Abstract
<title>Abstract</title> <p> <bold>Background</bold> <italic>Neopicrorhiza scrophulariiflora</italic> (Pennell) D. Y. Hong, a traditional medicinal plant adapted to extreme alpine environments, has a long history of use in Tibetan and Chinese medicine. The species was recognized as an endangered (EN) species in the IUCN Red List of Threatened Species in 1987. Despite its ecological and medicinal importance, its mitochondrial genome remains uncharacterized, limiting insights into its evolutionary adaptations and genomic architecture. <bold>Results</bold> In this study, we assembled and comprehensively analyzed the complete mitochondrial genome of <italic>N. scrophulariiflora</italic> . The genome spans 606,216 bp with a GC content of 44.31% and exhibits a multi-chromosomal architecture comprising four circular chromosomes, encompassing 56 genes, including 34 protein‑coding genes (PCGs), 3 rRNA genes, and 17 tRNA genes. Analysis of codon usage bias revealed a preference for A/U-ending synonymous codons, with 23 of the 30 preferentially used codons terminating in A or U, aligning with trends observed in other angiosperms. Repetitive sequence analysis identified 66 SSRs, 11 tandem repeats, and 557 dispersed repeat pairs (284 forward and 273 palindromic repeats), suggesting their involvement in genomic rearrangement and structural diversification. A total of 492 high‑confidence RNA editing sites (score ≥ 0.9) were predicted across all PCGs, all of which were C‑to‑U transitions, with nad5 harboring the highest number of editing sites (102) and rps14 the fewest (2). Comparative genomic analysis uncovered 49 chloroplast‑derived homologous fragments (MTPTs) totaling 37,452 bp, accounting for approximately 6.18% of the mitogenome, providing evidence of frequent inter-organellar DNA transfer. Phylogenetic reconstruction based on 32 conserved mitochondrial PCGs across 28 taxa placed <italic>N. scrophulariiflora</italic> within Plantaginaceae, clustering with Digitalis ferruginea with moderate bootstrap support (65%). Synteny analysis with 8 closely related species revealed extensive structural rearrangements, with homologous regions covering 34.57% of the mitogenome with PX523758.1. Additionally, Ka/Ks analysis of 33 shared PCGs indicated that most mitochondrial genes (24 genes) are under strong purifying selection, with 1 gene ( <italic>sdh3</italic> ) exhibiting a median Ka/Ks value ≥ 1, suggesting possible positive selection linked to high-altitude adaptation, while <italic>rps14</italic> showed particularly strong purifying selection reflecting its essential role in mitochondrial translation. <bold>Conclusions</bold> This study presents the first complete mitochondrial genome of <italic>N. scrophulariiflora</italic> , an endangered medicinal plant endemic to the Himalayan region, and offers a thorough characterization of its genomic architecture. Our findings expand the mitochondrial genomic resources for Plantaginaceae and elucidate the structural features, codon usage patterns, repetitive elements, RNA editing landscape, and inter-organellar gene transfer events of <italic>N. scrophulariiflora</italic> . Additionally, we establish its phylogenetic position within the family. The results confirm the placement of <italic>N. scrophulariiflora</italic> in Plantaginaceae, consistent with the APG IV system, and identify candidate genes under positive selection that may be linked to high-altitude adaptation. This work provides a valuable genomic foundation for future conservation genetics, evolutionary studies, and the sustainable utilization of this endangered medicinal species. </p>