Yuxue Liu, qiang zhang, Zhenhua Wu, Zhenping Shi, Shuo Wang
Background: The subtribe Pleurothallidinae is a diverse group within Orchidaceae with a complex taxonomic history. Comparative plastome analysis can provide insights into genome evolution and facilitate phylogenetic reconstruction. Methods: Here we analyzed 25 complete chloroplast genomes representing 15 genera, including 14 newly assembled genomes, to investigate plastome evolution in this subtribe. Results: All genomes exhibited the typical quadripartite structure (148, 246–158, 138 bp) with conserved gene content (128–134 genes). While most protein-coding genes were under purifying selection, we detected signatures of positive selection in specific lineages. Notably, ndhF in Lepanthes tachirensis showed a markedly elevated Ka/Ks ratio (3.65), which may be associated with adaptation to an extensive distributional range. ENC-plot analysis indicated that natural selection, rather than mutation pressure alone, shapes codon usage bias, with patterns varying among species from different geographic regions. Nucleotide diversity analysis identified eight hypervariable intergenic regions (psbK-psbI, atpI-rps2, petN-psbM, psbB-psbT, petD-rpoA, rpoA-rps11, rps3-rpl22, ccsA-ndhD) suitable as candidate molecular markers. Phylogenetic analysis confirmed that Lepanthes and Pleurothallis are non-monophyletic as traditionally defined. Conclusions: These findings expand plastome resources for Pleurothallidinae, reveal lineage-specific patterns of selection, and provide molecular markers for future taxonomic and evolutionary studies.