DOI: 10.5061/dryad.xd2547dr5
title: Data from: Trichromacy is insufficient for mate detection in a mimetic butterfly

description: Color vision is thought to play a key role in the evolution of animal
 coloration, while achromatic vision is rarely considered as a mechanism
 for species recognition. We examined the eyeshine and photopigments of
 adult Adelpha fessonia butterflies (Nymphalidae: Lepidoptera), and the
 ultraviolet, blue and long-wavelength opsin sequences of 22 species of
 Adepha and Limenitis butterflies. Opsin sequences were
 extracted from individual RNA-seq transcriptomes of 24 adult male and
 female butterflies (brain + eye), combined with previously published
 sequences, and aligned for phylogenetic analysis. We measured reflectance
 spectra of white, orange and brown dorsal wing color patches of individual
 wild-caught female and male Adelpha fessonia and co-mimetic female and
 male A. basiloides butterflies. We also measured in vivo long
 wavelength rhodopsin dark spectra for A.
 fessonia and A. californica, and pupillary
 sensitivity for A. fessonia. We then used these
 reflectance spectra data in color space models and calculations of
 chromatic and achromatic discriminabilities (JNDs) of birds and
 butterflies. Taken together, our data suggest that the dorsal wing color
 patches of Adelpha fessonia and A. basiloides are likely
 indiscriminable to both birds and butterflies using both chromatic and
 achromatic channels.
description: Twenty-four individual transcriptome fasta files were produced
 using the Trinity pipeline (trinityrnaseq_r2012-06-08v2) on the University
 of California’s Legacy High Performance Computing cluster (HPC2) (Grabherr
 et al. 2011; Haas et al. 2013) from paired-end RNA-seq data of individual
 heads of adult male and female <em>Adelpha</em> and
 <em>Limenitis </em>butterflies including: <em>Adelpha
 boreas </em>(n=1 female); <em>A. cocala </em>(n=1 female
 and n=1 male); <em>A. cytherea </em>(n=1 female and n=1
 male); <em>A. erotia </em>(n=1 female and n=1
 male); <em>A. fessonia </em>(n=1 female and n=1
 male); <em>A. heraclea </em>(n=1 female and n=1 male);
 <em>A. leucophthalma </em>(n=1 female and n=1
 male); <em>A. malea </em>(n=1 female); <em>A. naxia
 </em>(n=2 females and n=2 males); <em>A.
 phylaca </em>(n=2 males); <em>Adelpha serpa
 celerio </em>(n=1 female and n=1 male); and <em>Limenitis
 arthemis astyanax</em> (n=1 female and n=1 male). Raw reads were
 trimmed for quality and parsed using custom scripts. We detected minor
 contamination of the RNA-seq reads of 4 of 24 specimens
 (i.e., <em>Adelpha fessonia</em> (RIH102), <em>A.
 leucophthalma</em> (RIH4203), <em>A. naxia</em>
 (RIH4463), and <em>Limenitis arthemis astyanax</em> (AW0103))
 with other butterfly species' DNA when blasting these transcriptomes
 with opsin genes.  Nucleotide sequences for
 <em>Adelpha</em> and <em>Limenitis</em> UV, blue
 and long-wavelength opsins were retrieved from individual transcriptomes
 by blast searches using <em>Heliconius melpomene</em> opsins
 as query sequences.  Epi-microspectrophotometry on
 living eyes was used to collect the long wavelength rhodopsin dark spectra
 on <em>A. fessonia</em> (n=1) and <em>A.
 californica</em> (n=1). Optophysiolgy was used to determine the
 pupillary response of living <em>Adelpha
 fessonia </em>(n=1) eyes. Idealized rhodopsin templates were matched
 to experimental data using a least-squares fit.
 Wild-caught female (n=8) and male (n=8) <em>Adelpha
 fessonia</em> and female (n=8) and male (n=11) <em>A.
 basiloides </em>butterflies were collected in Michoacán and Oaxaca,
 Mexico, respectively. Wings were assessed for damage by eye and undamaged
 patches of wings (left and right forewing orange, left forewing and
 hindwing white, and forewing brown) were measured using and Ocean Optics
 UV-visible USB2000 spectrometer and Ocean Optics DH-2000 deuterium-halogen
 lamp. Wing reflectances were processed in pavo (Maia et al. 2019) and used
 to calculate achromatic and chromatic just noticeable differences (JNDs).
