Integrating morphology, molecular phylogeny and chemotaxonomy for the most effective authentication in Chinese Rubia with insights into origin and distribution of characteristic Rubiaceae-type cyclopeptides

The Rubia is one of important medicinal genera of the family Rubiaceae, widely distributed in the Old World with a great chemical diversity of quinones, cyclopeptides and triterpenoids. It has recently aroused us the most attention for its utilization as multi-origin resources and potential anti-tumor constituents of Rubiaceae-type cyclopeptides (RAs). But a thorough identification and discrimination of the Rubia species is challenging due to multi-species with very similar appearance. In this study, a synthesis of morphology, molecular phylogeny and chemotaxonomy was used to identification and discrimination of the Rubia species. DNA barcoding based on eight chloroplast markers and one nuclear ITS region was firstly applied to reconstruct its phylogenetic history. Sect. Rubia and sect. Oligoneura were clearly supported, but could not completely resolve species relationships within sect. Oligoneura. UHPLC-QTOF-MS technique was further optimized for non-targeted metabolomic analysis with multivariate statistical analysis in both supervised and unsupervised manners to distinguish patterns among the eight main populations. Variable importance in projection (VIP) revealed that triterpenes and RAs were the main specific chemotaxonomic markers. The origin and distribution center of the characteristic RAs in the Rubia species was inferred to be in Southwest (SW) China, which is also the hotspot for Rubia plants biodiversity. Our results highlighted a synthesis of morphology, molecular phylogeny and chemotaxonomy is the most effective strategy for authentication of complex plants (such as Rubia), and also provided insights into the exploration and optimization of high-quality germplasm resource for RAs.