The genus Berberis is a case in which DNA barcoding using only a few regions has had limited success (Roy et al., 2010). Similarly, a phylogeny of Berberis based on ndhF and ITS loci failed to resolve boundaries of several species (Adhikari et al., 2015). Berberis aristata is a medicinal plant that has been in traditional use in India for centuries and is nowadays traded throughout the world (Srirama et al., 2017). Local market studies suggest that several species are traded under the same vernacular name (Srivastava and Rawat, 2013), including B. aristata and B. asiatica. B. aristata is described in several pharmacopoeias (Ayurvedic Pharmacopoeia of India, 2001; British Pharmacopoeia, 2016). Chemical and anatomical tests are deficient and conventional macro-morphological and microscopic examination do not distinguish the traded materials (Chandra and Purohit, 1980; Srivastava et al., 2004) therefore there is a strong incentive for the development of a DNA barcoding method for their identification.
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With the emergence of new sequencing technologies, whole plastid sequencing has been proposed as an extension of the current barcoding concept (Coissac et al., 2016). It has been shown that whole plastid sequences increase phylogenetic resolution (Parks et al., 2009) and simultaneously increase the effectiveness of discriminating between species. In this study, we show how whole plastid next-generation sequencing can be used to investigate sequence variability patterns for the discovery of informative DNA barcodes. We confirm the difficulty of barcoding Berberis species as suggested by Roy et al. (2010), even when whole plastid sequences are used for comparison. Although the sampling was limited, with only a few of the species represented with multiple samples, the low resolution of the plastid phylogeny at shallow phylogenetic levels and the presence of polyphyletic species (e.g., B. aristata) indicates evolutionary reasons for the failure of barcoding this genus to species level (Mutanen et al., 2016). DNA barcoding is challenging in groups where frequent hybridization occurs in conjunction with plastid capture or where lineage sorting has not yet been completed (Fazekas et al., 2009). A salient point arising from our study is that the pharmacopoeial species, B. aristata, is polyphyletic. One explanation for this finding is hybridization, a phenomenon documented in Berberis (Adhikari et al., 2012). Low resolution among the closely related species of Berberis as reported in the whole plastid phylogeny, could point toward retention of ancestral polymorphism or incomplete lineage sorting (Naciri and Linder, 2015). Misidentification of B. jaeschkeana, B. karnaliensis and/or B. mucrifolia is unlikely, since these have been included in recent revisionary work (Adhikari et al., 2012). Polyphyletic species are likely to persist where they are morphologically robust entities, and the development of methods for their identification, in this case for pharmacopoeia, benefits from understanding of their evolutionary history. The case of barcoding medicinal Berberis species provides an example of how barcoding for regulatory purposes in an evolutionarily complex group can be approached. Phylogenies can be essential for formulating adequate barcoding hypotheses; the whole plastid phylogeny reveals that at least three species are nested in the clade with the main species. The polyphyly of B. aristata indicates that universal barcodes are unlikely to delineate these species, and haplotype analysis shows this is the case for three of the most variable regions. Furthermore, several clades show low resolution at terminal branches. We have therefore adapted our classification scheme and defined meaningful OPUs that do not correspond to existing species limits. OPUs are the entities that can be discriminated by the barcodes put forward. The OPUs in this study are delimited using an integrative approach based on the interpretation of a whole plastid phylogeny, coupled with the detection of diagnostic nucleotides in relatively short barcodes for well-supported groups. These DNA barcodes can be targeted by PCR and Sanger sequencing and therefore offer a simple and fast identification test for regulatory purposes and quality control. Appropriate OPUs would be identified on a case-by-case basis for other evolutionarily complex groups for regulatory purposes. This is because for evolutionarily complex groups barcodes do not confirm species identity. The novelty of our approach lies in using whole plastid phylogeny to identify of short, easily amplified markers that incorporate clade-specific SNPs, and although we expect it to be more widely applicable it is only appropriate when the non-pharmacopoeial species belonging to the OPU are neither candidate adulterants nor substitute species, as is the case here.
Change is coming because the "war on drugs" is being convincingly won by drugs, and the powerful criminal gangs who deal in them. Since 1998, when the UN held an event entitled "A drug-free world: we can do it", consumption of cannabis (marijuana) and cocaine has risen by about 50%; for opiates, it has more than trebled. And a swelling pharmacopoeia of synthetic highs is spinning heads in dizzying new ways. The UN reckons that 230m people used illegal drugs in 2010. They and their suppliers (usually the humblest ones) fill prisons in rich and poor countries alike. Drug convictions account for almost half of American prisoners in federal jails. 2ff7e9595c
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