Coding-sequence evolution does not explain divergence in petal anthocyanin pigmentation between Mimulus luteus var. luteus and M. l. variegatus

Biologists have long been interested in understanding genetic constraints on the evolution of development. For example, noncoding changes in a gene might be favored relative to coding changes due to being 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, transfecte..., A 1-mL disposable syringe with the needle removed was used to deliver A. tumefaciens cells containing overexpression transgenes to the leaves of young (1–3 months) N. tabacum. The top of the leaf was held firmly while the leaf underside was injected until liquid had visibly spread past the site of injection (100–200μL). The M. l. luteus and M. l. variegatus alleles of transcription factor gene MYB5a were infiltrated in pairs, alternating which transgene was injected into the left versus the right side of each leaf. pGFPGUSPlus negative controls were evenly distributed between the two sides of other leaves.
Leaves were imaged 3–12 days after infiltration in a dark room with a Nikon D3500 DSLR camera with an 18–55mm lens, illuminated by a Sylvania Ceramic Metal Halide bulb. VGG Image Annotator was used on .jpeg images to demarcate the infiltration boundaries and injection site.
A custom Python program imported RAW image files for processing, using a modified version of the MacDuff color c..., No special software is required to open the data files, but data may be analyzed using the image analysis code at https://github.com/WhitmanOptiLab/PigmentSpotting, # Data from: Coding-sequence evolution does not explain divergence in petal anthocyanin pigmentation between Mimulus luteus var. luteus and M. l. variegatus

Anthocyanin pigmentation values obtained from Nicotiana tabacum leaves that had been infiltrated with one of three overexpression transgenes (containing the coding regions of the MYB5a gene from Mimulus luteus var. luteus or from M. l. variegatus; or a negative control of pGFPGUSPlus).

## Description of the data and file structure

Damaged leaves were excluded from the dataset prior to analysis.

**Dataset1_Sgreen.csv:** Infiltrated areas of the leaf were digitally defined and analyzed for color.*
Pair: *Each pair of luteus and variegatus transgenes that were infiltrated into the same leaf also share an ID number.*
Treatment: *Leaf infiltrated with the coding regions of the MYB5a gene from Mimulus luteus var. luteus (\"luteus\") or from M. l. variegatus (\"variegatus\"); or a negative control of pGFPGUSPlus (\"pGFP\").*
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