Role of subfunctionalized MYB paralogs in the evolution of pigmentation patterning in Clarkia

Color has long served as a model trait for understanding questions at the intersection of genetics, development, and evolution. But recent advances in genomic and gene editing technologies are finally allowing for the in-depth study of the intricate spatial patterning of pigmentation, for example zebra stripes, butterfly wings, and flower spots. The study of plant pigmentation patterning is still in its infancy, but one surprising preliminary result is that patterning, specifically with anthocyanin pigments, always involves subfunctionalized MYB paralogs that each control the initiation of pigmentation in a specific pattern element. These early studies have mostly focused on the mechanism for patterning, though, and few have looked at the evolution and diversification of this complex trait.

Clarkia is a genus of 42 species that display intricate and widely varied pigmentation patterns on their petals while other species in the family have for the most part unpigmented and unpatterned petals, suggesting that pigmentation patterning first evolved at the origin of the Clarkia genus and then has diversified in extant species. In addition, the mechanism for patterning has been attributed to the function of three recently duplicated MYB paralogs in Clarkia gracilis, which makes this genus a tractable system for further understanding the evolution of pigmentation patterning between species. My research aims to answer three fundamental questions about the evolution of pigmentation patterning, using Clarkia as the focal system. 1) Did the duplication of the MYB paralogs coincide with the evolution of pigmentation patterning? 2) Did a patterning ground plan, with each MYB paralog regulating one specific pattern element, evolve in the ancestor of Clarkia and remain generally conserved across the species? 3) Do independent mutations to the MYB paralogs underlie the repeated evolution of similar phenotypes in distantly related species?

I used a combination of phylogenomics, transcriptomics, and classical genetics to answer these three questions. First, I inferred a well-supported species tree for 27 of 32 diploid species in the genus, and then explored a novel method for elucidating the putative parents for the 10 allopolyploid species. Then I used this species tree and whole petal transcriptomes from a majority of Clarkia species and six outgroup species in the Onagraceae family to explore the origin and role of these duplicated MYBs. Presence of these MYB paralogs in the petal transcriptomes of the outgroup species indicate that they duplicated long before the origin of Clarkia, and while all three seem to be integral for patterning across the genus, there is little evidence of a conserved ancestral ground plan to patterning.

I then focused on three distantly related species and used transcriptomics and classical genetics to disentangle the exact role that each paralog plays in patterning in these species, as well as the genetic basis for intraspecies variation in the presence or absence of each pattern element. I find that these paralogs have been repeatedly co-opted for different roles in patterning in the repeated evolution of similar pattern elements in distantly related species. I also find that intraspecies variation in patterning has likely evolved recently and often involves both cis-regulatory and coding mutations to the MYB paralogs. These findings, when combined with previous studies of plant pigmentation patterning, suggests that the modularity of the duplicated R2R3 MYB genes, with specific and less pleiotropically constrained function, may play a central role in pattern diversification in plants