## 4 Discussion  
Universal DNA barcoding is a widely used and effective tool for distinguishing diverse groups of plants and animal species [24]. This method can quickly and accurately identify plant species, and has improved our understanding of their genomic and phylogenetic relationships. However, land plants barcoding is more puzzling as the substitution rates within plant genomes are considerably lower than those of animals [25; 26]. Still, there are exciting examples where the barcoding genes have been remarkably successful in resolving relationships of taxonomically complex groups or species-level molecular systematics [27]. The same principle is proposed for customs officers where the use of DNA barcodes might identify plant samples or species where trade is governed or prohibited by international agreements such as CITIES [28]. Herein, for gaining insights into the diversity, potential origin and putative ancestry of _U. villosa_, universal barcoding genes, i.e., _rbcl_, _matK_ and _trnH_ regions were used (Table 1). More recently, the ITS as well as the _rbcl_ regions of plants have provided a baseline for comparing other genes and intergenic spacers to be used as DNA barcodes. Similarly, the _matK_ region has the potential to speed up the exploration and there are a number of studies where DNA barcoding have enhanced conservation efforts [26]. With all four barcode regions assessed here, at least one sequence of _Zelkova_ species was selected as a reference (outgroup). Phylogenomic analysis based on chloroplast genome have revealed the close relationships of _Z. serrata_ and _Z. schneideriana_ to the genus _llmus_, and it forms a well-supported monophyletic clade sister to genus _llmus_[29]. Therefore, in all barcodes investigated here, _Zelkova_ sequences were used as outgroup and it radiated out separately, this highlights the importance and robustness of DNA barcode at species or population level identification. For the current study, four barcode regions were selected, and universal primers were used to amplify these loci [20,27]. The success rate of amplification was 100% and all primers were able to amplify genomic regions of the expected lengths (Figures S1 and S2). By and large, there was agreement to the previously published literature regarding size of the amplicons [27,28]. However, sequencing results were not uniform, and out of 27 samples of _U. villosa_ for sequencing (Table 2), only six, four, four and 14 high quality reads were obtained for _rbcl_, _matK_ XF + 5R, _matK_ 390F + 1326R and _trnH-psbA_, respectively (Figures 2-5). For sequencing, samples collected from at least 50 m of elevation were used; interesting, the barcode loci indicated enough variability within the _U. villosa_ regions. Indels and SNPs were found across the whole length of the regions used (Figures 2-5). In addition to standard DNA barcodes (i.e., _rbcl_, _matK_ and of _trnH-psbA_), other single-copy genes such as leafy and waxy genes are gaining importance at species-level systematics [25]. However, for these regions, lack of universal primers and the low success rate of the existing oligonucleotides have significantly reduced their potential use as barcodes [28]. Furthermore, a low success rate of amplification of these genes with existing primers is likely due to their lower copy number and DNA extraction of poor quality from degraded plant samples. Additionally, from a number of plant species, DNA extraction with the general CTAB method is not ideal as it results in low quality DNA with cellular contaminants, and this requires cloning of the PCR products before such regions could be sequenced [30,31]. Constraints imposed by time, and more specifically by resources, did not allow us to exploit the above regions or endeavor cloning, and thus chloroplast and mitochondrial genes were investigated in the current study.