Cities are emerging as models for addressing the fundamental question of whether populations evolve in parallel to similar environments. Here, we examine the environmental factors that drive parallel evolutionary urban-rural clines in a Mendelian trait -- the cyanogenic antiherbivore defense of white clover (Trifolium repens). We sampled over 700 urban and rural clover populations across 16 cities along a latitudinal transect in eastern North America. In each population, we quantified the

more »

... ncy of genotypes that produce hydrogen cyanide (HCN), and in a subset of the cities we estimated the frequency of the alleles at the two genes (CYP79D15 and Li) that epistatically interact to produce HCN. We then tested the hypothesis that winter environmental conditions cause the evolution of clines in HCN by comparing the strength of clines among cities located along a gradient of winter temperatures and frost exposure. Overall, half of the cities exhibited urban-rural clines in the frequency of HCN, whereby urban populations evolved lower HCN frequencies. The weakest clines in HCN occurred in cities with the lowest temperatures but greatest snowfall, supporting the hypothesis that snow buffers plants against winter frost and constrains the formation of clines. By contrast, the strongest clines occurred in the warmest cities where snow and frost are rare, suggesting that alternative selective agents are maintaining clines in warmer cities. Additionally, some clines were driven by evolution at only CYP79D15, consistent with stronger and more consistent selection on this locus than on Li. Together, our results demonstrate that both the agents and targets of selection vary across cities and highlight urban environments as large-scale models for disentangling the causes of parallel evolution in nature.