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This study investigates the adaptive evolution of the CONSTANS-like (COL) gene family inArabidopsis thaliana(L) Heynh., focusing on the molecular and structural consequences of episodic positive selection. Using CODEML-based site and branch-site models, I identified statistically significant adaptive substitutions in COL3 and COL5. Ancestral sequence reconstruction revealed that COL3 evolved from serine to alanine at residue 212, while COL5 transitioned from glycine to serine at residue 343. To assess structural impacts, I retrieved modern COL protein structures from the AlphaFold Protein Structure Database, introduced reverse mutations, and generated ancestral models using AlphaFold3. Stability analyses with DynaMut indicated that the G343S mutation in COL5, located within a unique proteomic domain, resulted in slight destabilization and reduced flexibility, with ChimeraX revealing the loss of hydrogen bonding. In contrast, the S212A mutation in COL3, positioned upstream of its CCT domain, showed a stabilizing effect despite reduced flexibility. GO enrichment analysis revealed that positively selected COL genes are significantly associated with red and far-red light signaling and developmental regulation. These findings suggest that episodic positive selection fine-tunes COL protein function, warranting further studies using molecular dynamics simulations and in vivo functional assays.