Data from: Floral color variation across life history and geography in Mimulus ringens (Phrymaceae)
Creators: name: Royer, Anne
name: White, Sarah
Publisher: name: Zenodo
Year: 2023
Subjects: Mimulus ringens
Speciation
floral evolution
Evolutionary ecology
flower color
flowering time
latitudinal cline
Citizen science

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             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 > adjust > color balance > all colors. Using the "auto" balance function, we balanced the image's color, saved the image as a new file, and used analyze > 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 > 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.