 Wing reflectances were also plotted in the trichromatic color space
 of <em>A. fessonia </em>and the tetrachromatic color space of
 the UV and violet bird visual systems
description: # Trichromacy is insufficient for mate detection in a mimetic butterfly
 Authors: Andrew Dang, Gary D. Bernard, Furong Yuan, Aide Macias-Munoz,
 Ryan I. Hill, J.P. Lawrence, Aline G. Rangel Olguin, Armando
 Luis-Martinez, Sean P. Mullen, Jorge Llorente-Bousquets, Adriana D.
 Briscoe *Communications Biology,* 8:189,
 [https://doi.org/10.1038/s42003-025-07472-7](https://doi.org/10.1038/s42003-025-07472-7). ## Description: **Wing reflectance spectra folder**: This folder includes one .csv file for wing voucher specimen collection locality data and eighteen .csv files for wing reflectance spectra measurement data, separated by species (*A. basiloides* or *A. fessonia*), left (L) or right (R) wing, hind or forewing, orange or white patch, and male (M) or female (F) specimens. Individual wing reflectance spectra file includes measurements from 300 to 700 nm wavelengths (wl) and columns corresponding to individuals with IDs indicating species (ABA for *A. basiloides* or AFE for *A. fessonia*), number, and sex (M and F). **Adult head transcriptomes folder**: This folder includes one .csv file for the collection locality data for individual *Adelpha* and *Limenitis* butterflies used in the RNA-Seq experiments along with the 24 transcriptomes .fasta files. In addition there is one .csv file for the *Adelpha fessonia* opsin gene log transcripts per million (TPMs) as quantified using RNA-seq data and kallisto. **Opsin nucleotide sequences folder**: This folder contains three .fasta files used in the phylogenetic analysis, one for each opsin gene: *UVRh*, *BRh* and *LWRh*. GenBank accession numbers for each sequence are included as part of the sequence name and also listed in the .csv file in the transcriptomes folder. **Blue rhodopsin absorbance spectra folder**: This folder contains one .csv file with absorbance spectra data for three experiments in which three different Limenitis blue opsins were expressed in HEK293 cells and reconstituted with 11-*cis*-retinal in the dark to obtain a dark spectrum: Wildtype *Limenitis lorquini* blue opsin, wildtype *L. arthemis* blue opsin and mutated *L. arthemis* blue opsin with the Y195F substitution. BRh=blue rhodopsin; WT=wildtype **In vivo** **absorbance spectra and spectral sensitivities folder**: This folder contains two .csv files with 1) normalized absorbance spectra of *Adelpha fessonia* and *A. californica* long wavelength-absorbing rhodopsins measured by epi-microspectrophotometry and 2) normalized pupillary sensitivity of *A. fessonia*. The normalized absorbance spectra sheet contains an idealized rhodopsin absorbance spectra (template) for rhodopsin that peak at 530 nm, while the normalized pupillary sensitivity sheet contains idealized sensitivity curves (templates) for rhodopsins that peak at 355 nm and 530 nm, respectively.  **Just noticeable differences (JNDs) and color space models folder**: Just noticeable differences (JNDs) and color space models: This folder contains six .csv files for three types of visual system: *Adelpha fessionia*, UV bird, and violet bird. For each visual system, the JNDs file gives the 1000 individual bootstrap replicate achromatic or chromatic contrasts (JNDs) obtained when comparing *Adelpha fessonia* orange or white wing color patches with the equivalent color patch in *Adelpha basiloides*. Columns correspond to the patches being compared and are identified as either left (L) or right (R) side, fore- (FW) or hindwing (HW), orange (Or) or white (wh) patch, and chromatic (c) or achromatic (a). **Rstudio folder**: This folder contains three subfolders: Figure_6B_WingReflectanceSpectra, Figure_7_ColorSpaces_VisualModels, and Supplementary_Figure1_SexComparison. * **Figure_6B_WingReflectanceSpectra subfolder**: The subfolder Figure_6B_WingReflectanceSpectra contains one .csv file and one .R file for R code used to run the analysis shown in figure 6B. The .csv file contains spectra measurements of all the wing patches processed for use in the provided script. Both left and right side orange patches and fore- and hindwing white stripes have been pooled together as either orange patches or white patches. Columns correspond