Experimental designs for testing the interactive effects of temperature and light in ecology: The problem of periodicity

1. Temperature and light cues interact to control many biological processes. Experiments give researchers the ability to manipulate these environmental cues independently and can be designed to robustly quantify their individual and interactive effects on any particular biological activity. Such experiments have produced important insights into the environmental controls on numerous biological processes in both plant and animal taxa across terrestrial and aquatic environments. Testing the interactive effects of multiple environmental cues, however, requires experimental treatments to be fully independent; any unmeasured experimental covariation among treatments can result in incorrect conclusions. 2. Using a database of controlled environment experiments on the spring phenology of woody plants as a case study, we highlight how a common experimental set-up, designed to parse the interactive effects of temperature and photoperiod on time to budburst, introduces a latent experimental covariation of these treatments by coupling photo- and thermoperiodicity. Using data simulations, algebraic corrections and a comparative analysis of published experiments, we demonstrate how this unmeasured experimental covariation biases statistical inference regarding the relative contribution of light and temperature cues to phenological variation. 3. We identify this experimental covariation in more than 40% of published phenology studies that manipulate photoperiod. Our analyses demonstrate that the coupling of thermo- and photoperiodicity results in the overestimation of the effect of photoperiod, the underestimation of forcing effects, and misleading conclusions about their interactions on phenology. This may, in part, explain why the significance of photoperiod cues for spring phenology is currently debated in the literature. 4. Accurate forecasting of how varying environmental conditions will impact the dynamics of biological events requires accurately quantifying cue responses. To this end, we present several options for statistical corrections and alternative experimental designs that can provide more robust estimates of the relative effects of temperature and photoperiod on phenology and many other biological processes controlled by temperature and light.

Automated summary

Temperature and light cues interact to control biological processes, but the experimental coupling of photoperiod and thermoperiod introduces unmeasured covariation, leading to biases in statistical inference regarding the effects of these cues.