Predictable patterns of constraint among anthocyanin-regulating transcription factors in Ipomoea

• Transcription factors (TFs) may play a central role in plant morphological evolution. Variation in the nonsynonymous to synonymous nucleotide substitution rate (dN/dS) ratio among TFs can be attributed to either differences in constraint or the frequency of adaptive substitution. However, the relative contribution of these forces to the variation in dN/dS ratios is unknown. • We synthesize current and previous results comparing the variation in dN/dS ratios among members of the MYB-bHLH-WDR complex of TFs that regulates floral anthocyanin pigmentation in Ipomoea. • Low values of dN/dS in a WDR gene are the result of exceptionally strong purifying selection, with no evidence of positive selection. bHLH and MYB genes also fail to show evidence for positive selection, but have higher dN/dS ratios, indicating reduced selective constraint. • Differences in constraint are consistent with expectations based on the intrinsic features and regulatory network properties among these proteins. Significantly elevated dN/dS ratios in the MYB gene suggest that mutations experience reduced magnitudes of deleterious pleiotropy compared with the rest of the complex. Although reduced constraint may account for the observation that Myb mutations disproportionately contribute to differences in floral pigmentation, the lack of detectable positive selection in any of these TF proteins suggests that amino acid substitutions contribute little to flower colour evolution.