Comparative genomic and seasonal expression analyses highlight HSPA12A as a candidate gene associated with winter physiology in the Chinese alligator (Alligator sinensis)

Background The HSP70 family maintains cellular proteostasis and mediates vertebrate responses to environmental stress. The Chinese alligator ( Alligator sinensis ), an ectothermic crocodilian, undergoes winter dormancy characterized by cessation of feeding, reduced activity, and metabolic depression. However, the evolutionary patterns of the HSP70 family across vertebrates and their potential relevance to winter dormancy in crocodilians remain poorly understood. We characterized the HSP70 family across 24 vertebrate species, focusing on the molecular evolution of HSPA12A along crocodilian lineages and its seasonal expression in the hypothalamus of the Chinese alligator. Results Using HMMER- and BLAST-based searches, we identified 355 HSP70 family members, with 11–28 members per species. Phylogenetic, synteny, and subcellular localization analyses indicated that the family is broadly conserved but exhibits subfamily- and lineage-associated divergence. Under two foreground schemes in which the terminal branches of two winter-dormant crocodilians or all four crocodilian species were jointly designated as foreground, HSPA12A showed an elevated relative evolutionary rate and significant evidence of positive selection. HyPhy aBSREL supported episodic diversifying selection on the A. sinensis HSPA12A terminal branch. Public RNA-seq data showed that HSPA12A expression was highest in the hypothalamus among sampled female tissues and was higher in winter than in summer (600.0 and 329.2 TPM, respectively). Western blotting and immunofluorescence analyses showed descriptive differences between the examined specimens, with higher HSPA12A protein abundance and a higher density of cells double-positive for anti-HSPA12A antibody immunoreactivity and NeuN in winter. Because each RNA-seq, western blotting, and immunofluorescence comparison included only one biological specimen per season, these observations were treated as descriptive, and no inferential statistical comparisons were performed. Conclusions The vertebrate HSP70 family is broadly conserved but shows differentiation among subfamilies and lineages. The evolutionary analyses indicate a broader crocodilian-lineage-associated pattern of HSPA12A evolution that cannot be attributed specifically to winter dormancy. Separately, the descriptive transcriptomic and preliminary protein-level observations provide a basis for considering HSPA12A as a candidate for further investigation in relation to winter physiology in the Chinese alligator. These findings do not establish winter dormancy as the selective driver of HSPA12A evolution, and its biological function and causal role remain to be determined.