Coding‐Sequence Evolution Does Not Explain Divergence in Petal Anthocyanin Pigmentation Between <i>Mimulus luteus</i> Var <i>luteus</i> and <i>M. l. variegatus</i>

Biologists have long been interested in understanding genetic constraints on the evolution of development. For example, noncoding changes in a gene might be favored over coding changes if they are less constrained by pleiotropic effects. Here, we evaluate the importance of coding-sequence changes to the recent evolution of a novel anthocyanin pigmentation trait in the monkeyflower genus Mimulus. The magenta-flowered Mimulus luteus var. variegatus recently gained petal lobe anthocyanin pigmentation via a single-locus Mendelian difference from its sister taxon, the yellow-flowered M. l. luteus. Previous work showed that the differentially expressed transcription factor gene MYB5a/NEGAN is the single causal gene. However, it was not clear whether MYB5a coding-sequence evolution (in addition to the observed patterns of differential expression) might also have contributed to increased anthocyanin production in M. l. variegatus. Quantitative image analysis of tobacco leaves, transfected with MYB5a coding sequence from each taxon, revealed robust anthocyanin production driven by both alleles. Counter to expectations, significantly higher anthocyanin production was driven by the allele from the low-anthocyanin M. l. luteus, a result that was confirmed through both a replication of the initial study and analysis by an alternative method of spectrophotometry on extracted leaf anthocyanins. Together with previously published expression studies, our findings support the hypothesis that petal pigment in M. l. variegatus was not gained by protein-coding changes, but instead solely via noncoding cis-regulatory evolution. Finally, while constructing the transgenes needed for this experiment, we unexpectedly discovered two sites in MYB5a that appear to be post-transcriptionally edited-a phenomenon that has been rarely reported, and even less often explored, for nuclear-encoded plant mRNAs.