to individuals’ measurements from 300 to 700 nm wavelengths (wl) and identified as ADE (number) as a unique ID, *A. basiloides* (Ba) or *A. fessonia* (Fe), and orange (Or) or white (Wh). * **Figure_7_ColorSpaces_VisualModels subfolder**: The subfolder Figure_7_ColorSpaces_VisualModels contains 16 .csv files and one .R file used to generate Figure 7. ‘Adelpha_wings.csv’ contains all of the wing measurements to be used for JND calculations. There are eight .csv files that are derived from ‘Adelpha_wings.csv’ that group the 300-700 nm wavelength (wl) measurements based off of species (ABA for *A. basiloides* and AFE for *A. fessonia*), left (L) or right (R) wing, fore- (Fw) or hindwing (Hw), and orange (Or) or white (Wh) that are used map those groupings for color spaces. ‘Adelpha_dark.csv” contains wing reflectance measurements for the dark brown patches of *Adelpha* wings to be used as the background in our models. Columns in these .csv files correspond to identification as ADE(number) as a unique ID, *A. basiloides* (Ba) or *A. fessonia* (Fe), and orange (Or) or white (Wh). This folder also includes three (one for each model type, *A. fessonia*, UV bird, or Violet bird) trimmed .csv files to plot JND bootstrap replicates. The columns indicate left (L) or right (R) wing, fore- (FW) or hindwing (HW), orange (Or) or white (wh) patch, and chromatic (c) or achromatic (a) JND measurements. Another three (one for each model type) processed .csv files include the mean bootstrap values and the upper and lower values for the 95% confidence intervals. Rows indicate left (L) or right (R) wing, fore- (FW) or hindwing (HW), orange (Or) or white (wh) patch, and chromatic (c) or achromatic (a) JND measurements. * **Supplementary_Figure1_SexComparison subfolder:** The Supplementary_Figure1_SexComparison subfolder contains eight .csv files and one .R file for code used to run the analysis shown in Supplementary Figure 1. File names indicate the species, left (L) or right (R), fore- or hindwing, and color patch measurements from 300-700 nm wavelengths (wl) in the file. Columns in each file include a identifier and sex (F for female, M for male) of each sample. Code/Software R and Rstudio software is required to run the various provided R scripts. R scripts generated in R 4.4.1 and Rstudio 2024.04. Libraries used in the R scripts were Pavo (2.9.0) and ggplot2 (3.5.1). R scripts have been annotated throughout the scripts describing general workflow and necessary input files provided in the Rstudio folder. Data Sources: Links to the NCBI SRA BioProject PRJNA1152103 archive of the fastq files used to make the 24 transcriptomes: [https://www.ncbi.nlm.nih.gov/sra/PRJNA1152103](https://www.ncbi.nlm.nih.gov/sra/PRJNA1152103) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630529](https://www.ncbi.nlm.nih.gov/sra/RUN:39630529) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630528](https://www.ncbi.nlm.nih.gov/sra/RUN:39630528) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630527](https://www.ncbi.nlm.nih.gov/sra/RUN:39630527) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630526](https://www.ncbi.nlm.nih.gov/sra/RUN:39630526) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630525](https://www.ncbi.nlm.nih.gov/sra/RUN:39630525) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630524](https://www.ncbi.nlm.nih.gov/sra/RUN:39630524) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630523](https://www.ncbi.nlm.nih.gov/sra/RUN:39630523) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630522](https://www.ncbi.nlm.nih.gov/sra/RUN:39630522) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630521](https://www.ncbi.nlm.nih.gov/sra/RUN:39630521) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630520](https://www.ncbi.nlm.nih.gov/sra/RUN:39630520) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630519](https://www.ncbi.nlm.nih.gov/sra/RUN:39630519) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630518](https://www.ncbi.nlm.nih.gov/sra/RUN:39630518) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630517](https://www.ncbi.nlm.nih.gov/sra/RUN:39630517) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630516](https://www.ncbi.nlm.nih.gov/sra/RUN:39630516) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630515](https://www.ncbi.nlm.nih.gov/sra/RUN:39630515) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630514](https://www.ncbi.nlm.nih.gov/sra/RUN:39630514) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630513](https://www.ncbi.nlm.nih.gov/sra/RUN:39630513) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630512](https://www.ncbi.nlm.nih.gov/sra/RUN:39630512) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630511](https://www.ncbi.nlm.nih.gov/sra/RUN:39630511) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630510](https://www.ncbi.nlm.nih.gov/sra/RUN:39630510) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630509](https://www.ncbi.nlm.nih.gov/sra/RUN:39630509) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630508](https://www.ncbi.nlm.nih.gov/sra/RUN:39630508) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630507](https://www.ncbi.nlm.nih.gov/sra/RUN:39630507) [https://www.ncbi.nlm.nih.gov/sra/RUN:39630506](https://www.ncbi.nlm.nih.gov/sra/RUN:39630506) [https://www.ncbi.nlm.nih.gov/biosample/43293976](https://www.ncbi.nlm.nih.gov/biosample/43293976) [https://www.ncbi.nlm.nih.gov/biosample/43293977](https://www.ncbi.nlm.nih.gov/biosample/43293977) [https://www.ncbi.nlm.nih.gov/biosample/43293978](https://www.ncbi.nlm.nih.gov/biosample/43293978) [https://www.ncbi.nlm.nih.gov/biosample/43293979](https://www.ncbi.nlm.nih.gov/biosample/43293979) [https://www.ncbi.nlm.nih.gov/biosample/43293980](https://www.ncbi.nlm.nih.gov/biosample/43293980) [https://www.ncbi.nlm.nih.gov/biosample/43293981](https://www.ncbi.nlm.nih.gov/biosample/43293981) [https://www.ncbi.nlm.nih.gov/biosample/43293982](https://www.ncbi.nlm.nih.gov/biosample/43293982) [https://www.ncbi.nlm.nih.gov/biosample/43293983](https://www.ncbi.nlm.nih.gov/biosample/43293983) [https://www.ncbi.nlm.nih.gov/biosample/43293984](https://www.ncbi.nlm.nih.gov/biosample/43293984) [https://www.ncbi.nlm.nih.gov/biosample/43293985](https://www.ncbi.nlm.nih.gov/biosample/43293985) [https://www.ncbi.nlm.nih.gov/biosample/43293986](https://www.ncbi.nlm.nih.gov/biosample/43293986) [https://www.ncbi.nlm.nih.gov/biosample/43293987](https://www.ncbi.nlm.nih.gov/biosample/43293987) [https://www.ncbi.nlm.nih.gov/biosample/43293988](https://www.ncbi.nlm.nih.gov/biosample/43293988) [https://www.ncbi.nlm.nih.gov/biosample/43293989](https://www.ncbi.nlm.nih.gov/biosample/43293989) [https://www.ncbi.nlm.nih.gov/biosample/43293990](https://www.ncbi.nlm.nih.gov/biosample/43293990) [https://www.ncbi.nlm.nih.gov/biosample/43293991](https://www.ncbi.nlm.nih.gov/biosample/43293991) [https://www.ncbi.nlm.nih.gov/biosample/43293992](https://www.ncbi.nlm.nih.gov/biosample/43293992) [https://www.ncbi.nlm.nih.gov/biosample/43293993](https://www.ncbi.nlm.nih.gov/biosample/43293993) [https://www.ncbi.nlm.nih.gov/biosample/43293994](https://www.ncbi.nlm.nih.gov/biosample/43293994) [https://www.ncbi.nlm.nih.gov/biosample/43293995](https://www.ncbi.nlm.nih.gov/biosample/43293995) [https://www.ncbi.nlm.nih.gov/biosample/43293996](https://www.ncbi.nlm.nih.gov/biosample/43293996) [https://www.ncbi.nlm.nih.gov/biosample/43293997](https://www.ncbi.nlm.nih.gov/biosample/43293997) [https://www.ncbi.nlm.nih.gov/biosample/43293998](https://www.ncbi.nlm.nih.gov/biosample/43293998) [https://www.ncbi.nlm.nih.gov/biosample/43293999](https://www.ncbi.nlm.nih.gov/biosample/43293999) Software: To create *de novo* transcriptome assemblies, we used the Trinity pipeline (trinityrnaseq_r2012-06-08v2) on the University of California's legacy High Performance Computing cluster (Gabherr et al. 2011; Haas et al. 2013). References: Bray NL et al. 2016. Near-optimal probabilistic RNA-seq quantification. Nature Biotechnology, 34: 525�527, [https://doi.org/10.1038/nbt.3519](https://doi.org/10.1038/nbt.3519) Grabherr MG et al. 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology, 29: 644-652, [https://doi.org/10.1038/nbt.1883](https://doi.org/10.1038/nbt.1883) Haas BJ et al. 2013. *De novo* transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nature Protocols, 8:1494-1512, [https://doi.org/10.1038/nprot.2013.084](https://doi.org/10.1038/nprot.2013.084) Maia R et al. 2019. pavo 2: New tools for the spectral and spatial analysis of colour in r. Methods in Ecology and Evolution, 10: 1097�1107, [https://doi.org/10.1111/2041-210X.13174](https://doi.org/10.1111/2041-210X.13174)