Lineage-Specific Retention of the Plastid tilS-trnI(CAU) Module and Its Variable Relationship With Codon Usage...

Plastid genomes retain a reduced but essential translation system inherited from cyanobacterial ancestors, but the evolutionary constraints shaping their diversification remain poorly resolved. One key component ensuring translational fidelity is the tRNAIle-lysidine system, mediated by tRNAIle-lysidine synthetase (TilS), which enables accurate decoding of the AUA isoleucine codon. Here, we characterized the complete plastid genome of the Antarctic green alga Micractinium simplicissimum and investigated the evolutionary distribution of the tilS-trnI(CAU) module across green algal plastomes. Phylogenomic analyses of 35 plastomes revealed consistent retention of this module in Chlorellales, whereas partial loss or structural fragmentation occurred in core Trebouxiophyceae lineages. Comparative analyses of codon usage showed that AUA frequencies varied widely among major green algal lineages but were not tightly associated with tilS retention, suggesting partial evolutionary decoupling between tRNA modification systems and synonymous codon usage. Structural comparisons further revealed lineage-specific insertions and domain rearrangements in plastid TilS proteins relative to their cyanobacterial homologs. Candidate nuclear-encoded TilS homologs were additionally identified in several plastid tilS-lacking taxa, suggesting possible intracellular relocation of decoding functions. Together, these findings suggest that plastid decoding systems evolve through modular, lineage-specific trajectories that allow structural plasticity while maintaining translational fidelity. This study provides new insights into the evolutionary dynamics of gene expression systems in endosymbiotic organelles.