Evidence of prezygotic isolation, but not assortative mating, between locally adapted populations ofFundulus heteroclitusacross a salinity gradient

Selection along environmental gradients can drive reproductive isolation and speciation. Among fishes, salinity is a major factor limiting species distributions and, despite its importance in generating species diversity, transitions between marine and freshwater are rare. Here, we tested for mechanisms of reproductive isolation between locally adapted freshwater and brackish water-native populations of killifish, Fundulus heteroclitus , from either side of a hybrid zone along a salinity gradient. There was evidence for pre-zygotic endogenous reproductive isolation with reduced fertilization success between crosses of freshwater-native males and brackish water-native females. Exogenous pre-zygotic isolation was also present where females had highest fertilization in their native salinity. We used a replicated mass spawning design to test for mate choice in both brackish and fresh water. After genotyping 187 parents and 2,523 offspring at 2,347 SNPs across the genome, 85% of offspring were successfully assign to their parents. However, no reinforcing mate choice was observed. These results therefore demonstrate emerging, yet limited, reproductive isolation and incipient speciation across a marine to freshwater salinity gradient and suggest that both endogenous and exogenous mechanisms, but not assortative mating, contribute to divergence.