## 4 Discussion
### DNA Barcoding Identification of Herbal Tea  
DNA barcoding identification can identify counterfeit species with highly similar biological traits that cause great difficulty in morphological identification. This technology can accurately and rapidly identify several species or even dozens of families based on one or a few selected gene segments. This technology has the advantages of simple operation and good repeatability of results. The method can be universally applied to build a unified database and evaluation platform, as well as to promote and standardize plant classification. DNA sequences have been extensively used in systematic taxonomic research for species identification [40; 41]. DNA barcoding technology has been increasingly applied in authenticating herbal tea [42; 43]. [PERSON] et al. [42] used _rbcl_ or _matK_ barcodes to test the ingredients of 146 commercial tea products and showed that a third of the identified DNA was not found on herbal tea labels. [PERSON] et al. [43] systematically collected 177 samples of raw materials and established DNA barcodes for the identified botanical sources of Chinese \"cooling\" beverages. In this study, the CH-17 and CH-18 are commercial tea samples and high-quality DNA in both of them were extracted from these samples and the NJ tree and BLAST result based on similarity showed that they were identified as the labeled species.  
China is the \"hometown\" of tea and the \"birthplace\" of tea culture. Chinese tea culture has a long history, and this culture has spread worldwide. Tea is edible and can be used as an antidote. Long-term tea-drinking can benefit health and prolong life-span. An ancient saying has stated that \"tea is the essence of heaven and earth, and is the foundation of life\" [44; 45; 46]. Shi-Liang tea is a health drink used by the She minority group for hundreds of years. The importance of standardization and quality control of Shi-Liang tea has increased because of its rapidly growing popularity and trade. To ensure the use of correct materials in tea production, this study demonstrated the use of DNA barcodes to identify the source species of Shi-Liang tea (_C. salicoflius_ and _C. zhejiangensis_) and other Calycanthaceae species using the ITS2 sequence. The results of sequence alignment demonstrated the significant differences in ITS2 sequences between the original species of Shi-Liang tea and closely related species. The nucleotide variation sites among these sequences reached up to 37. The NJ treeconstructed based on the ITS2 sequences demonstrated that the Shi-Liang tea species (_C. salicilius_ and _C. zhejiangensis_) formed one clade, which can be successfully distinguished from closely related species.  
Application of the Secondary Structure of ITS2 Sequences in Traditional Chinese Medicine Identification  
The ITS2 region of nuclear DNA is a powerful marker for DNA barcoding because of the abundant variation in primary sequences and secondary structures. The application of secondary structures of ITS2 sequences to reconstruct phylogenies could improve the stability of obtained trees and increase the accuracy of phylogenetic estimations [24; 25; 26]. [PERSON] et al. [26] built an ITS2 database web server, which facilitates the detection and prediction of secondary structure of ITS2 sequences. [PERSON] et al. [47] compared the secondary structures of the ITS2 sequences of _Acanthopanacis cortex_ and its adulterants, providing another dimensionality to species identification. Thus, we focused on the divergence of primary sequences of ITS2 and the use of variations in the secondary structures to identify the She medicine species. By comparing the secondary structure of Shi-Liang tea species and closely related species, the results show that the secondary structures of all analyzed species had four similar helices: Helices I, II, III, and IV. Major differences among these helices could more distinctly distinguish the original plants of Shi-Liang tea species from closely related species than primary sequences.