Lineage-specific expansion and functional divergence of β-keratin genes underlying shell evolution in turtles

β-Keratins are key structural proteins that contribute to the formation of keratinized epidermal structures such as scales, feathers, and shells in reptiles and birds. However, the evolutionary diversification and functional specialization of β-keratins in turtles remain poorly understood. Here, we combined comparative genomic, structural, and transcriptomic analyses across 10 turtle species representing five families, alongside four outgroup species (birds, crocodilians, lizards, and snakes), to systematically investigate the evolution of β-keratin genes in Testudines. We identified 835 β-keratin genes in turtles, which clustered into two major evolutionary lineages: conserved, non-specific β-keratins within the epidermal differentiation complex (EDC) and lineage-specific β-keratins located outside the EDC. Notably, these turtle-specific β-keratins were absent in squamates but shared evolutionary origins with avian feather keratins. Domain and protein structure analyses revealed that hard-shelled turtles retain β-keratins with conserved β-sheet structures and glycine-tyrosine enrichment, likely associated with shell rigidity. In contrast, soft-shelled turtles exhibit reduced β-sheet content and transcriptional silencing of these genes. Transcriptomic and semi-quantitative PCR further confirmed carapace-specific expression of turtle-specific β-keratins in hard-shelled turtles, but not in soft-shelled species. Together, our findings demonstrate that the expansion, structural modification, and tissue-specific expression of β-keratin genes underlie the evolutionary divergence of shell phenotypes in turtles, providing new insights into the molecular basis of epidermal adaptation and morphological innovation in reptiles.