Forsythia suspensa (Thunb.) Vahl, a pharmacopoeial medicinal plant, is valued for its therapeutic efficacy in heat-clearing detoxification, dispelling wind-heat, and promoting blood circulation to resolve stasis. Flavonoids, ubiquitous secondary metabolites in F. suspensa, are critically linked to pharmacological activities and exhibit diverse biological functions. To elucidate the chemotypic divergence and ecological drivers of its bioactive compounds, we conducted flavonoid metabolomic profiling across ten wild populations F. suspensa using UPLC-MS/MS. Results revealed significant inter-population variation in all twenty-nine flavonoid metabolites analyzed. Notably, Notably, Cinchonain Ic was significantly enriched in the JX population, Flavanomarein in the LT population, and Desmethylxanthohumol in the HX population. Association analysis with environmental variables further indicated that Sulfuretin, Apigenin-5-O-glucoside, and Flavanomarein were positively correlated with multiple precipitation-related variables (bio12-Annual Precipitation, bio14-Precipitation of Driest Month, bio17-Precipitation of Driest Month, and bio19-Precipitation of Coldest Quarter), whereas Vicenin 2 was negatively correlated with bio12, bio17, and bio19. Homoplantaginin showed a positive correlation with bio4 (Temperature Seasonality) and bio7 (Temperature Annual Range). Loureirin B was positively correlated with elevation but negatively correlated with high-temperature variables (bio5-Max Temperature of Warmest Month, bio8-Mean Temperature of Wettest Quarter, and bio10-Mean Temperature of Warmest Quarter). 5-Demethoxynobiletin was positively associated with both precipitation (bio12, bio17, bio19) and temperature variables (bio1-Annual Mean Temperature, bio6-Min Temperature of Coldest Month, bio9-Mean Temperature of Driest Quarter and bio11-Mean Temperature of Coldest Quarter). Cinchonain Ic was positively correlated with bio2 (Mean Monthly Temperature Range), and Oroxin A was negatively correlated with elevation. These findings demonstrated that flavonoids accumulation in F. suspensa was predominantly influenced by temperature heterogeneity, with precipitation serving as a secondary factor, while latitude and elevation play only limited roles. This study systematically investigates the divergence and environmental drivers of flavonoids in F. suspensa populations, clarifies the molecular ecological basis of its adaptation to environmental heterogeneity, and provides valuable insights for leveraging ecological factors to enhance medicinal potential, ultimately supporting targeted breeding and optimized field management strategies.

Introduction: Forsythia suspensa (Thunb.) Vahl,a pharmacopoeial medicinal plant,is valued for its therapeutic efficacy in clearing heat and detoxifying, dispelling wind-heat, and promoting blood circulation to resolve stasis. Phenolic acids, ubiquitous secondary metabolites in F. suspensa , are critically linked to pharmacological activities and exhibit diverse biological functions.It is therefore of significant interest to investigate whether environmental changes also alter the content distribution of phenolic acids in F. suspensa . Methods: To elucidate the chemical diversity and ecological drivers of its bioactive compounds, we conducted phenolic acids metabolomic profiling across 10 wild populations F. suspensa using UPLC-MS/MS. Results: Results showedsignificant inter-population variation in all thirty phenolic acids analyzed. Specifically, Verbascoside was significantly enriched in the AZ population, showing a positive correlation with Mean Monthly Temperature Range, Temperature Seasonality, and Temperature Annual Range, but a negative correlation with Annual Precipitation, Precipitation of Driest Quarter, Precipitation of Coldest Quarter and Min Temperature of Coldest Month. 4-Coumaroylshikimate, accumulated in WML and PS, was positively correlated with Precipitation of Wettest Month, Precipitation of Wettest Quarter, and Precipitation of Warmest Quarter, while negatively correlated with Min Temperature of Coldest Month. Gallotannin, enriched in LT, was negatively correlated with Mean Monthly Temperature Range, Temperature Seasonality, Temperature Annual Range, and Precipitation Seasonality, but positively correlated with Annual Precipitation. Isosalicin, accumulated in HX, showed a positive correlation with Max Temperature of Warmest Month and negative correlation with Annual Precipitation and Elevation. Discussion: These findings demonstrated that phenolic acids accumulation in F. suspensa was primarilydriven by temperature heterogeneity, with precipitation as a secondary factor, whereas adaptation to elevation plays a minimal role. This study systematically investigated the divergence and environmental drivers of phenolic acids in F. suspensa populations, clarifying the molecular ecological mechanisms behind its adaptation toenvironmental heterogeneity and thereby offering important insights into how ecological factors shape the medicinal potential of F. suspensa , ultimately informing targeted breeding and optimized field management.