Urbanization-induced environmental changes have been shown to drive the evolution of various organisms. Occurrences of urban evolutions mean that genetic variations in functional traits have been maintained within local populations. However, in the context of urban evolution, the mechanism by which such genetic variations are maintained is still unknown. One of the important mechanisms maintaining genetic variations is negative frequency-dependent selection (NFDS). NFDS can maintain genetic variations within populations by favoring rare alleles over common alleles. Previous studies have shown that genetic polymorphism in chemical trait (cyanogenesis) is maintained in a white clover population at various environments. Moreover, it has also been shown that frequency of cyanogenesis in white clover differs between urban and rural environments. If genetic polymorphism in cyanogenesis is maintained by NFDS in white clover, the urban-rural difference in cyanogenesis gene frequency can be a result of interactions between NFDS and urbanization-induced environmental changes, suggesting that the pattern of natural selection changes between urban and rural areas. To examine this hypothesis, manipulated field experiments examining frequency-dependent selection need to be conducted. However, no studies have ever tested such a hypothesis.

In this study, we aimed to empirically test whether NFDS contributes to maintaining polymorphism in cyanogenesis of white clovers, and whether the mode of natural selection differs between urban and rural environments. In particular, we focused on frequency-dependent selection on cyanogenesis in white clovers.

We conducted field manipulative experiments in combination with multi-site reciprocal transplanting in urban and rural environments. To examine the effects of frequency-dependence of cyanogenesis on fitness measurement (survival rate and biomass), we randomly manipulated the frequency of surrounding plants producing HCN (cyanogenic) around each individual plant. First, we found that herbivory pressure was higher in rural areas than in urban areas. Second, there were frequency-dependent selections on cyanogenesis. Third, NFDS occurred in rural environments mediated by higher herbivory pressure, whereas positive frequency-dependent selection (PFDS) occurred in urban environments mediated by lower herbivory pressure. These results highlight that the mode of natural selection changes between urban and rural environments. In other words, genetic variations in white clover are maintained by NFDS in rural areas, whereas urbanization causes PFDS, leading to a loss of genetic variations in urban areas.