Chloroplast genome analysis reveals intraspecific variation and phylogenetic conflicts in Bletilla.

The genus Bletilla Rchb.f., an endangered taxon of Orchidaceae Juss. with significant medicinal value, faces persistent controversies in taxonomic identification and phylogenetic relationships. In this study, we analyzed a dataset of 57 complete chloroplast genomes (CPGs) from five subfamilies of Orchidaceae, including five newly sequenced Bletilla CPGs and publicly available data. These CPGs were combined with corresponding nuclear ribosomal internal transcribed spacer (nrITS) sequences obtained from public databases to resolve taxonomic uncertainties and phylogenetic relationships within Bletilla. Our results revealed significant intraspecific variation in Bletilla CPGs with overlapping intraspecific and interspecific genetic distances (GDs), alongside non-monophyletic clades of B. striata, B. ochracea, and B. formosana in species-level phylogenies. Consequently, the CPG 'super barcodes'-proposed as high-resolution markers for plant identification-demonstrated limited discriminatory power in Bletilla, challenging their universal reliability. Phylogenetic analysis revealed conflicting placements between Bletilla and B. sinensis in CPG and nrITS trees: while nrITS data supported a monophyletic Bletilla within the subtribe Coelogyninae Benth., CPG data placed B. sinensis outside Bletilla, forming a sister clade with Calanthe triplicata and Tainia dunnii. Collectively, this study provides new evidence for the taxonomic identification and phylogenetic relationships of Bletilla through chloroplast-nuclear genome integration, offering a preliminary framework for taxonomic re-evaluation of complex plant groups.