DOI: 10.5061/dryad.37pvmcvr6
title: Data from: Floral color variation across life history and geography in Mimulus ringens (Phrymaceae)

description: With these data, we explore the possibility that both floral color and
 life history have shifted together in a recently described, genetically
 distinct group within the species Mimulus ringens. Using a large,
 range-wide citizen science dataset, we test for geographic trends in
 flower color and flowering time. We combine this with greenhouse studies
 in populations of known life history to test for differences in flower
 color with life history. We show that darker-flowered plants are more
 common at higher latitudes, that annual-like populations have darker
 flowers, and that flowering time varies with latitude only in the subset
 of populations that have lighter flowers. This suggests that annual-like
 populations (with the earlier flowering time typical of this life history)
 are restricted to the northern part of the species range, and likely arose
 there. 
description: Greenhouse study <br>            We assessed flower color
 in two ways: anthocyanin extractions and photographic analyses. We
 obtained 22 individual plants from six populations from the Sobel
 Laboratory of SUNY Binghamton University. Three populations had been
 previously identified as perennials and three populations as annual-like.
 Flowers were collected from September to November 2021.<br>         
   Plants were vernalized for six weeks at 4ºC and moved on August 9 2021
 to the greenhouse in the Loyola Science Center at the University of
 Scranton, Scranton, Pennsylvania, USA, where they were kept in 16 hour
 days (28ºC) and 8 hour nights (18ºC) until flowering was complete. Water
 levels were monitored daily and plants were bottom-watered when their
 trays appeared dry. If an individual’s leaves turned brown, the plant was
 immediately repotted to regulate soil pH. Plants were sprayed with Neem
 Oil when spider mites appeared.<br>            Flowers were sampled
 every other day throughout the flowering season using the freshest
 fully-opened flowers from each plant. During collection, we prioritized an
 even sampling and attempted to collect at least one fresh flower from each
 plant each sampling day. However, as plants flowered at different times,
 we collected fewer flowers from some plants. In addition, on days when
 only a few plants produced fresh flowers, we would collect up to four
 flowers from those plants on an individual day. A flower was removed with
 forceps and placed in a test tube holder. A unique label was created for
 each flower, which included plant identification number, sample number
 from that plant, collector’s initials, and date. <br>            We
 then photographed each flower from the side and the front using a Canon
 EOS Rebel XSi digital camera fitted with a macro lens, flash, and flash
 diffuser (attached to the manual focus) and mounted at 46.8 cm above the
 base of a copy stand. We set the camera to an AV setting with the white
 balance set to flash, F-stop of 10, ISO of 100, and picture style of
 “faithful.” Each photograph contained the flower of interest, the entirety
 of the unique label, and a corner of an X-Rite Classic Mini color checker
 (the white box, the first 3 grayscale boxes, and the first ~10mm of the
 scale). We used a Canon RS-60ES Remote Switch to take each
 photograph.<br>            After photographing each flower, we used
 a standard hole punch to remove a circle of tissue from the right lower
 lip of each flower with the standard corolla pigment (without any throat
 color). We extracted the anthocyanins in 1mL of methanol/1%HCl, and stored
 it for 12-24 hours. After centrifuging each sample, we removed 500mL of
 supernatant to evaluate absorbance. We used a Genesys 20 Thermo Scientific
 spectrophotometer to analyze absorbance at 520nm. Higher absorbance values
 indicate darker flowers, with more anthocyanin.<br>            To
 analyze the color from each photograph, we used the program FIJI, an
 ImageJ application. To standardize each image, we used the third grayscale
 square from the right, white side of the color checker. We used the first
 analyzed image as our standard. We loaded the image, then selected image
 &gt; adjust &gt; color balance &gt; all colors. Using the
 “auto” balance function, we balanced the image’s color, saved the image as
 a new file, and used analyze &gt; measure to assess the mean gray
 value of the square for standardizing. As the mean gray value was 183.493,
 we then standardized each image so that the third grayscale square had a
 mean gray value between 183 and 184. <br>            After saving
 each standardized image as a new file, we created a region of interest
 (ROI) that encompassed the entirety of the flower petals in the image. We
 then performed analyze &gt; color histogram to obtain red, green, and
 blue color values. We then split the image into stacks of hue, saturation,
 and brightness. For each stack, we obtained the mean gray value.
