Complex environmental cycles reveal evolution of circadian activity waveform and thermosensitive timeless splicing

Circadian clocks synchronise physiological and behavioural rhythms to environmental cycles such as light and temperature. In nature, temperature cycles lag light cycles, with the extent of lag varying seasonally. Thus, the extent of this delay is an essential aspect of seasonal cues. Yet, the combined effects of light and temperature cycles on circadian systems remain poorly understood. Using a series of environmental cycles with varying degrees of lag between temperature and light, we examined the response of circadian activity to complex environments and show that morning and evening activity exhibit differential temperature sensitivity in Drosophila melanogaster. We find that the expression of temperature-induced timeless splice variants is modulated by light cycles as well as the degree of lag between temperature and light. We also reveal rhythmic expression of the timeless splicing regulator Psi. Furthermore, we leveraged a laboratory-selection approach to reveal that the differential temperature sensitivity of morning and evening activity evolves upon selection. We observed selection-dependent differences in temporal expression of timeless splice variants thus linking circadian gene splicing to behavioural plasticity. Thus, our integrated behavioural, molecular, and evolutionary approach advances the understanding of how circadian systems integrate seasonal cues.