Animal coloration is a multifaceted trait with many ecological roles and related to a variety of developmental and physiological processes. Consequently, coloration is often subject to a variety of selective pressures, leading to the evolutionary maintenance of variation. In this study, we investigated hypotheses related to the maintenance of dorsal color variation in wood frogs (Rana sylvatica). First, we tested for multimodality, and whether color correlates with body size or condition or varies by sex or age-class. We combined behavioral trials with visual modeling to test for sex recognition. We also considered visual models for predators and tested for an interaction between discriminability indexes (JND) of color channel (chromatic vs. achromatic) and predator type (birds vs. snakes), as well as for a within individual trade-off between the JND of chromatic and achromatic coloration. Finally, we tested for disruptive viability selection on color using predation trials, and for antagonistic directional selection between viability selection and reproductive investment of females. We found that wood frogs present continuous color variation that does not correlate with body size or condition, but that changes with age. Wood frogs present subtle sexual dichromatism, but we found no evidence for a role of color in sex recognition. Instead, we discuss the possibility that sex differences might, at least in part, have a demographic explanation. Predator visual models indicated that wood frogs cannot solely rely on dorsal coloration for camouflage. Moreover, different predators might present selective pressures in different color channels, while individuals’ achromatic and chromatic coloration trade-off in JND. Therefore, different selective pressures caused by different predators might interact with ontogenetic changes and developmental/physiological trade-offs to maintain color variation. We found no relationship between color and survival or reproductive investment, suggesting further work is required to fully understand selection on color. Our results highlight the importance of understanding evolutionary trade-offs and developmental/physiological constraints in combination with one another, and suggest the potential for an interaction between these proximate and ultimate mechanisms in the evolutionary maintenance of variation. These results likely extend beyond color expression in amphibians, and exemplify a more general process for such evolutionary outcomes.

1) Digital images color data was obtained using MICA plugin in ImageJ (see manuscript).

 -- Raw files are named: "ResultsMICA" for the frogs, and "MICA_Standards_noOverexposed" for color callibration cards.

 -- Data used in analyses was transformed, and is saved as "HSVdata_Pictures"

2) Spectrophotometry files were imported into R using the package pavo (see manuscript).

 -- Data used in analyses was transformed, and is saved as "CombinedSpecs", "backgroundSpecs_NegValuestoMin", and "frogSpecs_NegValuestoMin"

3) Ocular media data:

 -- frogs: digitized from: Govardovskii VI, Zueva LV. 1974. Spectral sensitivity of the frog eye in the ultraviolet and visible region. Vision Research 14:1317–1321.

 -- kestrel: downloaded from: Lind O, Mitkus M, Olsson P, Kelber A. 2013. Ultraviolet sensitivity and colour vision in raptor foraging. J Exp Biol 216:3764–3764.

(Be careful, correct kestrel data is accessed through the correction note: https://jeb.biologists.org/content/suppl/2013/04/25/216.10.1819.DC1)

 -- snake: obtained from B. Simões, data from: Simões BF, Sampaio FL, Douglas RH, Kodandaramaiah U, Casewell NR, Harrison RA, Hart NS, Partridge JC, Hunt DM, Gower DJ. 2016. Visual pigments, ocular filters and the evolution of snake vision. Molecular Biology and Evolution 33:2483–2495.

- kestrel and snake ocular media data are not provided within data folders since obtained from external sources

- directory structure: codes and datasets are split into 3 main folders:

1) "Codes-Experiments" = codes and data in reference to behavioral experiments

2) "Codes-Pictures" = all codes and data for analyses in which color data obtained from digital images were used

3) "Codes-Specs" = all codes and data for analyses in which color data obtained from spectrophotometry were used

- additional analyses / figures for response to reviews are found in folder "revisions"