Developmental constraints mediate the summer solstice reversal of climate effects on European beech bud set

Reviewer #1 (Public review):

Summary:

This study provided key experimental evidence for the "Solstice-as-Phenology-Switch Hypothesis" through two temperature manipulation experiments.

Strengths:

The research is data-rich, particularly in exploring the effects of pre- and post-solstice cooling, as well as daytime versus nighttime cooling, on bud set timing, showcasing significant innovation. The article is well-written, logically clear, and is likely to attract a wide readership.

Weaknesses:

However, there are several issues that need to be addressed.

(1) In Experiment 1, significant differences were observed in the impact of cooling in July versus August. July cooling induced a delay in bud set dates that was 3.5 times greater in late-leafing trees compared to early-leafing ones, while August cooling induced comparable advances in bud set timing in both early- and late-leafing trees. The study did not explain why the timing (July vs. August) resulted in different mechanisms. Can a link be established between phenology and photosynthetic product accumulation? Additionally, can the study differentiate between the direct warming effect and the developmental effect, and quantify their relative contributions?

(2) The two experimental setups differed in photoperiod: one used a 13-hour photoperiod at approximately 4,300 lux, while the other used an ambient day length of 16 hours with a light intensity of around 6,900 lux. What criteria were used to select these conditions, and do they accurately represent real-world scenarios? Furthermore, as shown in Figure S1, significant differences in soil moisture content existed between treatments - could this have influenced the conclusions?

(3) The authors investigated how changes in air temperature around the summer solstice affected primary growth cessation, but the summer solstice also marks an important transition in photoperiod. How can the influence of photoperiod be distinguished from the temperature effect in this context?

(4) The study utilized potted trees in a controlled environment, which limits the generalization of the results to natural forests. Wild trees are subject to additional variables, such as competition and precipitation. Moreover, climate differences between years (2022 vs. 2023) were not controlled. As such, the conclusions may be overgeneralized to "all temperate tree species", as the experiment only involved potted European beech seedlings. The discussion would benefit from addressing species-specific differences.

Reviewer #2 (Public review):

In 'Developmental constraints mediate the summer solstice reversal of climate effects on European beech bud set', Rebindaine and co-authors report on two experiments on Fagus sylvatica where they manipulated temperatures of saplings between day and night and at different times of year. I enjoyed reading this paper and found it well written. I think the experiments are interesting, but I found the exact methods somewhat extreme compared to how the authors present them. Further, given that much of the experiment happened outside, I am not sure how much we can generalize from one year for each experiment, especially when conducted on one population of one species. I next expand briefly on these concerns and a few others.

Concerns:

(1) As I read the Results, I was surprised the authors did not give more information on the methods here. For example, they refer to the 'effect of July cooling' but never say what the cooling was. Once I read the methods, I feared they were burying this as the methods feel quite extreme given the framing of the paper. The paper is framed as explaining observational results of natural systems, but the treatments are not natural for any system in Europe that I have worked in. For example, a low of 2 {degree sign}C at night and 7 {degree sign}C during the day through the end of May and then 7/13 {degree sign}C in July is extreme. I think these methods need to be clearly laid out for the reader so they can judge what to make of the experiment before they see the results.

(2) I also think the control is confounded with the growth chamber experience in Experiment 1. That is, the control plants never experience any time in a chamber, but all the treatments include significant time in a chamber. The authors mention how detrimental chamber time can be to saplings (indeed, they mention an aphid problem in experiment 2), so I think they need to be more upfront about this. The study is still very valuable, but again, we may need to be more cautious in how much we infer from the results.

(3) I suggest the authors add a figure to explain their experiments, as they are very hard to follow. Perhaps this could be added to Figure 1?

(4) Given how much the authors extrapolate to carbon and forests, I would have liked to see some metrics related to carbon assimilation, versus just information on timing.

(5) Fagus sylvatica is an extremely important tree to European forests, but it also has outlier responses to photoperiod and other cues (and leafs out very late), so using just this species to then state 'our results likely are generalisable across temperate tree species' seems questionable at best.

(6) Another concern relates to measuring the end of season (EOS). It is well known that different parts of plants shut down at different times, and each metric of end of season - budset, end of radial expansion, leaf coloring, etc - relates to different things. Thus, I was surprised that the authors ignore all this complexity and seem to equate leaf coloring with budset (which can happen MONTHS before leaf coloring often) and with other metrics. The paper needs a much better connection to the physiology of end of season and a better explanation for the focus on budset. Relatedly, I was surprised that the authors cite almost none of the literature on budset, which generally suggests it is heavily controlled by photoperiod and population-level differences in photoperiod cues, meaning results may be different with a different population of plants.

(7) I didn't fully see how the authors' results support the Solstice as Switch hypothesis, since what timing mattered seemed to depend on the timing of treatment and was not clearly related to the solstice. Could it be that these results suggest the Solstice as Switch hypothesis is actually not well supported (e.g., line 135) and instead suggest that the pattern of climate in the summer months affects end-of-season timing?

Author Response:

We would like to thank the reviewers and editors for your consideration of our manuscript, your kind comments about the value of our study, and for providing constructive feedback. We intend to submit a revised version of the manuscript and address the concerns and recommendations. This will include improvements to the statistical analyses, text content, and text format.

Specifically, we will:

1. Revise the text to better explain the experimental methods, interpretation of results and how our findings are situated in the literature. Although we still believe that there is sufficient evidence to suggest that temperate tree species other than Fagus sylvatica may show similar patterns, we understand the reviewers concerns regarding these statements and will revise them.

2. Add a supplemetal analysis of leaf chlorophyll content data to use leaf discolouration as an alternative marker of the end of the growing season. On this we would like to make two important points. Firstly, we agree with the reviewers that bud set often occurs before leaf discolouration. In experiment 1, bud set occurred on average on day-of-year (DOY) 262, onset of leaf senescence (last day when leaf chlorophyll content fell below 90% of its measured maximum) occurred on average at the same time – DOY 261, and mid-senescence (50% leaf discolouration) occurred on DOY 320. We do not agree that this excludes the combined discussion of bud set and leaf senescence timing. Whilst environmental drivers can affect parts of plants differently, often responses from different end-of-season indicators (e.g. bud set and leaf discolouration) are similar, even if only directionally. Secondly, shifts in bud set timing will remain the key focus of the manuscript as we believe it has greater physiological relevence to plant development, whereas leaf discolouration may simply follow bud set as a symptom of the completion of growth (reduced sink activity).

3. Address points raised about potential additional drivers of our observed phenological shifts. For example, photoperiod effects and the Sosltice-as-Phenology-Switch hypothesis are not mutually exclusive, the annual progression of photoperiod is fundamental to how we suggest the switch is regulated (please see L66-68 in the original manuscript). The reviewers also comment on the significant differences in soil water content between the treatment groups in Fig. S1. However, all pots were watered sufficiently to avoid water deficit and all efforts were made to minimise differences in water availabiltiy. A provisional analysis shows only one treatment pair (6 - Late_July_Extreme vs. 7 - Early_August_Moderate) had significantly different soil water content, a pair whose differences are not discussed.