description: # Data from: Floral color variation across life history and geography in
 Mimulus ringens (Phrymaceae)
 [https://doi.org/10.5061/dryad.37pvmcvr6](https://doi.org/10.5061/dryad.37pvmcvr6) Give a brief summary of dataset contents, contextualized in experimental procedures and results. ## Description of the data and file structure GENERAL INFORMATION 1\. Title of Dataset: Data from: Floral color variation across life history and geography in Mimulus ringens \(Phrymaceae\) 2\. Author Information Corresponding Investigator Name: Dr. Anne Royer Institution: University of Scranton, Scranton, PA, USA 800 Linden St., Scranton, PA, 18510 570-941-7469 anne.royer@scranton.edu Co-investigator 1 Name: Sarah White Institution: University of Scranton, Scranton, PA, USA 3\. Date of data collection: 2020\-2022 4\. Geographic location of data collection: Scranton\, PA\, USA 5\. Funding sources that supported the collection of the data: University of Scranton 6\. Recommended citation for this dataset: White and Royer \(2023\)\, Data from: Floral color variation across life history and geography in Mimulus ringens \(Phrymaceae\, Dryad\, Dataset DATA & FILE OVERVIEW 1\. Description of dataset The data were generated to investigate associations between life history, floral color, geographic location, and flowering time in the species Mimulus ringens, which includes perennial and annual-like populations. Flowering time, floral color, and geographic location were analyzed using 1458 observations from a citizen science dataset from INaturalist, downloaded from the Global Biodiversity Information Facility (GBIF) database.The relationship between flower color and life history was studied by collecting floral color information from six populations of known life history (three annual, three perennial) grown in a common greenhouse at the University of Scranton. File 1 Name: WhiteRoyer\_Greenhouse.csv File 1 Description: Floral color observations from greenhouse-grown plants, including date of collection, plant ID, maternal plant ID, population, life history, and floral color measured from photos and using spectrophotometry on extracted pigments METHODOLOGICAL INFORMATION   Greenhouse study             We assessed flower color in two ways: anthocyanin extractions and photographic analyses. We obtained 22 individual plants from six populations from the Sobel Laboratory of SUNY Binghamton University. Three populations had been previously identified as perennials and three populations as annual-like. Flowers were collected from September to November 2021.             Plants were vernalized for six weeks at 4ºC and moved on August 9 2021 to the greenhouse in the Loyola Science Center at the University of Scranton, Scranton, Pennsylvania, USA, where they were kept in 16 hour days (28ºC) and 8 hour nights (18ºC) until flowering was complete. Water levels were monitored daily and plants were bottom-watered when their trays appeared dry. If an individual’s leaves turned brown, the plant was immediately repotted to regulate soil pH. Plants were sprayed with Neem Oil when spider mites appeared.             Flowers were sampled every other day throughout the flowering season using the freshest fully-opened flowers from each plant. During collection, we prioritized an even sampling and attempted to collect at least one fresh flower from each plant each sampling day. However, as plants flowered at different times, we collected fewer flowers from some plants. In addition, on days when only a few plants produced fresh flowers, we would collect up to four flowers from those plants on an individual day. A flower was removed with forceps and placed in a test tube holder. A unique label was created for each flower, which included plant identification number, sample number from that plant, collector’s initials, and date.             We then photographed each flower from the side and the front using a Canon EOS Rebel XSi digital camera fitted with a macro lens, flash, and flash diffuser (attached to the manual focus) and mounted at 46.8 cm above the base of a copy stand. We set the camera to an AV setting with the white balance set to flash, F-stop of 10, ISO of 100, and picture style of “faithful.” Each photograph contained the flower of interest, the entirety of the unique label, and a corner of an X-Rite Classic Mini color checker (the white box, the first 3 grayscale boxes, and the first \~10mm of the scale). We used a Canon RS-60ES Remote Switch to take each photograph.             