Complete Chloroplast Genomes of two medicinal orchids from the Himalaya Habenaria intermedia and Herminium edgeworthii: Implications for DNA barcoding, phylogenetics, and conservation

• We report the first complete chloroplast genome sequences of Habenaria intermedia and Herminium edgeworthii , two threatened Himalayan medicinal orchids from the Astavarga group. • The plastomes exhibit a typical quadripartite structure with 133–134 genes and conserved gene order, but notable variation in intergenic regions and repeat content. • Five highly variable regions ( matK–rps16, rps16–psbK, trnC–petN–psbM, ndhF–rpl32, ycf1 ) were identified as potential plastid DNA barcodes for Habenaria and related genera. • Phylogenomic analyses (ML and BI) confirmed the polyphyly of Habenaria and recovered H. intermedia and H. edgeworthii in distinct, well-supported clades. • This study contributes valuable genomic resources for DNA barcoding, phylogenetics, and conservation of Orchidinae taxa underrepresented in current plastome datasets. Habenaria intermedia and Herminium edgeworthii are ecologically and medicinally important terrestrial orchids of the Himalayas and key members of the Astavarga group, traditionally used in Ayurveda for their rejuvenating and immunomodulatory properties. Despite their significance, validated DNA barcodes and phylogenetic placements for these species remain unresolved due to morphological convergence and limited genomic data. In this study, we report the complete chloroplast genome sequences of H. intermedia (153,035 bp) and H. edgeworthii (152,937 bp), both exhibiting the typical quadripartite structure with LSC, SSC, and IR regions. The genomes encode 133 and 134 genes, respectively, including 86 protein- coding genes, 8 rRNA genes, and 39–40 tRNA genes, with GC content of 36.6 % ( H. intermedia ) and 36.7 % ( H. edgeworthii ). Comparative plastome analysis of ten Habenaria species identified five hypervariable regions ( matK–rps16, rps16–psbK, trnC–petN – psb M, ndhF–rpl32 , and ycf1 ) as potential plastid DNA barcodes. Due to the lack of available plastomes, comparative analyses could not be performed for Herminium . Phylogenomic analysis based on complete chloroplast genomes provided robust support for genus-level relationships within subtribe Orchidinae and highlighted the evolutionary distinctiveness of Habenaria and Herminium . This study represents the first plastome-level resource for these taxa, contributing to DNA barcode reference libraries and offering insights into their phylogenetic placement. These findings provide a foundation for accurate taxonomic identification, molecular authentication, and the prioritization of conservation strategies for these threatened Himalayan orchids.