The Tolerated functional decline Model: Relaxed constraint in the molecular evolution of mammalian master...

While most genes are subject to strong purifying selection to maintain essential functions, some master sex-determining genes (MSDGs) appear to evolve under unexpectedly relaxed constraints. To understand the molecular mechanisms and evolutionary rationale behind this phenomenon, we investigated the eutherian Sry gene, a top-level regulator of testis development that originated from Sox3. We focused on the high mobility group (HMG) box DNA-binding domain, analyzing its sequences across ten representative species. Comparative analyses revealed that Sry/SRY has a d_N/d_S ratio approximately 14 times higher than Sox3/SOX3, indicative of substantially relaxed purifying selection. In vitro DNA-binding assays revealed marked functional decline in five species, with particularly severe reductions in the Indian elephant and Aardvark. Ancestral sequence reconstruction and mutational analyses identified slightly deleterious amino acid substitutions, including F51L, Q58R, and R73C/H, that significantly impaired DNA-binding. Interestingly, the pattern of decline was convergent in two distantly related Afrotherians. These findings led us to propose the "Tolerated Functional Decline Model," in which MSDGs can accumulate mildly detrimental mutations as long as their function remains above a threshold necessary for sex determination. We suggest that SRY's limited number of essential targets, combined with the binary nature of its developmental role, permits evolutionary tolerance to reduced DNA-binding affinity. This study provides mechanistic insight into how functional robustness and relaxed constraint can coexist in key developmental regulators, shedding light on the evolution of sex-determining systems and the stability of genetic sex- determining pathways in mammals.