Characterization of the phenotypic differentiation and genetic basis of traits that can contribute to reproductive isolation is an important avenue to understand the mechanisms of speciation. We quantified the degree of prezygotic isolation and geographic variation in mating behaviour among four populations of Sepsis neocynipsea that occur in allopatry, parapatry, or sympatry with four populations of its sister species S. cynipsea. To obtain insights into the quantitative genetic basis and the

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... ole of selection against hybrid phenotypes we also investigated mating behaviour of F 1 hybrid offspring and corresponding backcrosses with the parental populations. Our study documents successful hybridisation under laboratory conditions, with low copulation frequencies in heterospecific pairings but higher frequencies in pairings of F 1 hybrids signifying hybrid vigour. Analyses of F 1 offspring and their parental backcrosses provided little evidence for sexual selection against hybrids. Longer copulation latencies in heterospecific pairings indicate species recognition, probably due to surface or volatile chemicals. The frequency of male mating attempts did not differ greatly among species or hybrid pairings, suggesting no male discrimination of mating partners. Female shaking duration, signifying female choice and/or reluctance to mate, differs strongly between the species and appears to contribute to avoiding heterospecific males; this trait is partially maternal inherited. Importantly, females of both species discriminated more strongly against males in areas of sympatry than allopatry indicating reinforcement. Shorter copulations in heterospecific parental pairings and longer copulations in F 1 hybrids suggest mechanistic difficulties with sperm transfer. Overall, our study highlights an important role of character displacement affecting mating behaviour of hybridising sepsid species in geographic areas of co-existence. Highlights • Successful but low rates of hybridisation • Females, but not males, discriminate against heterospecific mating partners • Maternal inheritance of female mating behaviour • Biogeographic variation in heterospecific mating behavior driven by reinforcement • Little evidence for sexual selection against hybrids