The genetic basis of replicated bullseye pattern reduction across the Hibiscus trionum complex

Colorful petal patterns fulfill important functions and constitute excellent systems to illuminate the evolutionary processes that generate morphological diversity or instead support the repetitive emergence of similar forms. Here, we combined phylogenomic approaches, genetic manipulations, molecular techniques, and bee behavioral experiments to (i) solve the species relationships across the Trionum complex, a small Hibiscus clade that displays bullseye petal patterns varying in size, hue, and composition, (ii) identify key genes involved in the production of bullseye pigmentation, and (iii) reveal molecular events underpinning pattern variation during the evolution of the group. We found that epidermal cell shape, texture, and pigmentation are genetically distinct and that pigmentation is the most labile feature across the group. We demonstrate that repetitive bullseye reduction events primarily occur through independent modifications of a single genetic locus encoding BERRY1, an R2R3 MYB (myeloblastosis) that regulates anthocyanin pigment production in petals. We also found that buff-tailed bumblebees discriminate against flowers with smaller bullseye sizes, suggesting that changing bullseye proportions impact plant-pollinator interactions. Our results demonstrate how repeated mutations in a single locus led to morphological variation in petal patterning, a trait shown to impact plant fitness in other species and contribute to angiosperm reproductive isolation and speciation.