Single gene mutation in a plant MYB transcription factor causes a major shift in pollinator preference

Understanding the molecular basis of reproductive isolation and speciation is a key goal of evolutionary genetics. In the South American genus Petunia, the R2R3-MYB transcription factor MYB-FL regulates the biosynthesis of UV-absorbing flavonol pigments, a major determinant of pollinator preference. MYB-FL is highly expressed in the hawkmoth-pollinated P. axillaris, but independent losses of its activity in sister taxa P. secreta and P. exserta led to UV-reflective flowers and associated pollinator shifts in each lineage (bees and hummingbirds, respectively). We created a myb-fl CRISPR mutant in P. axillaris and studied the effect of this single gene on innate pollinator preference. The mutation strongly reduced the expression of the two key flavonol-related biosynthetic genes but only affected the expression of few other genes. The mutant flowers were UV reflective as expected but additionally contained low levels of visible anthocyanin pigments. Hawkmoths strongly preferred the wild-type P. axillaris over the myb-fl mutant, whereas both social and solitary bee preference depended on the level of visible color of the mutants. MYB-FL, with its specific expression pattern, small number of target genes, and key position at the nexus of flavonol and anthocyanin biosynthetic pathways, provides a striking example of evolution by single mutations of large phenotypic effect. [Display omitted] •MYB-FL mutation strongly reduces floral UV color but increases visible color•The mutation reduces hawkmoth visitation but increases bee visitation•Bees discriminate between mutants with small intraspecific differences in visible color•Major shift in pollinator preference by a single mutation of large phenotypic effect UV color is a major determinant of pollinator preference. Lüthi et al. mutate MYB-FL, the key regulator of flavonol biosynthesis in Petunia. Hawkmoths prefer the UV-absorbent wild type, whereas bees prefer the mutant. This work represents a striking example of a single mutation of large phenotypic effect on pollinator-mediated reproductive isolation.