Lineage-Specific Adaptive Evolution of the Growth Arrest–Specific Protein 8 (GAS8) Domain Protein in Trypanosoma melophagium

The Growth arrest–specific protein 8 (GAS8) domain-containing gene family (Orthogroup 6127at5690) is a strictly single-copy, highly conserved orthologous group across the genus Trypanosoma. Present in all 16 examined species, this family encodes a microtubule-associated protein implicated in cytoskeletal organization and flagellar motility. We conducted a genus-wide evolutionary analysis of the GAS8-domain protein, focusing on the Trypanosoma melophagium ortholog LSM04 004638. Maximum likelihood phylogenetic reconstruction, branch-specific and site-level codon-based selection analyses, ancestral sequence reconstruction, and Rosetta-based energetic estimation were performed. Despite strong purifying constraint consistent with essential cytoskeletal function, branch-level models detected significant episodic diversifying selection along the T. melophagium lineage. Site-level analyses identified three codon positions under episodic selection, two of which correspond to derived non-synonymous substitutions relative to the reconstructed ancestor (P2G and V106L). Ancestral-state back-mutation and energetic estimation indicate that individual substitutions exert modest stability effects relative to cumulative divergence between ancestral and extant sequences. These results demonstrate that highly conserved, single-copy cytoskeletal regulators can undergo limited lineage-specific adaptive refinement without disruption of core structural architecture. Our findings highlight the evolutionary plasticity of essential microtubule-associated proteins under ecological specialization.