Background: Viola philippica Cav. is the only original plant for Violae Herba, as described in the Chinese Pharmacopoeia. The quality of this crude drug is affected by several adulterants from congeneric Viola species, and the authentic plant origin of Violae Herba is still controversial. Genome-based identification offers abundant genetic information and potential molecular markers that can be used for the authentication of closely related species. This study aims to investigate the certified origin of Violae Herba and to develop more effective markers for these easily confused species at the genetic level. Methods: We compared the morphology and chemical composition of 18 batches of commercial samples and six widespread medicinal Viola plants used as Violae Herba or its substitutes by TLC and HPLC-Triple-TOF–MS/MS analyses. The complete chloroplast genomes of these species were sequenced and analyzed, including the general features, repeat sequences, mutational hotspots and phylogeny. The complete chloroplast genomes used as superbarcodes and some specific barcodes screened from mutational hotspots were tested for their ability to distinguish Viola species. Results: A comparative study showed that Violae Herba is a multi-origin traditional Chinese medicine. Commercial decoction pieces and the standard reference drug were mainly derived from V. prionantha, clashing with the record in the Chinese Pharmacopoeia. Chloroplast genome analyses of V. philippica and five adulterants indicated that sequence divergence was relatively low within Viola species. By tree-based approaches, the complete chloroplast genomes showed a better discrimination ability and phylogenetic resolution for each Viola species. These results indicate that the whole chloroplast genomes can be used as superbarcodes to differentiate Viola medicinal plants. More specific DNA barcodes could be further developed from the Viola chloroplast genomes for more efficient and rapid identification of commercial Violae Herba and its adulterants. Conclusions: This study has implications for chloroplast genome-based phylogenetic analysis and the authentication of multiple Viola species used as Violae Herba. The legal origin recorded in the Chinese Pharmacopoeia should be further revised to V. prionantha, in line with the commercial Violae Herba in the TCM markets.

Polygonaceae is a large family of medicinal herbs that includes many species used as traditional Chinese medicine, such as Per sicaria per foliata. Here, we sequenced the complete chloroplast genome of P. per foliata using Illumina sequencing technology with the purpose of providing a method to facilitate accurate identification. After being annotated, the complete chloroplast genome of P. per foliata was compared with those of Fagopyrum tataricum, Per sicaria chinensis, Fagopyrum dibotrys, and Fallopia multiflora. The complete chloroplast genome of P. per foliata is 160 730 bp in length, containing a small single-copy region of 12 927 bp, a large single-copy region of 85 433 bp, and a pair of inverted repeat regions of 62 370 bp. A total of 131 genes were annotated, including 8 rRNA genes, 34 tRNA genes, and 84 protein-coding genes. Forty-two simple sequence repeats and 55 repeat sequences were identified. Mutational hotspot analyses indicated that five genes ( matK, ndhF, ccsA, cemA, and rpl20) could be selected as candidates for molecular markers. Moreover, phylogenetic analysis showed that all the Polygonaceae species formed a monophyletic clade, and P. per foliata showed the closest relationship with P. chinense. The study provides valuable molecular information to accurately identify P. per foliata and assist in its development and application.