jats:titleSummary</jats:title>jats:p <jats:list list-type="bullet">

jats:list-itemjats:pjats:italicGorteria diffusa</jats:italic> has elaborate petal spots that attract pollinators through sexual deception, but how jats:italicG. diffusa</jats:italic> controls spot development is largely unknown. Here, we investigate how pigmentation is regulated during spot formation.</jats:p></jats:list-item>

jats:list-itemjats:pWe determined the anthocyanin composition of jats:italicG. diffusa</jats:italic> petals and combined gene expression analysis with protein interaction assays to characterise R2R3‐MYBs that likely regulate pigment production in jats:italicG. diffusa</jats:italic> petal spots.</jats:p></jats:list-item>

jats:list-itemjats:pWe found that cyanidin 3‐glucoside pigments jats:italicG. diffusa</jats:italic> ray floret petals. Unlike other petal regions, spots contain a high proportion of malonylated anthocyanin. We identified three subgroup 6 R2R3‐MYB transcription factors (GdMYBSG6‐1,2,3) that likely activate the production of spot pigmentation. These genes are upregulated in developing spots and induce ectopic anthocyanin production upon heterologous expression in tobacco. Interaction assays suggest that these transcription factors regulate genes encoding three anthocyanin synthesis enzymes.</jats:p></jats:list-item>

jats:list-itemjats:pWe demonstrate that the elaboration of complex spots in jats:italicG. diffusa</jats:italic> begins with the accumulation of malonylated pigments at the base of ray floret petals, positively regulated by three paralogous R2R3‐MYB transcription factors. Our results indicate that the functional diversification of these jats:italicGdMYBSG6s</jats:italic> involved changes in the spatial control of their transcription, and modification of the duration of jats:italicGdMYBSG6</jats:italic> gene expression contributes towards floral variation within the species.</jats:p></jats:list-item> </jats:list> </jats:p>