A cis-regulatory point mutation at a R2R3-Myb transcription factor contributes to speciation by reinforcement in Phlox drummondii

The process of reinforcement, whereby selection favors the evolution of increased reproductive trait divergence to reduce costly hybridization between species, has been well documented in nature, yet we know very little about how this process evolves at the molecular level. In this study, we combine functional characterization and genetic association tests to identify the mutational basis of reinforcement in the Texas wildflower Phlox drummondii. P. drummondii evolved from light to dark flower color intensity by selection to stop hybridization with the closely related species P. cuspidata, and previous research suggests differential expression of a R2R3-Myb transcription factor underlies this phenotypic transition. Using gene-silencing experiments, we demonstrate expression of this transcription factor does control variation in flower color intensity. We then apply association mapping across a large genomic region flanking the R2R3-Myb gene and identified a point mutation within the genes promoter that is highly associated with flower color intensity in nature. Alleles at this mutation site match the expected patterns of dominance, create variation in predicted cis-regulatory motifs within the R2R3-Myb proximal promoter, and occur in the direction of evolution predicted for flower color variation in this system. By identifying the mutational basis of reinforcement in this system we demonstrate that, as predicted by theory, reproductive isolation can evolve despite gene flow through a very simple genetic basis.