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 hours). 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.

Hybridization can have a profound negative effect on population fitness when species exhibit divergence in adaptive traits. The Streamside salamander, Ambystoma barbouri, and the Smallmouth salamander, A. texanum, are closely related species differentiated primarily by breeding habitat and reproduction-related traits, but previous work suggests patterns of hybridization and introgression between them. Here we investigate whether 1) hybridization occurs between the two species in laboratory settings, 2) their divergent reproduction-related traits are retained under a common treatment, and 3) hybrid offspring exhibit similar fitness as their non-hybrid counterparts. We bred conspecific and heterospecific pairs of A. barbouri and A. texanum and raised hybrid and non-hybrid offspring to metamorphosis. Reproduction-related traits such as oviposition location and clutch size remained well differentiated between the two species. Heterospecific pairs hybridized readily, at similar rates to conspecific pairs. The hybrid larvae generally exhibited a maternal effect and, less frequently, intermediate phenotypes with respect to measured traits. Hybrid larvae did not exhibit reduced fitness as measured by survival to metamorphosis. Our results suggest that traits differentiating A. barbouri and A. texanum are likely genetically determined despite the lack of reproductive isolation between them. This suggests that the generally parapatric distribution of the two species, often paired with abrupt transition in traits, might be driven by selection on these traits in local habitats. Further, the maternal effect and consequent lack of intermediate phenotypes observed for several traits likely reduces the negative effect of hybridization in locally adapted populations.