Ongoing structural changes highlight the dynamic nature of chloroplast genomes

High-throughput sequencing has transformed chloroplast genome research by enabling cost-effective analyses from shallow whole-genome sequencing datasets generated using short-read platforms, yet standardized approaches often overlook hidden structural signals critical for understanding plastome evolution. To uncover such signals, we reanalyzed data from our previous study of the subfamily Aurantioideae, which includes Citrus and its relatives, and developed a rapid bioinformatic strategy to detect subtle but significant structural rearrangements. Our analysis revealed the emergence of a short inverted repeat pair, which initially destabilizes the plastome and subsequently serves as a substrate for intramolecular homologous recombination. This recombination generates a ∼22 kb inversion in the large single-copy region, producing two distinct genomic configurations. It uncovers structural heteroplasmy across multiple species, which has been maintained since the early Miocene (12.1-28.2 Ma), with the minor isoform becoming dominant in certain lineages through genetic drift. Such structural changes occur gradually rather than as discrete events, representing intermediate stages of plastome evolution. Detection of these stages highlights the dynamic and unstable nature of chloroplast genomes, uncovering a previously underappreciated mechanism generating plastome diversity. Overall, these findings demonstrate that chloroplast genomes are more fluid than previously recognized, with structural transitions actively driving plastome evolution and diversification.