Temperature and herbivory have no effect on cyanide production in white clover

Hanna Jackson

Department of Biological Sciences, Simon Fraser University, Canada

*This report is a class project NOT peer-reviewed science


Factors including temperature, herbivory pressure, and elevation have been posited to exert selective pressures on the ability of white clover to produce cyanogenesis. It has previously been found that there is genetic variability in cyanogenic ability and it is a heritable trait (Ochoa-López et al. 2018), so evolution is possible. The varying environmental selective pressures on clovers will determine whether it is advantageous or disadvantageous for an individual to have the phenotype that allows produces cyanide. However, the components that make up cyanide are energetically costly to make (Hayden and Parker 2002), meaning that any potential benefit will need to outweigh this cost.

The more herbivory a plant experiences, the lower its reproductive success, thus conferring a selective pressure to ward off potential herbivores. Cyanide presence in plants one such deterrent, so we hypothesise (H1) that herbivory will be associated with the percentage of white clover plants in a given population that are capable of cyanogenesis. Specifically, we predict that with increased herbivory, cyanogenic plants will make up a higher proportion of individuals.

When cyanogenic plants freeze, the substrate and enzyme can react to create cyanide, and it was though that this then caused damage to the plant thereby lowering reproductive success (Daday, 1965). However, more recent studies have found that clover plants likely use physiological mechanisms that regulate cyanide and preserve the function of plant cell function in the presence of cyanide (Kooyers et al. 2018). Because of this recent evidence, and the fact that at higher elevations plants will experience more frequent freezing, we hypothesize (H2) that elevation will not be associated with the percent of white clover plants that are capable of cyanogenesis.

To conduct these experiments, we sampled white clover (Trifolium repens) for percent herbivory and percent cyanogenic capability at high and low elevations to see which factors would significantly explain the variation in cyanogenic ability.


The average percent herbivory was 3.16% at high elevation sites and 3.45% at low elevations. The average percent cyanogenic plants was 10.0% at high elevation sites and 25.08% at low elevation sites. Percent herbivory and percent cyanogenic plants were not significantly associated with one another (R2=0.13, p=0.264, F1,9=1.419, Figure 1). There was no significant difference in percent cyanogenic plants between high and low sites (t-test: p=0.8803, t8.3=1.9312, Figure 2).

Figure 1 We sampled white clover at various sites and measured each site’s average percent herbivory and percent cyanogenic plants. Percent herbivory did not significantly explain percent cyanogenic plants (R2=0.13, p=0.264, F1,9=1.419)

Figure 2. We tested white clover plants at high and low elevations for their ability to produce cyanide. The difference in percent cyanogenic plants between high and low sites was marginally insignificant (t-test: p=0.8803, t8.3=1.9312). The average percent cyanogenic plants at high elevation sites was 10.0% and at low elevation sites it was 25.08%.


We sampled white clover at various elevations and measured percent herbivory and percent of plants with cyanogenic ability to see if elevation and herbivory were related to percent cyanogenic plants at any given site.

Our first hypothesis (H1) predicted that there would be a relationship between herbivory and percent cyanogenic plants. However, when tested, percent herbivory did not significantly explain differences in percent cyanogenic plants (R2=0.13, p=0.264, F1,9=1.419), which was counter to our hypothesis. This counterintuitive result may be explained in multiple ways. It is possible that there are other factors that make it disadvantageous to evolve cyanogenic ability, despite predation pressures. For example, it has previously been found that as distance from urbanized areas increases, the proportion of plants capable of cyanogenesis increases by 0.6% per kilometre away from urbanization (Johnson et al. 2018). Another possible explanation is that herbivory is transient, with varying degree of pressure exerted on any given population at a given time. This would lead to selective pressures that may change over time and across generations, leading to mixed effects even if the pressure does affect individual fitness. This hypothesis will need to be addressed in future work.

Our second hypothesis (H2) proposed that percent cyanogenic plans would be the same at high and low sites. The proportion of cyanogenic plants did not significantly differ between high and low sites (t-test: p=0.8803, t8.3=1.9312), which was consistent with our hypothesis. This result is consistent with a recent study that found that freezing-induced cyanide toxicity likely isn’t responsible for differing cyanogenic capacity in clover (Kooyers et al. 2018), in contrast to historical studies that proposed that freeze-induced cyanide toxicity induced the temperature clines in percent cyanogenic ability (Daday 1965). This recent study concluded that this is likely due to the presence of certain physiological mechanisms that can maintain plant cell function even in the presence of cyanide (Kooyers et al. 2018).

It is possible that the distance to urban areas was a confounding variable that affected the patterns of our results. It has been found that as distance from urban areas increases, proportion of plants capable of cyanogenesis increases as well (Johnson et al. 2018). Because of the nature of our study region, high elevation locations appear to be away from urbanization and this may have caused an increase in the amount of cyanogenesis that was seen in our high elevation samples compared to what we might see if the high and low sites were of equal distance to urban areas.

Future studies should look further into urban-rural differences that may affect cyanogenesis gene expression patterns, and try to uncover the relationship between temperature, elevation, urbanization and herbivory more clearly.


Daday, H. 1965. Gene friquencies in wild populations of Trifolium repens L. IV. Mechanisms of Natural Selection. Heredity 20:355–365.

Hayden, K. J., and I. M. Parker. 2002. Plasticity in cyanogenesis of Trifolium repens L: Inducibility, fitness costs and variable expression. Evolutionary Ecology Research 4:155–168.

Johnson, M. T. J., C. M. Prashad, M. Lavoignat, and H. S. Saini. 2018. Contrasting the effects of natural selection, genetic drift and gene flow on urban evolution in white clover (Trifolium repens). Proceedings of the Royal Society B: Biological Sciences 285.

Kooyers, N. J., B. Hartman Bakken, M. C. Ungerer, and K. M. Olsen. 2018. Freeze-induced cyanide toxicity does not maintain the cyanogenesis polymorphism in white clover (Trifolium repens). American Journal of Botany 105:1224–1231.

Ochoa-López, S., R. Rebollo, K. E. Barton, J. Fornoni, and K. Boege. 2018. Risk of herbivore attack and heritability of ontogenetic trajectories in plant defense. Oecologia 187:413–426.