## 4 Discussion
[PERSON] and [PERSON] have criticized _matK_ region mainly because no universal primers were available, in our case there was no issue with the amplification [20] and all samples have resulted in a PCR product of expected size (Figure S2B,C). Further, the _matK_ primers XF and 5R or 390F and 1326R [32] had the same success rate of sequencing, and both have amplified the same region with a 100% success rate (Figure S2). The _trnH-psbA_ loci is of low desire due to the extensive length variation and existence of pseudogene that are hosted by some species; this makes the alignment of sequences very difficult [32]. In some instances, trnH-psbA might indeed be problematic; however, in our case we found that it was one of the most useful regions with highest success rate of sequencing. On the contrary, ITS regions are the most frequently used barcode for plants, the success rate of sequencing here with ITS was the lowest and it was not accounted for even in the final analyses. Similarly, the success rate of sequencing using the same DNA quantity varied with every barcode. Thus, the simple looking task of selecting an appropriate locus to serve as a plant barcode has been much more complex, and there are no available DNA barcodes with likewise efficiency or that could work across all species [26]. Despite the current lack of consensus on a universal plant barcode, application of a genetic identifier to a wide set of research and applied programs has been projected [30; 31; 32]. More recently, it has been argued that single-locus DNA barcodes lack adequate variations in closely related taxa and might not serve the aim of precise species level identification; more stress is placed on the use of whole chloroplast genomes [29]. Over the years, chloroplast sequences are becoming more readily available because of improvements in sequencing technologies and cost [27; 29]. No doubt, the chloroplast genome sequencing can deliver a reliable barcode for accurate plant identification, though it is not yet resource effective, and cannot serve or replace the single-locus barcodes [26; 27; 28; 29; 30; 31; 32]. Nucleotide sequence analysis of the four genes revealed high levels of polymorphism. Additionally, the unconventional use of the assembly algorithm to group the most similar sequences that does not account for random nucleotide variations, and the phylogenetic trees built using the NJ algorithm revealed some important patterns of relationships within _Ullmus_ species. In addition, recombinant sequences (or at least very diverse) were identified (e.g., UV-RF-33 using \(matK\)) by the NJ algorithm and such features of sequence evolution would be extremely interesting to confirm it with additional markers. The overall phylogenetic results indicated that _Ullmus_ is not a monophyletic genus, and these results are in agreement with some of the most recent reports [26; 29]. Notably, no _U. parvifolia_ was found in the study area and only sequences were included from the NCBI, but within the NJ trees here, _U. villosa_ constituted a well-supported clade sister to _U. parvifolia_ indicating that in both trees, it resulted from \(matK\) region and indicative to their close relationships and possible shared ancestry. Further, it is reasoned that there have been multiple speciation events that have resulted in the evolution of _U. villosa_. We suggest that complete chloroplast genome of _U. villosa_ may provide important information for phylogenetic and evolutionary studies in _Ullmaceae_, as well as in other closely related families.