Complex evolution of novel red floral color in Petunia

Red flower color has arisen multiple times and is generally associated with hummingbird pollination. The majority of evolutionary transitions to red color proceeded from purple lineages and tend to be genetically simple, almost always involving a few loss-of-function mutations of major phenotypic effect. Here we report on the complex evolution of a novel red floral color in the hummingbird-pollinated Petunia exserta (Solanaceae) from a colorless ancestor. The presence of a red color is remarkable because the genus cannot synthesize red anthocyanins and P. exserta retains a nonfunctional copy of the key MYB transcription factor AN2. We show that moderate upregulation and a shift in tissue specificity of an AN2 paralog, DEEP PURPLE, restores anthocyanin biosynthesis in P. exserta. An essential shift in anthocyanin hydroxylation occurred through rebalancing the expression of three hydroxylating genes. Furthermore, the downregulation of an acyltransferase promotes reddish hues in typically purple pigments by preventing acyl group decoration of anthocyanins. This study presents a rare case of a genetically complex evolutionary transition toward the gain of a novel red color.