A field experiment assessing the roles of drought, herbivory, and local climate on cyanogenesis cline formation and local adaptation in Trifolium repens

Projecting how a population will adapt to environmental changes requires a mechanistic understanding of the specific biotic and abiotic factors that impose selection on that population. In white clover (Trifolium repens), clines in an antiherbivore defense mechanism, hydrogen cyanide (HCN), form via variation in selection imposed by the environment. However, the specific environmental factors that select for or against chemical defense phenotypes in white clover remain unresolved. We performed a field experiment in high and low latitude study sites, with a factorial manipulation of precipitation and herbivory at each site. These factors are hypothesized to be important in driving HCN cline formation, so we investigated their effects on fitness of a white clover F3 recombinant population, segregating for the alleles underlying the HCN chemical defense phenotype. Surprisingly, we found precipitation and herbivory either did not drive differential selection on HCN or its metabolic components, or did not impose selection in a manner consistent with the maintenance of observed HCN clines. Instead, we find that the production only one of the metabolic components of HCN, cyanogenic glycosides, resulted in a fitness advantage, even when lacking the ability to produce HCN. This was most prominent at the northern study site, which is again contrary to expectations. These results suggest additional physiological roles that cyanogenic glycosides may play in adaptation and the evolutionary ecology of white clover. This study highlights the importance of experimental manipulations of environmental factors to understand how selection acts on genes underlying important phenotypic traits, often in unexpected ways.