Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: MECHANISMS AND POTENTIAL IMMUNE TRADEOFFS OF ACCELERATED CORAL GROWTH INDUCED BY MICROFRAGMENTATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: All appropriate data and scripts are reported in the text, available online, and any sequence projects submitted to the proper databases.

Figures.
S2. It is unclear whether these are all the fragments produced from one colony or a subset without a caption. Also, not sure what the arrows represent.

S3&S4. Perhaps add Ofav into the title so readers know which species the cluster analysis is for.

S5 does not have ‘A’ or ‘B’ as panel titles. Would also prefer to see a genet pvalue somewhere on the figure (and in S6), but would be redundant if in both panels, so maybe report in the caption or just in panel A?

Text.
Lines 54 & 65. Needs space after the semicolon in citations.

Lines 75-77. Unclear what success means in this context, survival, increased growth after outplanting?

Lines 78-83. This is the crux of the study and could be expanded upon with similar studies investigating growth rates and potential tradeoffs. See Edmunds 2017 (Ecology) and Denis et al. 2011 (MEPS) and 2013 (PLoS); although these investigate growth tradeoffs correlated with environmental stress, there are relevant implications of fast growth/wound repair under stressors that can be applied here.

Line 120-121. “the same protocols on 12 Mar 2016” reads like you used the same protocol from that date, not the start date 10 Nov.

Line 154. Peroxidase activity is given the acronym PO but is defined/used as POX in the introduction and in the discussion.

Line 255. As in a sufficient amount? What does “high-quality tissue” mean?
Experimental design: Line 108. The “successively smaller fragments” make it difficult to glean relative fragment size. Did you have relative size bins? What is the sample size per fragment size per genet per species? Also, should refer to the supplemental figure here.

Line 126. Although the methods and analyses cannot be re-done, should take into consideration in the discussion that you airbrushed from the entire fragment, but gene expression was from the growing edge of fragments. Do you think you’d find different immune activity if you only airbrushed from the growing edge as well? Larger fragments would, in theory, have a smaller edge area so potentially reduced enzymatic activity (or dampened activity due to the presence of more ‘center’ tissue) driven by the rest of tissue instead of just the growing edge?

Line 200. In the same vein as above, could the association between growth and area of growing edge relative to total surface area (instead of or in addition to) be an additional factor influencing growth rate?
Validity of the findings: Line 231. Again, hard to understand the relevance of findings without knowing sample size, even if relative.

Line 242-243. A great demonstration of how growth, especially in smaller sizes, can be so variable.

Line 259. The sentence is slightly misleading when reviewing the figure. Although it is the only enzyme activity that was not significantly different from coral size, stating “declines in activity” is counterintuitive to the figure and the findings. Larger fragments exhibited higher activities which are reflected in the panels, so why would activity decline with coral size? Or did you mean to say growth rate, if smaller fragments grow more, they have lower enzymatic activity. Please clarify.

Line 340. Stated that “target of ~1cm2 fragments” but readers did not know until now that you had fragments that small based on experimental design.

Paragraph 351-356. Weak in discussing the tradeoffs, as enzymatic activity and any immune-related transcripts are not discussed here. Tie in these results for comparison and support of statement or combine with the following paragraph to provide evidence-based research for both positive and negative associations.

Lines 374-376. Is any of this related to the actual size of outplants, or attributed to nursery conditions (husbandry effects, like ‘healthier’ and ‘yummier’ corals), reef site (more corallivores than others), or an effect of time since outplant? How do these studies relate to your findings?

Line 386. Do they still grow as ‘rapidly’ (or more so) on the reef vs. nursery? This has implications on whether to grow out in land-based versus field nursery if growth rates are indeed more rapid in one setting versus another.

