DOI: 10.5061/dryad.7d7wm3814
title: Data from: Coding-sequence evolution does not explain divergence in petal anthocyanin pigmentation between Mimulus luteus var. luteus and M. l. variegatus

description: 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, transfected with MYB5a
 coding sequence from each taxon, revealed robust anthocyanin production
 driven by both alleles. Counter to expectations, significantly higher
 anthocyanin production was driven by the allele from the low-anthocyanin
 M. l. luteus. Together with previously-published expression studies, this
 supports the hypothesis that petal pigment in M. l. variegatus was not
 gained by protein-coding changes, but instead solely via non-coding
 cis-regulatory evolution. Finally, while constructing the transgenes
 needed for this experiment, we unexpectedly discovered two sites in MYB5a
 that appear to be post-transcriptionally edited – a phenomenon that has
 been rarely reported, and even less often explored, for nuclear-encoded
 plant mRNAs.
description: A 1-mL disposable syringe with
 the needle removed was used to deliver <em>A. tumefaciens</em>
 cells containing overexpression transgenes to the leaves of young (1–3
 months) <em>N</em>. <em>tabacum</em>. 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
 <em>M. l. luteus </em>and<em> M. l.
 variegatus</em> alleles of transcription factor gene
 <em>MYB5a</em> 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 chart
 detection algorithm (https://github.com/mathandy/python-macduff-colorchecker-detector) to automatically detect a reference color chart. Image pixel values were converted into normalized reflectance values based on a linear fit of the red, green, and blue signal strengths to the color chart.<br>
description: 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
description: # 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").* Vis**-red:* 1 indicates pigment intensity higher than
 the average for the negative controls and represents plants where red
 pigment was visibly produced. A value of 0 corresponds to no visible
 pigment produced. "n/a" corresponds to the negative controls
 which were not assessed. *Sgreen:* Obtained by digital image analysis of
 the infiltrated region as described in the manuscript. A lower Sgreen
 value corresponds to a greater intensity of red (anthocyanin)
 pigmentation. *Redness:* 1-Sgreen of the infiltrated region.
 **Dataset2_Infiltrated_Leaves_Data_Summer_2024_ForDataDryad.csv:** Leaf
 discs of uniform size were excised from infiltrated leaves and were
 analyzed in two ways: digitally, as in Dataset 1, and also by extracting
 their anthocyanins and quantifying anthocyanin concentration via
 spectrophotometry. *Leaf ID: Each pair of luteus and variegatus transgenes
 that were infiltrated into the same leaf also share an ID number.
 Type:* The line of Nicotiana tabacum plant into which the transgenes were
 infiltrated. *LeafNumber:* Which leaf was infiltrated, counting from the
 bottom up with the first true leaf assigned the number 1.
 *LeafSize:* Length in centimeters. *Treatment: *Leaf infiltrated with the
 coding regions of the MYB5a gene from Mimulus luteus var. luteus
 ("l") or from M. l. variegatus ("v"); or a negative
 control of pGFPGUSPlus ("n"). Infiltration:* Indicates which
 infiltration number the data were obtained from, with the first four
 infiltrations used as pilots to establish methodology and excluded from
 the final dataset. *A530:* Absorbance at 530 nm of the anthocyanin
 extraction from a leaf disc. *A653:* Absorbance at 653 nm of the
 anthocyanin extraction from a leaf disc. *Anthocyanin concentration
 estimated by spectrophotometry, in Absorbance Units:* A530-(0.24A653).
 *Visible Anthocyanin in leaf?* 0 = none, 1 = faint, 2 = dark *Sgreen of
 Punch: *Obtained by digital image analysis of a leaf disc as described in
 the manuscript. A lower Sgreen value corresponds to a greater intensity of
 red (anthocyanin) pigmentation. *Redness of punch:* 1-Sgreen of the leaf
 disc.
 **Dataset1_All_Tobacco_leaf_images_Brightness_30percent.pptx:** Whole-leaf
 images from Dataset 1, with brightness uniformly increased 30% in
 PowerPoint for better clarity to the human eye.
 **Dataset2_LeafPunchImages.zip.** Leaf disc images from Dataset 2,
 unmodified. **Dataset2_WholeLeafImages.zip.** Whole-leaf images from
 Dataset 2, prior to punching out a leaf disc, unmodified. ## Code/Software
 Image analysis code is at
 [https://github.com/WhitmanOptiLab/PigmentSpotting](https://github.com/WhitmanOptiLab/PigmentSpotting).