Transitions to a new floral color 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 P. exserta retains a nonfunctional copy of the floral anthocyanin activating MYB transcription factor AN2, and additionally the genus cannot synthesize monohydroxylated red anthocyanins. We show that moderate up-regulation and a shift in tissue specificity of an AN2 paralog, DEEP PURPLE (DPL) restores anthocyanin biosynthesis in P. exserta, a case of regulatory neofunctionalization. An essential shift in anthocyanin hydroxylation occurred through re-balancing the expression of three hydroxylating genes. Furthermore, the down-regulation of an acyltransferase promotes reddish hues in typically purple delphinidin pigments by preventing acyl group decoration of anthocyanins. Finally, additional mutations of relatively small effect are also involved. This study demonstrates that commonly observed phenotypic transitions, such as in floral color, can involve many more loci than predicted. Repeated evolution of red flowers has occurred in the Solanaceae and in the same biosynthetic pathway, yet the precise mechanism is less predictable and complex.

Supplemental Dataset 1. Fasta alignment of MYB Subgroup 6 and PH4 clade (P. exserta, P. axillaris, P. secreta)

Supplemental Dataset 2. Newick tree file for MYB Subgroup 6 and PH4 clade (P. exserta, P. axillaris, P. secreta)

Supplemental Dataset 3. Fasta alignment of entire MYB tree (P. exserta, P. axillaris)

Supplemental Dataset 4. Newick tree file for entire MYB tree (P. exserta, P. axillaris)

Supplemental Dataset 5. Normalized read counts for flavonoid and phenylpropanoid gene expression across Petunia species.

Additionally:

Supplemental protein alignments for Petunia DPL and PH4 transcription factors

Supplemental protein alignments for Petunia flavonoid pathway biosynthetic genes

Supplemental nucleotide alignments of AN4 and AN4-like genes detected in the draft P. exserta genome

Supplemental R/qtl datafile for RILs

Genome annotation file for P. secreta genome assembly