GENERAL COMMENTS

Lebrun et al. present a timely and well-designed study investigating the response of four habitat-building kelp species to a warming Arctic by means of a multi-factorial mesocosm experiment, in which they simulate predicted temperature increases along with decreased irradiance and decreased salinity under increased glacial melt scenarios over six weeks. They identify species-specific responses in physiology (growth, chlorophyll a, carbon:nitrogen ratio) and gene expression, indicating specific acclimation mechanisms and responses, which mainly respond to temperature as a key driver. In contrast to the majority of studies in this field, which often observe short-term stress responses, Lebrun et al. performed a 6-week experiment which allows for organismal acclimation to the novel environment, as evident in the lack of stress responses and stable growth rates across treatments. They therefore show that the kelps’ physiology is capable of acclimating to these interactive environmental effects, potentially allowing their range expansion into newly ice-free areas. The manuscript is well written and the conclusions are drawn based on a thorough analysis of the data. My main suggestions are to include a short description of the significance of the fixed factors in the results section before reporting the pairwise comparisons, and to consider the effect of temperature on enzyme reaction rates in the discussion. Apart from this, I only have minor questions and suggestions. This manuscript will be a valuable contribution to the field and I’m looking forward to seeing the paper published!

SPECIFIC COMMENTS

ABSTRACT

The abstract provides a concise overview over the premise, study design, key results and implications. Only the growth rates are not mentioned in the abstract. I would suggest to mention that growth remained stable for each species across treatments, which in my eyes is one of the most important results showing that Arctic warming may not be detrimental per se.

INTRODUCTION

The introduction is short but concise and presents Arctic kelp forests and the changing environment they are facing. A more general audience might appreciate a short taxonomic classification of kelps, e.g. as large brown algae (Phaeophyceae, Laminariales). Do all four tested species occur together in mixed assemblies or are they restricted to different depths in situ?

METHODS

The methods are described in detail and allow replication, provided that the study by Miller et al., which contains the detailed setup of the mesocosm experiment, will be published before this manuscript. Before the start of the experiment, were the holding tanks maintained at in situ conditions? Why were S. latissima and H. nigripes chosen for the transcriptomic analysis?

RESULTS

The results are presented in a clear and focused manner with good statistical support. However, throughout the reports of the physiological responses, the authors only report pairwise comparisons. I would prefer the paragraphs to begin with an overview of the fixed factor significance (Chi-square tests) to assess significant differences between species and treatments in general, and their interaction, i.e. whether species respond differently to the different treatments, before moving on to pairwise comparisons. The Figures are clear and intuitive, except for the display of significant differences in Figure 3 (A. esculenta), which could be improved. Figure 2C seems to be missing the light blue control treatment.

DISCUSSION

The authors provide a detailed placement of their data within the literature, especially macroalgae physiology, and relate the different responses between species to their resilience towards Arctic warming. A key point to be added to the discussion is the relationship of enzyme activity and temperature, especially with respect to the RNAseq results (down-regulation of stress responses and fundamental cellular machinery). For instance, the combination of cold temperature and high irradiance can induce photoinhibition due to slower reaction rates of crucial enzymes such as RuBisCO, triggering stress responses. In general, to maintain cellular functions, slower reaction rates at cold temperature can be compensated by higher protein expression. The authors mention that future warming may reduce stress responses (ln. 361), to which I would like to add that the tested high temperatures are close to the physiological optima described for the species. Regarding the reduced C:N ratio in S. latissima it may make sense to relate this to the increased growth rate in this species.

ADDITIONAL

At the moment there is no data availability statement. I would advise the authors to archive at least the RNAseq data in a public repository.

MINOR COMMENTS

Ln. 29 – gene expression patterns

Ln. 94 – Here it is described how temperature and salinity were adjusted, so I think it makes sense to add that irradiance was adjusted using filters here, too.

Ln. 109 – with a PAR sensor (LI-COR xyz)

Ln. 111 – the difference between the inner and outer

Ln. 120 – consider adding that the meristem is located above the stipe-blade transition zone for a more general audience

Ln. 125 – only on tfinal?

Ln. 129 – Were the samples kept frozen during extraction?

Ln. 132 – What is the Fa fluorescence? I would prefer if Lorenzen’s formula was reproduced in the text to put F0 and Fa into context.

Ln. 144 – Suggestion: 2 cm above the stipe-blade transition. Base of the stipe to me sounds like it is basally located near the holdfast.

Ln. 149 – with dist0: […] to the hole

Ln. 155 – it might be useful to mention that the protocol combines a CTAB extraction followed with a commercial Qiagen kit.

Ln. 167 – using rnaSPAdes

Ln. 191 – Table C1

Ln. 217 – Consider replacing “different” by “higher than”

Ln. 235 – According to the Chi-square test (Table F1), they are strongly affected. This is likely due to the much faster growth of S. latissima in general, but this should at least be acknowledged shortly.

Ln. 238 – It might be worth mentioning that the growth of S. latissima is higher by an order of magnitude

Ln. 245 – Principal component analysis of global gene expression revealed …

Ln. 248 – classified = functionally annotated?

