Episodic positive selection structurally stabilizes the Arabidopsis CONSTANS-like gene COL5 indicating adaptive evolution.

The CONSTANS-like (COL) transcription factors integrate photoperiod cues with developmental regulation in plants, yet the evolutionary forces shaping their structural diversity remain poorly understood. Here, the evolutionary history of COL5 was reconstructed across 31 Brassicaceae genomes using a curated set of 284 high-confidence orthologs validated for domain architecture, alignment quality, and absence of substitution saturation. Branch-specific codon models identified a single episodically selected lineage within Arabidopsis thaliana, and site-level analyses mapped two non-synonymous amino-acid replacements uniquely acquired along this branch. Ancestral sequence reconstruction recovered the historical residues at both positions with posterior probability 1.0, enabling controlled reverse-evolution mutagenesis. Reintroduction of these ancestral states into the modern COL5 protein revealed a profound biophysical impact, Rosetta ΔΔG values indicated strong destabilization, and 100-ns molecular dynamics simulations showed large increases in structural deviation, compaction, loss of flexibility, and significantly elevated potential energy. These results demonstrate that the derived residues stabilize the contemporary COL5 fold, whereas the ancestral residues are incompatible with the evolved structural background. The findings provide direct mechanistic evidence that episodic positive selection on COL5 produced a lasting shift in protein stability and conformational dynamics, illustrating how adaptive molecular evolution can reshape protein energy landscapes and entrench derived states through historical contingency.