Metabolomic Differentiation and Chemotaxonomic Insights into Three Korean Alnus Species Revealed by UPLC-QTOF/MS

Alnus species distributed on Ulleung Island, South Korea exhibit persistent taxonomic ambiguity due to high morphological similarity and limited genetic divergence within the green alder complex. To establish chemically discriminatory criteria for species delimitation, UPLC-QTOF/MS-based untargeted metabolomic profiling was applied to Alnus alnobetula subsp. fruticosa (AA), Alnus incana subsp. hirsuta (AI), and Alnus firma (AF). Multivariate statistical analysis revealed reproducible metabolome-level differentiation among the three taxa. Among 47 annotated secondary metabolites, statistically significant discriminant features were defined as marker metabolites. Biosynthetic interpretation showed that these markers were structurally concentrated within distinct metabolite classes and selected discriminant markers were structurally confirmed. Alnustic acid-related secodammarane-type triterpenoid markers were consistently detected in AA and AF but absent in AI, providing a chemical criterion for exclusion of AI. In contrast, separation between AA and AF was driven by quantitative differences in conserved diarylheptanoid and flavonoid markers. Functional evaluation further demonstrated metabolite class-dependent biological activities. Collectively, integration of statistical prioritization, biosynthetic interpretation, structural validation, and functional assessment establishes a marker-based chemotaxonomic framework for resolving taxonomic ambiguity within Korean Alnus taxa.