Chloroplast Phylogenomics and Barcode Discovery in Medicinal <i>Stachys</i> Species Reveal Evolutionary Relationships and Adaptive Signatures

The genus <i>Stachys</i>, a large and taxonomically intricate group within the Lamiaceae family, includes numerous species valued in traditional medicine, yet their evolutionary relationships are often obscured by morphological complexity. To resolve these persistent difficulties, we sequenced and assembled the complete chloroplast genomes of two medicinally important Iranian species, <i>Stachys persica</i> and <i>Stachys germanica</i>, and compared them against seven congeneric genomes. Despite an overall picture of strong structural conservation across the genus, we discovered that evolutionary variation concentrates in specific genomic hotspots. Several non-coding intergenic spacers, most notably <i>trnL-trnF</i>, along with the <i>matK</i> and <i>ycf1</i> genes emerged as exceptionally variable regions, making them promising candidates for species-level DNA barcoding. In a more surprising finding, we detected clear signatures of positive selection acting on the photosynthetic gene <i>petB</i>, suggesting that even core energy pathways have experienced adaptive refinement within this lineage. Phylogenetically, whole-plastome data placed <i>S. germanica</i> as sister to <i>S. byzantina</i>, with <i>S. persica</i> as their closest relative, a relationship that individual barcode loci failed to recover reliably. Taken together, this study provides a robust evolutionary framework for <i>Stachys</i>, identifies practical molecular tools for authenticating medicinal materials, and demonstrates the power of whole-plastome sequencing to untangle taxonomically intricate plant groups.