Larval Pigmentation Reveals Environmental and Genetic Influences in Hybridizing Ambystoma Salamanders

Closely related species may produce hybrids, and these hybrids often display intermediate traits that can influence fitness and reproductive isolation. In this study, we examine Ambystoma barbouri and Ambystoma texanum, two sister salamander species that breed in contrasting aquatic habitats with differing levels of ultraviolet radiation (UVR) exposure. Although these species can hybridize, it remains unclear whether hybrid pigmentation responses to UVR confer an advantage, a disadvantage, or are simply intermediate between parental forms. Pigmentation in salamanders is regulated by chromatophores, melanophores, and xanthophores, which help mitigate UVR-induced damage. Given previous knowledge on the species, habitats, and behaviors, we hypothesized that: (1) A. barbouri would show stronger UVR-induced pigmentation (i.e., greater darkening) than A. texanum, due to its exposure to clearer, shallower streams; (2) hybrids would exhibit intermediate responses; and (3) xanthophore coverage would remain stable or decrease under UVR as melanophores expand. To test these hypotheses, we conducted a fully factorial breeding experiment using pure and reciprocal hybrid crosses, exposing larvae to four UVR durations (0, 1, 4, and 12 h). We quantified skin darkness and chromatophore-specific pigmentation using standardized digital image analysis, Bayesian beta regression, and Bayesian compositional regression. Our results supported all three hypotheses. A. barbouri showed greater darkening under UVR exposure than A. texanum, and hybrids exhibited intermediate responses, but with a greater similarity toward their maternal phenotype. Xanthophore coverage remained stable or declined with increasing UVR exposure, evidencing that their main purpose is different from UVR protection. These findings raise the possibility that hybrids could experience reduced performance if intermediate pigmentation is suboptimal, but that maternal effects may offset some of these disadvantages.