DNA Barcoding and Untargeted Metabolomics Reveal Phylogenetic and Chemotaxonomic Relationships Among 11 Ferula Species from Xinjiang, China

Background/Objectives: Morphological similarity among Ferula species, particularly during the vegetative stage, complicates the reliable authentication of leaf materials. The correspondence between phylogenetic relationships and leaf chemotaxonomic patterns among wild and cultivated Ferula resources remains insufficiently characterized. This study addressed three questions in 11 Ferula species from Xinjiang, China: whether combined DNA barcodes support species authentication and broad phylogenetic grouping; whether leaf metabolic profiles reveal taxon-associated differentiation; and to what extent phylogenetic and metabolic groupings correspond. Methods: Four DNA barcode loci, ITS2, trnH–psbA, rps11, and atpA–trnR, were combined for molecular authentication and phylogenetic analysis. Ultrasound-assisted extraction conditions were optimized using response surface methodology to establish standardized sample preparation, followed by LC–MS-based untargeted metabolomic profiling of 33 leaf samples representing 11 species. Multivariate analyses were used to characterize taxon-associated metabolic patterns and chemotaxonomic relationships. Results: The combined DNA barcode dataset generally supported the authentication of most investigated samples and revealed four broad phylogenetic groups, although several closely related accessions showed limited resolution. Untargeted metabolomics resulted in the putative annotation of 1972 metabolites, predominantly terpenoids, flavonoids, phenolic acids, and coumarins. PCA revealed taxon-associated metabolic patterns, while HCA grouped the samples into three major metabolic clusters. Notably, F. sinkiangensis, F. fukanensis, F. lehmannii, F. teterrima, and F. conocaula occurred within the same broad phylogenetic group and the same metabolic group, indicating partial correspondence between molecular relationships and metabolic phenotype. Conclusions: DNA barcoding and untargeted metabolomics provided complementary genetic and chemical evidence for the authentication and comparative characterization of Ferula leaves. The observed partial correspondence between phylogenetic relationships and metabolic profiles provides an exploratory basis for understanding chemical differentiation among the investigated Ferula species.