Many species of Schisandraceae are used in traditional Chinese medicine and are faced with contamination and substitution risks due to inaccurate identification. Here, we investigated the discriminatory power of four commonly used DNA barcoding loci (ITS, trnH-psbA, matK, and rbcL) and corresponding multi-locus combinations for 135 individuals from 33 species of Schisandraceae, using distance-, tree-, similarity-, and character-based methods, at both the family level and the genus level. Our results showed that the two spacer regions (ITS and trnH-psbA) possess higher species-resolving power than the two coding regions (matK and rbcL). The degree of species resolution increased with most of the multi-locus combinations. Furthermore, our results implied that the best DNA barcode for the species discrimination at the family level might not always be the most suitable one at the genus level. Here we propose the combination of ITS+trnH-psbA+matK+rbcL as the most ideal DNA barcode for discriminating the medicinal plants of Schisandra and Kadsura, and the combination of ITS+trnH-psbA as the most suitable barcode for Illicium species. In addition, the closely related species Schisandra rubriflora Rehder & E. H. Wilson and Schisandra grandiflora Hook.f. & Thomson, were paraphyletic with each other on phylogenetic trees, suggesting that they should not be distinct species. Furthermore, the samples of these two species from the southern Hengduan Mountains region formed a distinct cluster that was separated from the samples of other regions, implying the presence of cryptic diversity. The feasibility of DNA barcodes for identification of geographical authenticity was also verified here. The database and paradigm that we provide in this study could be used as reference for the authentication of traditional Chinese medicinal plants utilizing DNA barcoding.

DNA barcoding is a biological technique that uses short and standardized genes or DNA regions to facilitate species identification. DNA barcoding has been used successfully in several animal and plant groups. Ligustrum (Oleaceae) species occur widely throughout the world and are used as medicinal plants in China. Therefore, the accurate identification of species in this genus is necessary. Four potential DNA barcodes, namely the nuclear ribosomal internal transcribed spacer (ITS) and three chloroplast (cp) DNA regions (rbcL, matK, and trnH-psbA), were used to differentiate species within Ligustrum. BLAST, character-based method, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capabilities of the chosen markers for discriminating 92 samples representing 20 species of this genus. The results showed that the ITS sequences have the most variable information, followed by trnH-psbA, matK, and rbcL. All sequences of the four regions correctly identified the species at the genus level using BLAST alignment. At the species level, the discriminating power of rbcL, matK, trnH-psbA, and ITS based on neighbor-joining (NJ) trees was 36.8%, 38.9%, 77.8%, and 80%, respectively. Using character-based and maximum parsimony (MP) tree methods together, the discriminating ability of trnH-psbA increased to 88.9%. All species could be differentiated using ITS when combining the NJ tree method with character-based or MP tree methods. Overall, the results indicate that DNA barcoding is an effective molecular identification method for Ligustrum species. We propose the nuclear ribosomal ITS as a plant barcode for plant identification and trnH-psbA as a candidate barcode sequence.

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