Petal anthocyanin evolution by R2R3 MYBs in Mimulus cupreus and Mimulus luteus var. variegatus

Repeated evolution refers to the independent gain of a single trait by multiple taxa. Studying examples of repeated evolution can elucidate patterns in how molecular mechanisms cause phenotypic change. In Chilean monkeyflowers (Mimulus), petal lobe anthocyanin pigmentation has been gained independently by three taxa: Mimulus luteus var. variegatus, Mimulus cupreus, and Mimulus naiandinus. Anthocyanins give the flowers of these taxa an orange or purple color, while other taxa in the same section of Mimulus have yellow flowers. One hypothesized mechanism for the gain of petal anthocyanin pigmentation is through modifications to R2R3 MYB transcription factors, which are activators in the anthocyanin biosynthetic pathway. This work explores how the expression of one R2R3 MYB candidate gene in M. l. variegatus and two in M. cupreus contribute to petal color phenotype. In M. l. variegatus, expression of a MYB5 splice variant is known to be necessary for petal anthocyanin biosynthesis. This study outlines the assembly of a MYB5 overexpression vector to be used to test the sufficiency of the splice variant’s expression to induce anthocyanin production in related yellow-flowered taxa. Two sequence variants of the MYB5 splice variant were discovered, and further studies are needed to investigate if they are alleles, genes, or transcripts, as well as to determine their roles in anthocyanin biosynthesis. In M. cupreus, RNAi knockdowns of candidate genes MYB2b and MYB3a were used to determine the necessity of each for petal anthocyanin pigmentation. The knockdown of each candidate gene resulted in a range of petal phenotypes, from yellow (completely lacking petal anthocyanins) to the wild type dark orange. To understand the significance of these results, a further study that determines the success of the knockdowns by testing transgene and target gene expression is needed. While research to determine the exact mechanisms underlying petal pigment evolution in M. l. variegatus and M. cupreus is still underway, the past and current work presented here suggests that in both taxa, a change in a MYB transcription factor gene has contributed to anthocyanin phenotypic change.