DNA barcoding and phylogenetic insights into the selected endemic flora of the Western Himalayas

The Himalayan region is recognized as one of the world’s major biodiversity hotspots due to its remarkable altitudinal variation, high species richness, and exceptional endemism. Despite its ecological significance, the endemic flora of the western Himalayas remains insufficiently explored at the molecular level. This study aimed to evaluate the performance of multiple nuclear and chloroplast DNA barcode loci (ITS2, rbcL, trnH-psbA, trnA, trnV, rpoB, ycf3, and rbcLa) for the identification and phylogenetic assessment of 32 endemic and threatened plant species representing 31 genera and 24 families from the western Himalayan region of Pakistan, such as Thalictrum secundum Edgew. Aquilegia pubiflora Wall. ex Royle, Anemone obtusiloba D.Don and Delphinium cashmerianum Royle of family (Ranunculaceae), Pimpinella stewartii (C.B. Clarke) Nasir (Umbeliferaceae), Bistorta amplexicaulis (D.Don) Greene, and Bistorta affinis (D.Don) Greene (Polygonaceae) Abelia triflora R.Br. ex Wall. And Lonicera japonica Thunb. (Caprifoliaceae), Aesculus indica (Wall. ex Cambess.) Hook. (Sapindaceae), Arisaema utile Nakai (Araceae), Coriaria nepalensis Wall. (Coriaraceae), Desmodium elegans DC (Papilionacea), Daphne papyracea Wall. ex Steud. (Thymelaceae), Epimedium elatum C.Morren. (Berberidaceae), Galium tetraphyllum. Nazim. & Ehrend. (Rubiaceae), Bupleurum lanceolatum Wall. ex August. and Heracleum polyadenum Franchet (Apiaceae), Impatiens edgeworthii Hook.f. (Balsaminaceae), Incarvillea emodi Chatterjee (Bignoniaceae), Potentilla erecta (L.) Raeusch., Cotoneaster frigidus var. affinis (Lindl.) Wenz., and Spiraea hazarica R.Parker (Rosaceae), Phytolacca latbenia (Moq.) H.Walter. (Phytolaccaceae), Rhamnus parvifolius Bunge. (Rhamnaceae), Thymus linearis Benth. (Lamiaceae), Jasminum leptophyllum Wall. ex G.Don (Oleaceae), Rhododendron lepidotum Wall. ex Hook.f. (Ericaceae), Swertia ciliata Roxb. ex Fleming. (Gentianaceae), Zanthoxylum armatum DC. (Rutaceae), Dioscorea balcanica Kosanin (Dioscoreaceae), and Deutzia staminea R.Br. ex Wall. (Hydrangeaceae). Among the tested markers, rbcL showed the highest PCR amplification (100%) and sequencing success (96%), followed by trnH-psbA (100% amplification, 80% sequencing success), whereas ITS2 exhibited comparatively lower sequencing efficiency (52%). BLAST analysis demonstrated ≥ 97% sequence identity for most taxa, and best Match/Best Close Match analysis indicated higher discrimination efficiency for rbcL and trnH-psbA compared with other loci. Multilocus phylogenetic analysis further confirmed taxonomic placement at the family, genus, and species levels. Overall, this study demonstrates that a multilocus DNA barcoding approach provides reliable species authentication and phylogenetic resolution for endemic Himalayan flora, contributing valuable molecular reference data to global databases and supporting conservation and taxonomic efforts in this biodiversity-rich yet understudied region.