Plastome evolution in annual Brachypodium species reveals widespread heteroplasmy and chloroplast capture, lineage-specific codon usage bias, and low positive selection

Comparative genomics and plastome phylogenomics have advanced significantly in recent years, highlighting the diversity, possible admixture, and non-neutral evolution of the predominantly considered non-recombinant chloroplast genomes in angiosperms. The grass genus Brachypodium serves as a powerful model for studying evolutionary processes in monocots. We analyzed 287 plastomes across the native circum-Mediterranean range of the three annual Brachypodium species (B. distachyon, B. stacei, B. hybridum), focusing on their structural variation, selection patterns and phylogenomic relationships. Our analyses confirmed the differentiation of the S and D plastomes, inherited respectively from the diploid progenitor species B. stacei and B. distachyon. We identified novel structural rearrangements and indels, and unique repeat motifs, along with widespread heteroplasmy, particularly in ancestral B. hybridum-D plastotypes. SNP diversity varied among plastotypes, reflecting population dynamics and evolutionary histories, with B. hybridum-D plastotypes showing the highest normalized diversity and B. hybridum-S the lowest. Positive selection was detected in 29 plastid genes by Tajima’s neutrality test, and in nine genes by site and branch-site evolutionary models, including matK, ndhF, rbcL, and rpoC2. Phylogenomic analyses revealed well-supported clades corresponding to the S and D plastome lineages, with frequent chloroplast capture events and long-distance dispersals shaping their evolutionary trajectories. These findings highlight the evolutionary complexity of the Brachypodium panplastome, emphasizing the roles of heteroplasmy, structural variation, and adaptive evolution in shaping plastome diversity in this group of model grasses. While the ancestral plastomes of B. hybridum-D show evidence of heteroplasmy caused by introgressive hybridization and recombination, most of the heteroplasmic patterns in the other plastome groups can be attributed to somatic mutations. The topological congruence of phylogenies based on neutrally evolving whole plastome sequences and the majority of the plastid encoding gene sequences, including positively selected genes like ndhF, underscores the robustness of plastome-based phylogenies even under selective pressures. Together, these results shed new light on the evolutionary complexity of plastid genomes in a model polyploid system, demonstrating the interplay of structural divergence, heteroplasmy, hybridization, and selection in shaping plastome diversity. Our work thus provides a valuable comparative framework for future evolutionary, ecological, and functional genomic studies in Brachypodium and other grasses.