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

The Runt-related transcription factor (Runx) family governs a range of developmental processes through sequence-specific DNA recognition and transcriptional regulation. To investigate the evolutionary dy namics and functional roles of Runx genes in turtles, genomic and transcriptomic datasets spanning 29 Testudines species were analyzed using homology prediction, sequence alignment, phylogenetic reconstruction, evolutionary rate estimation, and spatiotemporal expression profiling. Comparative genomic analyses revealed strong Runx gene conservation across Testudines, while lineage-specific divergence was evident in Trionychidae (soft-shelled turtles), characterized by expanded intronic regions, unique single-nucleotide variants, and accelerated evolutionary rates. Notably, Runx2 exhibited clade-specific variation in polyglutamine/polyalanine (QA) repeat length and the Q:A ratio, which significantly correlated with morphological traits, including plastron length and cervical scute patterning. Transcriptomic data revealed stage- and tissue-specific expression of Runx genes in embryonic gonads and carapacial ridges (CR), with divergent upstream regulatory inputs and downstream targets implicated in carapace morphogenesis and sex determination pathways. These findings identify QA repeat modulation in Runx2 as a previously unrecognized molecular mechanism driving morphological innovation while maintaining core function in turtles. This work advances understanding of the conserved and lineage-specific roles of Runx genes and clarifies the molecular basis of morphological and reproductive trait evolution in Testudines.