Regulatory diversification of conserved Runx genes drives morphological innovation in soft-shell turtles

The Runt-related (Runx) gene family encodes transcription factors that regulate gene expression through sequence-specific DNA binding and play essential roles in development. To investigate the evolutionary dynamics and regulatory functions of Runx genes in turtles, we analyzed genomic and transcriptomic data from 29 Testudines species, integrating homology prediction, sequence alignment, phylogenetic reconstruction, evolutionary rate estimation, and expression profiling. Comparative genomic analyses revealed strong conservation of Runx genes across Testudines. Notably, Trionychidae (soft-shelled turtles) exhibit lineage-specific intronic expansions and unique single-nucleotide variants, accompanied by elevated evolutionary rates. A key finding is that variations in the polyglutamine/polyalanine (QA) repeat length and Q:A ratio within the Runx2 domain are strongly associated with morphological traits, including cervical scute loss, reduced marginal ossification, and variation in plastron length across turtles. Transcriptomic profiling revealed stage- and tissue-specific expression of Runx genes in the gonads and carapacial ridges (CR) during embryogenesis. Distinct sets of upstream regulators and downstream targets were identified in these tissues, implicating Runx genes in both carapace morphogenesis and sex determination pathways. These results highlight QA repeat evolution as a novel molecular mechanism driving morphological innovation in turtles. Subtle changes in Runx2 protein structure appear to modulate phenotypic diversity while maintaining core functional constraints. This study provides new insights into the evolutionary conservation and functional diversification of Runx genes and elucidates molecular mechanisms underlying morphological and reproductive evolution in Testudines.