Trichromacy is insufficient for mate detection in a mimetic butterfly

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.

Methods
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 Adelpha and Limenitis butterflies including: Adelpha boreas (n=1 female); A. cocala (n=1 female and n=1 male); A. cytherea (n=1 female and n=1 male); A. erotia (n=1 female and n=1 male); A. fessonia (n=1 female and n=1 male); A. heraclea (n=1 female and n=1 male); A. leucophthalma (n=1 female and n=1 male); A. malea (n=1 female); A. naxia (n=2 females and n=2 males); A. phylaca (n=2 males); Adelpha serpa celerio (n=1 female and n=1 male); and Limenitis arthemis astyanax (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., Adelpha fessonia (RIH102), A. leucophthalma (RIH4203), A. naxia (RIH4463), and Limenitis arthemis astyanax (AW0103)) with other butterfly species' DNA when blasting these transcriptomes with opsin genes. 

Nucleotide sequences for Adelpha and Limenitis UV, blue and long-wavelength opsins were retrieved from individual transcriptomes by blast searches using Heliconius melpomene opsins as query sequences. 

Epi-microspectrophotometry on living eyes was used to collect the long wavelength rhodopsin dark spectra on A. fessonia (n=1) and A. californica (n=1). Optophysiolgy was used to determine the pupillary response of living Adelpha fessonia (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) Adelpha fessonia and female (n=8) and male (n=11) A. basiloides 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 A. fessonia and the tetrachromatic color space of the UV and violet bird visual systems