Supplementary material from "Contrasting the effects of natural selection, genetic drift and gene flow on urban evolution in white clover (Trifolium repens)"

Urbanization is a global phenomenon with profound effects on the ecology and evolution of organisms. We examined the relative roles of natural selection, genetic drift and gene flow in influencing the evolution of white clover (Trifolium repens), which thrives in urban and rural areas. Trifolium repens exhibits a Mendelian polymorphism for the production of hydrogen cyanide (HCN), a potent antiherbivore defence. We quantified the relative frequency of HCN in 490 populations sampled along urban–rural transects in 20 cities. We also characterized genetic variation within 120 populations in eight cities using 16 microsatellite loci. HCN frequency increased by 0.6% for every kilometre from an urban centre, and the strength of this relationship did not significantly vary between cities. Populations did not exhibit changes in genetic diversity with increasing urbanization, indicating that genetic drift is unlikely to explain urban–rural clines in HCN frequency. Populations frequently exhibited isolation-by-distance and extensive gene flow along most urban–rural transects, with the exception of a single city that exhibited genetic differentiation between urban and rural populations. Our results show that urbanization repeatedly drives parallel evolution of an ecologically important trait across many cities that vary in size, and this evolution is best explained by urban–rural gradients in natural selection.