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Phenotypic variation is common across life history and among populations occupying different environments, yet the molecular mechanisms underlying these axes of divergence remain poorly understood. Much work has focused on gene expression as a link between genetic variation, environmental variation, and phenotypes, but post-transcriptional processes such as alternative splicing—which affect how transcripts are assembled rather than how much of a transcript is produced—are increasingly recognized as additional modulators of plasticity and adaptation. Here, we examined gene expression and alternative splicing together in the wood frog (Rana sylvatica), an amphibian with a complex life cycle whose populations differ across replicated gradients of road adjacency and associated pollution. We found extensive transcriptomic differences between hatchlings and adults, with thousands of genes differentially expressed or spliced. Individuals clustered strongly by population for both expression and splicing. Differences at the habitat level were less extensive, but revealed two differentially expressed genes (HSP70 and Gpsm2) and one differentially spliced gene (Cd82) that consistently distinguished roadside and woodland populations. Overall, genetic differentiation between populations was low, suggesting that phenotypic and transcriptomic differences likely emerge in the presence of gene flow and reflect plastic responses. Together, these results highlight transcriptomic plasticity as an important mechanism shaping variation across both development and population differentiation. © 2025 The Author(s). Ecology and Evolution published by British Ecological Society and John Wiley & Sons Ltd.
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Human impacts on wild populations are numerous and extensive, degrading habitats and causing population declines across taxa. Though these impacts are often studied individually, wild populations typically face suites of stressors acting concomitantly, compromising the fitness of individuals and populations in ways poorly understood and not easily predicted by the effects of any single stressor. Developing understanding of the effects of multiple stressors and their potential interactions remains a critical challenge in environmental biology. Here, we focus on assessing the impacts of two prominent stressors affecting many organisms across the planet – elevated salinity (an increasingly common pollutant in freshwater habitats) and elevated temperature. We examined a suite of physiological traits and components of fitness across populations of wood frogs originating from ponds that differ in their proximity to roads and thus their legacy of exposure to road salt pollution. When experimentally exposed to road salt, wood frogs showed reduced survival, especially those from ponds adjacent to roads, and delayed time to metamorphosis. Family level effects mediated these outcomes, but high salinity generally eroded family level variance. When combined, exposure to both temperature and salt resulted in very low survival, and this effect was strongest in roadside populations. Taken together, these results suggest that temperature is an important stressor capable of exacerbating impacts from a prominent contaminant confronting many freshwater organisms in salinized habitats. More broadly, it appears likely that toxicity might often be underestimated in the absence of multi-stressor approaches.
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