populations diverged some 100,000 years ago, at the beginning of the last glacial cycle in Europe. Nuclear data show that contractions at multiple glacial refugia, and post-glacial expansions have facilitated genetic differentiation between lineages that today interact in hybrid zones. We find extensive introgression throughout the sampled species range, irrespective of the current strength of reproductive isolation. Populations exhibiting hybrid male sterility in two hybrid zones show repeatable patterns of genomic differentiation, consistent with shared genomic constraints affecting ancestral divergence or with the role of those regions in reproductive isolation. Together, our results suggest that reproductive barriers that characterize late stages of species formation can evolve relatively quickly, particularly when associated with strong demographic changes. Moreover, we show that such barriers persist in the face of extensive gene flow, allowing future studies to identify associated genomic regions.…