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

Projecting how populations will adapt to environmental changes requires a mechanistic understanding of selection imposed by various biotic and abiotic factors. 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 including a factorial manipulation of precipitation and herbivory at high and low latitude study sites. 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 defense phenotype. We found precipitation and herbivory did not drive differential selection on HCN or its metabolic components, although the strength of our conclusions about precipitation was impacted by the limited effectiveness of this treatment. Independent of the manipulations, HCN phenotypes did not exhibit the expected latitudinally-dependent fitness advantages based on observed HCN clines. Instead, we found that an individual metabolic component of HCN, cyanogenic glycosides, resulted in a fitness advantage, even when lacking the ability to produce HCN, and that this advantage was most prominent at the northern study sites, where cyanogenic glycosides are rarer These results suggest additional physiological roles of cyanogenic glycosides in adaptation of white clover to its environment. This study highlights the importance of experimental manipulations to understand the, often unexpected, ways selection acts on phenotypic traits in natural populations.