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

Reviewer #1 (Public review):

Summary:

This manuscript addresses an important question: how do circadian clocks adjust to a complex rhythmic environment with multiple daily rhythms? The focus is on the temperature and light cycles (TC and LD) and their phase relationship. In nature, TC usually lags the LD cycle, but the phase delay can vary depending on seasonal and daily weather conditions. The authors present evidence that circadian behavior adjusts to different TC/LD phase relationships, that temperature-sensitive tim splicing patterns might underlie some of these responses, and that artificial selection for preferential evening or morning eclosion behavior impacts how flies respond to different LD/TC phase relationship

Strength:

Experiments are conducted on control strains and strains that have been selected in the laboratory for preferential morning or evening eclosion phenotypes. This study is thus quite unique as it allows us to probe whether this artificial selection impacted how animals respond to different environmental conditions, and thus gives hints on how evolution might shape circadian oscillators and their entrainment. The authors focused on circadian locomotor behavior and timeless (tim) splicing because warm and cold-specific transcripts have been described as playing an important role in determining temperature-dependent circadian behavior. Not surprisingly, the results are complex, but there are interesting observations. In particular, the "late" strain appears to be able to adjust more efficiently its evening peak in response to changes in the phase relationship between temperature and light cycles, but the morning peak seems less responsive in this strain. Differences in the circadian pattern of expression of different tim mRNA isoforms are found under specific LD/TC conditions.

Weaknesses:

These observations are interesting, but in the absence of specific genetic manipulations, it is difficult to establish a causative link between tim molecular phenotypes and behavior. The study is thus quite descriptive. It would be worth testing available tim splicing mutants, or mutants for regulators of tim splicing, to understand in more detail and more directly how tim splicing determines behavioral adaptation to different phase relationships between temperature and light cycles. Also, I wonder whether polymorphisms in or around tim splicing sites, or in tim splicing regulators, were selected in the early or late strains.

I also have a major methodological concern. The authors studied how the evening and morning phases are adjusted under different conditions and different strains. They divided the daily cycle into 12h morning and 12h evening periods, and calculated the phase of morning and evening activity using circular statistics. However, the non-circadian "startle" responses to light or temperature transitions should have a very important impact on phase calculation, and thus at least partially obscure actual circadian morning and evening peak phase changes. Moreover, the timing of the temperature-up startle drifts with the temperature cycles, and will even shift from the morning to the evening portion of the divided daily cycle. Its amplitude also varies as a function of the LD/TC phase relationship. Note that the startle responses and their changes under different conditions will also affect SSD quantifications.

For the circadian phase, these issues seem, for example, quite obvious for the morning peak in Figure 1. According to the phase quantification on panel D, there is essentially no change in the morning phase when the temperature cycle is shifted by 6 hours compared to the LD cycle, but the behavior trace on panel B clearly shows a phase advance of morning anticipation. Comparison between the graphs on panels C and D also indicates that there are methodological caveats, as they do not correlate well.

Because of the various masking effects, phase quantification under entrainment is a thorny problem in Drosophila. I would suggest testing other measurements of anticipatory behavior to complement or perhaps supersede the current behavior analysis. For example, the authors could employ the anticipatory index used in many previous studies, measure the onset of morning or evening activity, or, if more reliable, the time at which 50% of anticipatory activity is reached. Termination of activity could also be considered. Interestingly, it seems there are clear effects on evening activity termination in Figure 3. All these methods will be impacted by startle responses under specific LD/TC phase relationships, but their combination might prove informative.

Reviewer #2 (Public review):

Summary:

The authors aimed to dissect the plasticity of circadian outputs by combining evolutionary biology with chronobiology. By utilizing Drosophila strains selected for "Late" and "Early" adult emergence, they sought to investigate whether selection for developmental timing co-evolves with plasticity in daily locomotor activity. Specifically, they examined how these diverse lines respond to complex, desynchronized environmental cues (temperature and light cycles) and investigated the molecular role of the splicing factor Psi and timeless isoforms in mediating this plasticity.

Major strengths and weaknesses:

The primary strength of this work is the novel utilization of long-term selection lines to address fundamental questions about how organisms cope with complex environmental cues. The behavioral data are compelling, clearly demonstrating that "Late" and "Early" flies possess distinct capabilities to track temperature cycles when they are desynchronized from light cycles.

However, a significant weakness lies in the causal links proposed between the molecular findings and these behavioral phenotypes. The molecular insights (Figures 2, 4, 5, and 6) rely on mRNA extracted from whole heads. As head tissue is dominated by photoreceptor cells and glia rather than the specific pacemaker neurons (LNv, LNd) driving these behaviors, this approach introduces a confound. Differential splicing observed here may reflect the state of the compound eye rather than the central clock circuit, a distinction highlighted by recent studies (e.g., Ma et al., PNAS 2023).

Furthermore, while the authors report that Psi mRNA loses rhythmicity under out-of-sync conditions, this correlation does not definitively prove that Psi oscillation is required for the observed splicing patterns or behavioral plasticity. The amplitude of the reported Psi rhythm is also low (~1.5 fold) and variable, raising questions about its functional significance in the absence of manipulation experiments (such as constitutive expression) to test causality.

Appraisal of aims and conclusions:

The authors successfully demonstrate the co-evolution of emergence timing and activity plasticity, achieving their aim on the behavioral level. However, the conclusion that the specific molecular mechanism involves the loss of Psi rhythmicity driving timeless splicing changes is not yet fully supported by the data. The current evidence is correlative, and without spatial resolution (specific clock neurons) or causal manipulation, the mechanistic model remains speculative.

This study is likely to be of significant interest to the chronobiology and evolutionary biology communities as it highlights the "enhanced plasticity" of circadian clocks as an adaptive trait. The findings suggest that plasticity to phase lags - common in nature where temperature often lags light - may be a key evolutionary adaptation. Addressing the mechanistic gaps would significantly increase the utility of these findings for understanding the molecular basis of circadian plasticity.

Reviewer #3 (Public review):

Summary:

This study attempts to mimic in the laboratory changing seasonal phase relationships between light and temperature and determine their effects on Drosophila circadian locomotor behavior and on the underlying splicing patterns of a canonical clock gene, timeless. The results are then extended to strains that have been selected over many years for early or late circadian phase phenotypes.

Strengths:

A lot of work, and some results showing that the phasing of behavioral and molecular phenotypes is slightly altered in the predicted directions in the selected strains.

Weaknesses:

The experimental conditions are extremely artificial, with immediate light and temperature transitions compared to the gradual changes observed in nature. Studies in the wild have shown how the laboratory reveals artifacts that are not observed in nature. The behavioral and molecular effects are very small, and some of the graphs and second-order analyses of the main effects appear contradictory. Consequently, the Discussion is very speculative as it is based on such small laboratory effects