Volume 21, Issue 8, Pages 700-704 (April 2011) Gene Duplication in Mimulus Underlies Parallel Floral Evolution via Independent trans- Regulatory Changes  Arielle M. Cooley, Jennifer L. Modliszewski, Megan L. Rommel, John H. Willis  Current Biology  Volume 21, Issue 8, Pages 700-704 (April 2011) DOI: 10.1016/j.cub.2011.03.028 Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 1 Spatial Distribution of Cyanidin Has Diversified in the luteus Group of Mimulus (A) Within the luteus group (black branches), increased anthocyanin production yields the orange of M. cupreus (in combination with yellow carotenoids) and the purple of M. l. variegatus and M. naiandinus (on a white, carotenoid-free background). The M. guttatus and M. glabratus species complexes (gray branches) are speciose but have constant floral pigmentation, in which anthocyanin pigment is restricted to the throat and lower central petal. See Figure S1 for possible paths to the evolution of petal lobe anthocyanin (PLA) in M. cupreus, M. l. variegatus, and M. naiandinus. Genotype at pla1 is sufficient to explain PLA presence versus absence between the orange and yellow morphs of M. cupreus, whereas genotype at pla2 controls PLA presence versus absence between M. l. variegatus and M. l. luteus [4]. (B) The red pigment in the luteus group is caused by cyanidin, one of several types of red anthocyanin pigment [20]. Cyanidin is the product of the precursor compounds listed in regular type, acted on by the six enzymes shown in boldface. Current Biology 2011 21, 700-704DOI: (10.1016/j.cub.2011.03.028) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 2 Flower Color Cosegregates with Expression of Anthocyanin Enzyme-Encoding Genes mRNA expression is higher in +PLA than in −PLA plants for all eight assayed genes in M. cupreus (A) and for all three assayed genes in M. l. luteus, M. l. variegatus, and their F2 hybrid progeny (B). Black columns indicate the mean of +PLA F2 progeny; white columns indicate the mean of the −PLA parent and F2 progeny; error bars show standard error. “h1” (homeolog 1) and “h2” (homeolog 2) were arbitrarily assigned based on order of discovery. Asterisks indicate significance of the gene expression difference between +PLA and −PLA phenotypes: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. The +PLA parent was used as the reference. Sample sizes for M. cupreus were n = 6 (+PLA F2s) and n = 7 (−PLA F2s plus the −PLA parent). Sample sizes for M. l. luteus × M. l. variegatus were n = 6 (+PLA F2s) and n = 6 (−PLA F2s plus the −PLA parent). Assays were performed on early-stage floral buds. See Figure S2 for a time course of gene expression during bud development and Tables S1 and S2 for additional qPCR details. Current Biology 2011 21, 700-704DOI: (10.1016/j.cub.2011.03.028) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 3 Five Anthocyanin-Related R2R3 MYBs Are Found in M. guttatus (A) Neighbor-joining tree of amino acid data, including all 57 R2R3 MYBs from M. guttatus as well as MYBs from other angiosperm species, constructed to reveal the R2R3 MYBs involved in anthocyanin pigmentation. Branches are labeled with bootstrap support values > 50%. The clade containing R2R3 MYBs known to regulate anthocyanin biosynthesis is shown in red. (B) Expanded view of the anthocyanin-related R2R3 MYBs. Bootstrap support values > 50% are shown. Species are indicated by the following two-letter abbreviations: Am, Antirrhinum majus; At, Arabidopsis thaliana; In, Ipomoea nil; Ip, Ipomoea purpurea; Le, Lycopersicon esculentum; Ma, Mimulus aurantiacus; Mg, Mimulus guttatus; Ph, Petunia hybrida; Vv, Vitis vinifera. Abbreviated gene names as listed in GenBank are given following each species designation. See Figure S3 for gene trees of MYBs 1–5 from M. guttatus, M. cupreus, M. l. variegatus, and M. l. luteus. Current Biology 2011 21, 700-704DOI: (10.1016/j.cub.2011.03.028) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 4 Candidate Genes in M. guttatus that Surround Markers Linked to Flower Color (A) The pla1 locus on scaffold 11 of M. guttatus. (B) The pla2 locus on scaffold 132 of M. guttatus. MYB genes are shown in black; numbers 1–5 indicate MgMYB1–MgMYB5. The coding sequence of each MYB is represented as an arrow pointing toward the 3′ end, with spaces representing the two introns. Two truncated MYBs are labeled as “partial.” Other candidate genes are shown as gray arrows; gene size and orientation are indicated but exon/intron boundaries are not. Numbers above each hash mark indicate nucleotide position along each scaffold. Although 1 Mb regions were scanned for gene sequences, only 300 kb of each scaffold are shown here, because no likely candidates were discovered outside of this section. See Figure S4 for MYB homologs and homeologs from M. cupreus, M. l. variegatus, and M. l luteus and Table S3 for preliminary functional tests of the MYBs. Current Biology 2011 21, 700-704DOI: (10.1016/j.cub.2011.03.028) Copyright © 2011 Elsevier Ltd Terms and Conditions