Comparative plastome analysis reveals structural variation, selection, and phylogenetic relationships in Verbascum...

The chloroplast genomes of Verbascum speciosum and V. sinuatum reveal lineage-specific structural variations and hypervariable loci (ycf3_1-trnS-GGA, rps15, matK) that serve as powerful markers for species identification. Signatures of positive selection in ndhB, ycf3, and ycf4 suggest adaptive evolution in plastid genes, while phylogenomic analyses confirm that plastome-scale data are essential for resolving species-level relationships in this medicinally valuable genus. The genus Verbascum (Scrophulariaceae) is both species-rich and notoriously difficult to classify, with many members valued for their medicinal properties but hampered by vague morphological boundaries and a lack of reliable molecular data. To address this, we sequenced and assembled the complete chloroplast genomes of two Iranian medicinal species, Verbascum speciosum and Verbascum sinuatum. The two plastomes measured 153,325 bp and 153,038 bp, respectively, each containing the typical quadripartite structure and 131 genes. Comparative work uncovered lineage-specific shifts at the inverted repeat boundaries, most notably involving the rpl23 gene, as well as clear differences in simple sequence repeat abundance, with V. sinuatum showing far fewer SSRs than its relative. Scanning across the genomes, we identified several hypervariable spots (rps15, rps16, matK, ycf1, and even some tRNA genes) that could serve as useful barcoding markers. While most coding regions are under strong purifying selection, we found signs of positive selection at specific codon sites within ndhB, ycf3, and ycf4. Phylogenomic analysis using whole plastome data strongly supported the monophyly of Verbascum and confidently placed both new species within the genus. In contrast, standard single-locus barcodes failed to resolve species-level relationships. This work provides the first complete cp genomes for these two species and demonstrates that plastome-scale data offer a real step forward for untangling Verbascum systematics, identifying informative markers, and guiding future taxonomic work.