Lucas C Wheeler, Joseph F. Walker, Julienne Ng, Rocio Deanna, Amy Dunbar-Wallis, Alice Backes, Pedro H. Pezzi, M. Virginia Palchetti, Andrew Monaghan, Loreta Brandão de Freitas

Evolutionary transitions in flower color often trace back to changes in the flavonoid biosynthetic pathway and its regulators. In angiosperms, this pathway produces a range of red, purple, and blue anthocyanin pigments. Transcription factor (TF) complexes involving members of the MYB, bHLH, and WD40 protein families control the expression of pathway enzymes. Here, we investigate flavonoid pathway evolution in the Petunieae clade of the tomato family (Solanaceae). Using transcriptomic data from 69 species of Petunieae, we estimated a new phylogeny for the clade. For the 65 species with floral transcriptomes, we retrieved transcripts encoding homologs of 18 enzymes and transcription factors to investigate patterns of evolution across genes and lineages. We found that TFs exhibit faster rates of molecular evolution than their targets, with the highly specialized MYB genes evolving fastest. Using the largest comparative dataset to date, we recovered little support for the hypothesis that upstream enzymes evolve slower than those occupying more downstream positions. However, expression levels inversely correlated with molecular evolutionary rates, while shifts in floral pigmentation were weakly related to changes affecting coding regions. Nevertheless, shifts in floral pigmentation and presence/absence patterns of MYB transcripts are strongly correlated. Intensely pigmented and patterned species express homologs of all three main MYB anthocyanin activators in petals, while pale or white species express few or none. Our findings reinforce the notion that regulators of the flavonoid pathway have a dynamic history, involving higher rates of molecular evolution than structural components, along with frequent changes in expression during color transitions.