After photographing each flower, we used a standard hole punch to remove a circle of tissue from the right lower lip of each flower with the standard corolla pigment (without any throat color). We extracted the anthocyanins in 1mL of methanol/1%HCl, and stored it for 12-24 hours. After centrifuging each sample, we removed 500mL of supernatant to evaluate absorbance. We used a Genesys 20 Thermo Scientific spectrophotometer to analyze absorbance at 520nm. Higher absorbance values indicate darker flowers, with more anthocyanin.             To analyze the color from each photograph, we used the program FIJI, an ImageJ application. To standardize each image, we used the third grayscale square from the right, white side of the color checker. We used the first analyzed image as our standard. We loaded the image, then selected image &gt; adjust &gt; color balance &gt; all colors. Using the “auto” balance function, we balanced the image’s color, saved the image as a new file, and used analyze &gt; measure to assess the mean gray value of the square for standardizing. As the mean gray value was 183.493, we then standardized each image so that the third grayscale square had a mean gray value between 183 and 184.             After saving each standardized image as a new file, we created a region of interest (ROI) that encompassed the entirety of the flower petals in the image. We then performed analyze &gt; color histogram to obtain red, green, and blue color values. We then split the image into stacks of hue, saturation, and brightness. For each stack, we obtained the mean gray value. DATA-SPECIFIC INFORMATION FOR: WhiteRoyer\_Greenhouse\_csv 1\. Number of variables: 39 2\. Number of rows: 233 3\. Variable List: * FlowerID: unique ID for each sampled flower. A series of four numbers: first, the population ID, then the maternal family, then the individual plant, then the flower number (e.g. “f2” is the second flower sampled from that plant) * PlantID: unique ID for each plant in the greenhouse. A series of three numbers: first, the population ID, then the maternal family, then the individual plant. * Population number: unique ID for each of the six populations studied * LifeHistory: identifying each plant as a member of an annual-like (“annual”) or perennial population * DateFlowerCollected: the date of collection * TimeSamplePlacedInMethanol: the time that the floral punch was actually placed in liquid for pigment extraction to begin * DatePigmentAnalyzed: the date the extracted pigment was measured * TimePigmentAnalyzed: the time the extracted pigment was measured * Absorbance: spectrophotometer measure of pigment absorbance in extracted sample * Orientation: The angle of the floral photo (‘top” - face-on; or “side”) * Area: the number of pixels included in the color analysis * Mean: the mean absorbance of pixels from photos measured in ImageJ * Mode: the mode absorbance of pixels from photos measured in ImageJ * Min: the minimum absorbance of pixels from photos measured in ImageJ * Max: the maximum absorbance of pixels from photos measured in ImageJ * RedMean: the mean red value of pixels from photos measured in ImageJ * RedMode: the mode red absorbance of pixels from photos measured in ImageJ * RedStandardDeviation: the standard deviation of red absorbance of pixels from photos measured in ImageJ * GreenMean: the mean Green value of pixels from photos measured in ImageJ * GreenMode: the mode Green absorbance of pixels from photos measured in ImageJ * GreenStandardDeviation: the standard deviation of Green absorbance of pixels from photos measured in ImageJ * BlueMean: the mean Blue value of pixels from photos measured in ImageJ * BlueMode: the mode Blue absorbance of pixels from photos measured in ImageJ * BlueStandardDeviation: the standard deviation of Blue absorbance of pixels from photos measured in ImageJ * ROIArea: the number of pixels included in the hue and saturation analysis * HueMeanGray: the mean gray hue value of pixels included in the ROI * HueModeGray: the mode gray hue value of pixels included in the ROI * HueMaxGray: the maximum gray hue value of pixels included in the ROI * SaturationMeanGray: the mean gray Saturation value of pixels included in the ROI * SaturationModeGray: the mode gray Saturation value of pixels included in the ROI * SaturationMaxGray: the maximum gray Saturation value of pixels included in the ROI * BrightnessMeanGray: the mean gray Brightness value of pixels included in the ROI * BrightnessModeGray: the mode gray Brightness value of pixels included in the ROI * BrightnessMaxGray: the maximum gray Brightness value of pixels included in the ROI * Notes: notes highlighting a few photos with unusual color 4\. Missing data codes: NA NaN, no absorbance values 5\. Abbreviations used: None 6\. Other relevant information None