Morphological profiling and DNA barcoding revealed genetic diversity and phylogeny of Mentha species cultivated in Pakistan

Mentha (family—Lamiaceae), known as mint is a perennial aromatic herb rich in antioxidant phenolic compounds. Genus Mentha comprises around 30 species, many interspecific hybrids, and selected varieties. In Pakistan, five Mentha species have been found and utilized for essential oils, culinary applications, food and pharmaceuticals. Understanding plant diversity across species, ecosystems and genetic levels is a prerequisite for conserving and sustainably utilizing native plants, as outlined in international and national strategies. This study aimed to identify 10 different Mentha species based on morphological characteristics and DNA barcoding. Morphological traits, including leaf blade shape, leaf margin, leaf apex, and flower color, were assessed to identify species-specific characteristics. Quantitative analysis revealed significant variation in stem length (ranging from 20 to 65 cm), leaf area (15–45 cm²), and chlorophyll index (25–45 SPAD units). Molecular characterization involved DNA barcoding using the Internal Transcribed Spacer (ITS) region of rDNA. The results revealed significant morphological diversity, classified into three distinct groups using Unweighted Pair Group Method with Arithmetic Mean (UPGMA) analysis. ITS markers proved effective in assessing genetic diversity and phylogeny, confirming the species' identities with 95–100% similarity to Gene Bank sequences. Results indicated significant differences among the studied Mentha species, leading to their clustering into three distinct groups according to UPGMA analysis. This research highlights the importance of genetic characterization for conserving Mentha species and underscores the potential of DNA barcoding in overcoming the limitations of traditional taxonomic methods.