Comparative plastome analyses reveal life-form-associated selection signatures in Orchidaceae

Orchidaceae, one of the largest and most ecologically diverse families of angiosperms, exhibits remarkable variation in life form and includes numerous species with significant medicinal and economic value. However, comparative analyses of plastomes across orchid genera—particularly those explicitly linking selection signatures to life-form divergence and evaluating their evolutionary implications—remain scarce. Therefore, systematic characterization of plastome structural variation and life-form-associated selective pressures has the potential to advance orchid conservation strategies, sustainable cultivation practices, and fundamental evolutionary understanding. We conducted comparative plastome and phylogenetic analyses of 67 orchid species representing 15 genera to characterize plastome structure, selective pressures, and evolutionary relationships. The plastomes exhibited GC contents ranging from 24.75% to 39.16% and a distinct codon usage bias toward A/U-ending codons. Our analyses revealed lineage-specific variation in genome size, ndh gene loss, repeat content, and codon usage, with fully mycoheterotrophic orchids generally exhibiting more extensive genome reduction. Positive selection analyses uncovered life-form-associated patterns: positively selected genes in terrestrial orchids included atpI and rpl22, whereas those in epiphytic orchids included rpl22, rpoA, and rps2; no positively selected genes were detected in mycoheterotrophic orchids. Site‑level analyses further indicated that amino acid substitutions at residues 122 and 124 of Rpl22 may be associated with life-form divergence. Structural analyses revealed inversions, IR boundary shifts, and lineage-specific disruptions in genomic synteny. Phylogenetic reconstructions largely recovered the expected generic relationships, whereas anomalous placements—such as Dendrobium chrysotoxum Lindl. nested within Pholidota—may reflect historical hybridization or introgression. This study provides insights into the potential effects of life form on plastid gene evolution in orchids, highlights molecular selection patterns associated with ecological strategies, and establishes a comprehensive genomic framework for research on orchid systematics, conservation, and sustainable cultivation.