Ln. 258 – 458 down-regulated genes […] classified down-regulated

Ln. 268 – no negative impacts

Ln. 283 – and lower irradiance treatment

Ln. 290 – Short-term acclimation may not be the right term, it may rather be an existent adaptation to effectively use lower irradiance? Niedzwiedz & Bischof (2023; doi.org/10.1002/lno.12312) show that Arctic A. esculenta has a lower compensation irradiance than S. latissima at 3-7°C.

Ln. 295 – Diehl and Bischof (2021)

Ln. 296 – the chl a content of S. latissima

Ln. 305 – Might the nitrogen limitation be related to the 10x higher growth in S. latissima?

Ln. 314 – higher in the T2 treatment (1.68 …)

Ln. 332 – in the control indicating

Ln. 348 – potentially because increased enzyme reaction rates compensate for the reduced expression (see general comment above)

Ln. 384 – and biotic interactions, see the reduced competition of sporophyte recruitment against A. esculenta in Zacher et al. (2019)

Ln. 393 – acclimation

Ln. 395 – So growth is plastic only in specific seasons? Or does this sentence refer to general seasonal growth patterns?

Ln. 405 – optimum

Ln. 406 – acclimation

Ln. 426 – Is it really more than one co-author on the editorial board?

Ln. 703 / Figure 7 – what is the meaning of the shading behind the bars for T2?

General comments:

This is a well-written and well-structured article that makes an important contribution to the growing field of study around the potential impacts of climate change on macroalgae in the Arctic region. The authors investigated the physiological and transcriptomic responses of four kelp species to the combined effects of increased temperature, reduced salinity, and reduced light availability in a six-week mesocosm experiment. The authors describe their methodology clearly. They provide evidence that the species studied here were able to acclimatize effectively to the experimental treatments, and suggest that these species will likely be resilient to future environmental changes in the Arctic. I have some minor questions around the study’s methods (chiefly the low number of replicates, and the fact that different species were analyzed for the physiological and transcriptomic portions of the paper), and I think it would be useful to provide a more detailed list of differentially expressed genes. Overall, this is a strong article that makes a novel and valuable contribution to science, and I would be very happy to see it published.

The abstract is generally well-written, but a bit too long. Perhaps the experimental treatments, results and discussion could be described more briefly?

The introduction is concise, and does a very effective job of explaining how environmental conditions in the Arctic are predicted to change, and why it is valuable to investigate how these changes might impact kelp species. The knowledge gap that this study addresses is identified clearly. A little more information on the study species, and any previous findings about their environmental tolerance, would have been useful.

The methods are logical, and explained clearly enough to be replicable. Can you explain why physiological measurements were not taken for Hedophyllum nigripes, and why gene expression was not investigated in Laminaria digitata and Alaria esculenta?

The results section is concise and clear.

The discussion section places the results well within the wider context of research on stress responses in kelp. Some reasonable hypotheses are provided regarding the cellular and physiological mechanisms that could underlie the observed changes in chlorophyll a content, C:N ratio, and gene expression. There are some good suggestions for other physiological responses that could be measured in follow-up studies to validate these hypotheses. The low number of independent replicates (i.e. mesocosms) used, as well as the low sample size for L. digitata, make it harder to be confident about the significance of some trends. This is an understandable choice, given the high spatial and economic costs of mesocosm experiments, but should be made clear in the discussion.

Specific comments:

Abstract:

Line 19-22 - The rationale behind the experimental treatments is explained very clearly in the Introduction and Methods sections, so perhaps it isn’t necessary to go into so much detail here.

Line 26-27 - Nitrate concentration was not one of the factors deliberately manipulated in this experiment, so be more specific about which experimental treatments were linked to this change in C:N ratio.

Line 29-30 - The statement about gene expression is not very specific. What patterns of gene expression were found at different temperatures, and what “ability” does this reflect? Consider giving more details, or leaving this out of the abstract.

Introduction:

Line 54-55: Be more specific about how temperature and salinity impact kelp physiology. Is there anything known about the tolerance ranges of these study species, and was this taken into account when developing hypotheses?

Methods:

Line 108 - Units of salinity?

Line 120 - I assume samples were taken using a scalpel or similar. What steps were taken to prevent cross-contamination between samples?

Line 136 - Mass or molar C:N ratio?

I believe it is best practice to use quotation marks when citing R packages, e.g. “EnvStats” (Line 195, 198, 201)

Results:

Line 211-212 - Units of salinity?

Line 240 - “significant differences in growth over time were only found in the T3 treatment” might be clearer.

It would be worthwhile to include a more detailed list of DEGs (perhaps as supplementary material).

Discussion:

Line 268 - “no negative impacts”

Line 313 - “activity of nitrate reductase”

Line 350 - “involved in reducing”

Line 368 - “increased chlorophyll a content”

Line 372 - Citation would be useful.

Line 410 - “barren” would be more accurate than “bare”

Figures:

The figures are generally easy to interpret. Removing background gridlines would make Figures 2-5 cleaner, and reducing the width of the bar outlines in Figure 7 would make it easier to interpret.

Figure 2 - Providing this data is helpful and transparent. There are some large fluctuations in salinity and PAR - what could have caused these? They are mostly brief enough not to affect the overall results. However, PAR increased steadily in the light-limited treatments during the latter three weeks, and did not appear to be significantly lower than T3 by the end of the experiment, which could be of concern.