Transitioning from a freshwater to saltwater habitat requires major physiological changes which may be necessary for freshwater organisms facing habitat salinization. Contemporary understanding of the various pathways that give rise to salt tolerance is limited, especially for salt-sensitive taxa such as amphibians. This study investigates mechanisms of salt tolerance in coastal populations of a treefrog, Hyla cinerea, that are locally adapted to brackish wetlands. Using a common garden experiment we (a) determine the extent that environment (i.e., saltwater exposure during early life) or genotype (i.e., location of origin) affects developmental benchmarks and transcriptome expression across salt-adapted (coastal) and non-salt-adapted (inland) Hyla cinerea and (b) identify genes that may underpin differences in saltwater tolerance. Our analysis revealed five novel findings: First, coastal populations exhibited higher survival and higher plasma osmolality than inland populations regardless of the salinity of the developmental environment. Second, despite a high degree of overlap in transcript expression profiles, location of origin was the predominant driver of differential expression of annotated genes. Third, coastal populations differentially expressed osmoregulatory genes that encode ion transporters (e.g., atp1b1, slc26a, atp6V1g3), cellular adhesion components (e.g., cdh26, cldn1, gjb3, and ocln), and cytoskeletal components (e.g. odc1-a, oaz, tgm3) that likely contribute to enhanced salt tolerance in coastal populations. Fourth, coastal Hyla cinerea highly expressed glycerol-3-phosphate dehydrogenase 1 (gpd1) which suggests that coastal tadpoles use glycerol as a compatible osmolyte to reduce water loss in hyperosmotic environments. If confirmed, glycerol is a novel mechanism of saltwater tolerance in frogs. Fifth, gene expression patterns in coastal populations are not an amplification of the responses made by freshwater-acclimated, inland populations, but instead follow patterns similar to those of marine-acclimated euryhaline teleost fish, such that coastal Hyla cinerea populations may be considered a salt-tolerant ecotype. Finally, our findings highlight several candidate pathways that may allow other freshwater organisms facing habitat salinization to persist.
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