Cyanogenesis variation in white clover is decoupled from regional climate and human footprint across a longitudinal gradient

Latitudinal clines in adaptive traits are well documented, yet how such patterns translate across longitudinal gradients, where climatic variation is reduced, remains poorly understood. We investigated spatial variation in hydrogen cyanide (HCN) - a well-characterized anti-herbivore defense polymorphism governed by two independently segregating loci ( Ac and Li ), in white clover ( Trifolium repens ) across nine Midwestern U.S. cities. A total of 269 populations (4,035 individuals) were sampled along urban-rural transects, and cyanotype (cyanogenesis phenotypes) frequencies were quantified alongside regional and site-level environmental variables, including vegetation cover (NDVI), land surface temperature (LST), snow cover, impervious surface intensity, aridity, and potential evapotranspiration. Despite substantial variation in cyanogenic frequency among cities (6.2–19.0%), minimum winter temperature varied by only 7.9 °C, markedly less than continental latitudinal gradients (21–35 °C). Accordingly, cyanogenic frequency showed no consistent relationship with environmental predictors: model selection supported a null-model, and within-city associations were heterogeneous in direction, indicating that regional effects do not scale predictably across the longitudinal gradient. In contrast, analyses of individual cyanotypes revealed locus-specific environmental associations. Site-level NDVI and LST linked to the linamarase-only cyanotype ( acLi ), suggesting increased frequency in warmer, less vegetated urban habitats. Model selection identified environmental predictors for individual cyanotypes but not for total HCN frequency. These results indicate that cyanogenesis across this regional gradient is decoupled from broad-scale climatic drivers. Instead, locus-specific responses to heterogeneous environments generate compensatory dynamics among cyanotypes, obscuring patterns at the phenotypic level. Our findings highlight the importance of genetic architecture in adaptive polymorphisms and support fine-scale selective mosaics as key drivers of evolutionary dynamics.