Project Summary Natural selection and gene flow are important during species formation, and yet there is still much to be learned about how these forces shape the evolution of mutations that cause reproductive isolation. The overall vision for the proposed research program is to use functional genetic and genomic investigations to determine when and how selection and gene flow contributed to divergence during speciation. This research investigates reinforcement, which is the evolution of reproductive isolation in response to selection to decrease costly hybridization. Reinforcement can be a critical step during the speciation process, and yet there is little known about this process at the molecular level. The mutations causing reinforcement have not been identified for any organism, and the effect that reinforcement has on patterns of genetic variation throughout the genome has not been previously investigated. Three major goals of the proposed research are to 1. identify the mutations causing reinforcement; 2. infer the evolutionary history of the mutations underlying reinforcement; and 3. determine the extent of and gene flow during reinforcement. Mutations causing reinforcement will be identified using genetic association mapping near candidate genes and transgenic functional validation. Once the causal mutations are identified, population genetic analyses quantifying and describing variation surrounding the causal mutations will be used to infer how the mutations evolved through time. Specifically, this research will examine target genomic regions for evidence of selection, and determine if the causal mutations were from standing genetic variation or arose under selection. Finally, this research will use innovative comparative genomic analyses to quantify introgression across geographic space, throughout time, and along the genome of two species involved in the reinforcement. This research will be accomplished in the wildflower Phlox drummondii, arguably the best-studied case of reinforcement. Previous work provides a clear ecological and evolutionary understanding of how and why reinforcement occurred. Field experiments have confirmed that reinforcement caused divergence in flower color and two candidate genes causing this divergence have been identified. This system offers a unique opportunity to identify the mutations causing reinforcement and the signature of variation this process leaves across the genome. Understanding selection and gene flow during the evolution of functional genetic variants is critical to determining how all organisms, including humans, adapt to changing environments. This work is transformative because it develops a novel genomic framework for understanding selection and gene flow during reinforcement.