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The plant family Lamiaceae (Mints) contains ca. 7173 species distributed among 273 genera worldwide. It has highly varied phenotypic characters. Many species are of horticultural and economic significance and most importantly the family represents a wealth of medicinaly important aromatic herbs e.g Lavandula (Lavender), Mentha (Mint), Origanum (Oregano, Marjoram), Salvia (Sage), Rosmarinus (Rosemary), Melissa (Lemon balm) and Thymus (Thyme). There are 60 genera and about 212 species of Lamiaceae reported from Pakistan. This is the first ever investigation from Pakistan based on DNA barcoding identification of herbal medicinal products (HMPs) of family Lamiaceae and its subsequent phylogenetic assessment based on DNA sequence analysis. The study is divided into three sections: DNA barcoding of HMPs belonging to Lamiaceae is conducted for their correct identification and to fix the problem of adulteration in the first section of present work. It can help in shielding consumers from the health hazards connected with the potential contamination and substitution of herbal medicinal products. HMPs representing 32 Lamiaceae plant samples were collected from three Pansar stores (herb stores) located at Islamabad and a herbal pharmaceutical industry. The extraction of total genomic DNA was carried out from these HMPs. Three plastid barcoding loci (rbcL, matK and trnH-psbA) were selected for PCR amplification and nucleotide sequencing was carried out. The comparison of DNA sequences obtained from these loci was carried out to ascertain the taxonomic identification of the plant material. We identified four mislabeled samples including one industry sample (Salvia haematodes) and three pansar store samples (Leucas linifolia, Lycopus europaeus and Salvia moorcroftiana II). Additionaly two product substitutions are also found in which Hyssopus officinalis is replaced by Nepeta bracteata and Nepeta ruderalis is substituted by Salvia spp. The identification of three HMPs (Lallemantia royleana, Origanum vulgare and Salvia aegyptiaca) is highly ambiguous because of lack of reference sequences available in GenBank. In Lamiaceae the overall amplification success for rbcL is 87% and for matK it is 81% while trnH-psbA showed 69%. Post sequencing analysis showed that trnH-psbA and matK have been able to discriminate the species relatively better with 40% success rate than rbcL (16%). On the whole a total of 22 sequences are genus-level barcodes (78%) and 12 sequences are species-level barcodes (44%). The nucleotide sequence data produced from the current study has been published in GenBank under the accession numbers KP172036-KP172082, KP218929- KP218945. We performed a comparative analysis of rbcL, matK and trnH-psbA to evaluate their performance for Lamiaceae barcoding. Our findings suggest matK as the potential barcode for Lamiaceae HMPs. The species-level identification was considerably challanging due to insufficient reference data and selection of plastid markers. Therefore, it is recommended for herbal pharmaceutical industries to develop a local (regional) herbal barcode library for their species of interest. The method of DNA barcoding can greatly assist the regulatory authorities and herbal industries to devise a mechanism for quality control and customer care. It can largely support the herbal pharmaceutical industries to restore the eroded consumer confidence. In second part of the study, phylogenetic utility of three barcoding regions (rbcL, matK & trnH-psbA) is estimated. The data sets were comprised of 245 rbcL ingroup taxa, 235 for matK and 259 for trnH-psbA. This sequence data was acquired from GenBank including the accessions produced in the first part of work. We found that among these three selected markers matK seems to be the best gene region which is able to resolve the subfamilies and provide strongly supported monophyletic genera. rbcL was not able to resolve the clades with a strong bootstrap (BS) support. It was difficult to align the spacer region trnH-psbA across the whole family; as a result it affected the tree topology and could not produce the well resolved clades of subfamilies. The third section of present work is the phylogenetic analysis based on plastid trnL-trnF and nuclear ribosomal internal transcribed spacer (nrITS) regions. A sum of 89 taxa was collected by visiting different wild areas and two herbaria of Pakistan. Additional publicly available data was downloaded from GenBank and the data sets were constructed. The data sets constitute 398 trnL-trnF taxa and 413 ITS ingroup taxa. The phylogenetic analyses were performed on the trnL-trnF and ITS data matrices by utilizing methods of ML (Maximum Likelihood) and BI (Bayesian Inference). We observed the Bayesian consensus trees showed more resolved nodes in comparison to ML consensus trees. The subfamilies received strong bootstrap values in the BI as compared to the ML results. In Pakistan’s Lamiaceae species, hybridization was observed, particularly evident in the nuclear ITS analysis. The analysis of taxa collected from Pakistan revealed that these species are undergoing possible radiation in place instead of dispersal. The taxonomic position of some species from Pakistan which were originally based on morphological characters did not corroborate with the findings of current molecular analysis. Therefore, it would be interesting to explore more plastid and nuclear loci with increased number of species for each group. Such approach will provide improved insight into relationships of mints from Pakistan. The intense studies more focused on each group (each subfamily) may draw a better picture of Pakistan’s Lamiaceae.
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