Paragraph 438-445. Previous studies are documenting wild coral symbiont types, and it is likely that these corals could harbor different symbiont types on land using a different water source than reef water. The argument for post-stress shuffling isn’t clear unless the shift came about as a stress response to microfragmenting (unlikely). What is more interesting is Durusdinium typically leads to lower growth rates (Cunning et al. 2015, Coral Reefs), and you didn’t see significant differences between the two species as M. cavernosa harbored both Cladocopium and Durusdinium.
Additional comments: This study is a great foundation for what we know and do not know about the potential tradeoffs associated with the microfragmentation process. Able to generate sufficiently sized outplants much faster than using larger fragments, restoration practitioners can upscale to potentially replace the biomass that’s being lost. The reproducibility of results regarding smaller sizes grow faster is important to the restoration field, but the group also introduced applicable future experiments to further understand the potential repercussions of a corals energy budget. For example, it would be worthwhile to track immune activity and gene expression along the growing edge alone (not the entire fragment) at multiple time points, as it is likely the wound healing response occurs within the first month of lesion/fragmenting (see Denis et al. 2013, PLoS) and it could be possible that the immune activity was dampened by looking at the whole fragment instead of just the growing edge. Moreover, coupling the experiment with a disease exposure component could elucidate the potential immunity tradeoff further and provide critical implications for direct outplanting post-fragmentation.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: MECHANISMS AND POTENTIAL IMMUNE TRADEOFFS OF ACCELERATED CORAL GROWTH INDUCED BY MICROFRAGMENTATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overview:
Schlecker et al. conducted a manipulative study that aimed to quantify the growth rate, immune protein, and gene expression of Orbicella faveolata and Montastraea cavernosa four months after being fragmented into varying sizes. The authors conclude that smaller fragments grow faster, but there is a negative association between fragment size and immune competence suggesting that there may be tradeoffs between accelerated growth and immunity. Overall, the paper is well written and poses timely questions, which are both highly relevant and novel. I thoroughly hope that this group continues to ask and answer important questions, such as those posed within this paper, in collaboration with groups conducting boots on the ground coral restoration.
One challenge I had with the paper though was the presentation and tone suggests that coral restoration practitioners haven’t considered potential tradeoffs or the threats like disease on corals created through microfragmentation. However, this practice has been going on for almost a decade and although not widely published, the overall survival rates are quite high even when outplanting within the endemic areas of SCTLD (see references listed below). Placing the paper within the current precautionary tone ignores about a decade’s worth of that information. I suggest that this paper should not be focused as a precautionary tail to beware when outplanting microfragments; perhaps rephrase the message to include restoration practitioners’ common knowledge that  “there may be a reduced immune competency associated with microfragmentation, yet these corals are still surviving after outplanting. How/Why is that happening?!”.
Also, the important implications of the present study could help guide further care when corals are in nursery settings or guide when corals should be outplanted to increase survival. For example, Orbicella faveolata microfrags are typically grown for 8 months prior to outplanting and M. cavernosa is grown for 12 months or more. What does the immune status look like at the time of outplanting? Additionally, corals are not outplanted until they completely cover the plug. It is difficult to tell, but only a few of the corals in the photograph from March 2016 would actually have been ‘outplantable sized’. Does this reduced immune competence found in the study further support the long grow out period current practitioners use?
One caveat I am also struggling with…how do we know that the differences in immune related activity are associated with fragmentation process and not simply related to the size of the corals? For example small sized corals (whether fragmented or not) may have reduced immune capacity. We don’t have a control in this study so perhaps there are comparative measurements from other studies? What do we know about the measurements in the present study and those taken from ‘wild’ corals of comparable sizes or just any ‘wild’ corals in general? A comparison to field collected samples would be really interesting component to add to the discussion.

Additional References to Consider Including or reading for context:
White paper to consider integrating (and also cite instead of E. Muller personal observation Line 388):
https://floridadep.gov/rcp/coral/documents/can-sctld-susceptible-species-be-outplanted-florida%E2%80%99s-coral-reef-acceptable

Another resource on outplanting survival of microfragments (incorporating predation effects):
Smith, Kylie M., Devon M. Pharo, Colin P. Shea, Brian A. Reckenbeil, Kerry E. Maxwell, and C. Sharp. "Recovery from finfish predation on newly outplanted boulder coral colonies on three reefs in the Florida Keys." Bulletin of Marine Science 97, no. 2 (2021): 337-350.

We also showed that microfragmented and fused corals can spawn a few years after outplanting: https://www.the-scientist.com/features/restored-corals-spawn-hope-for-reefs-worldwide-68368
I think the references listed above (although I realize not all peer reviewed) should be included if possible and help guide a more balance discussion.
Experimental design: Abstract:
I think the gene expression results are specific to only one species, Orbicella faveolata, please make that clear in lines 39 – 42, or ignore me if I am incorrect.
Lines 43 – 45: I think this sentence could be broader. SCTLD is just the latest disease threat to the Florida Reef Tract and all of the other diseases certainly haven’t gone away. Disease has been a major issue restoration practitioners have had to consider since the beginning of restoration within this region.
Lines 47 – 48: revisit the concluding sentence after reconsidering broad presentation of the paper.

Introduction:
Line 63: I’m not sure Dobbelaere et al. 2020 is the best reference here since it focuses on FL hydrodynamics and particle modeling, perhaps include Meilling et al from USVI or Sharp et al. 2020, Williams et al. 2021 from FL
Line 66: several other studies showed antibiotics work to treat SCTLD (Neely et al. 2020 PeerJ, Aeby et al. 2019)
General: Recommend including a paragraph on gene expression association with growth/immune competency in corals. There is a section on immune-related proteins but not gene expression.
Line 75 – 76: seems to be a word missing in this sentence; also, what does ‘success’ mean here?
Line 96 – 98: I would re-write this to include the use of gene expression as a tool for characterizing both growth and immune competency since that is what you actually use for conclusions.
Methods:
Line 104: provide more details of the coral nursery…this was likely the NOAA Rescue Nursery, where corals were removed from construction projects (seawalls etc – extremely shallow areas) and preserved in the nursery for research purposes instead of being lost in the demolition process.
Line 108: please explain how you determined the size the fragment the colonies. Looking at the size distributions there were many more around the 1cm mark compared with the larger fragments and Figure 1A appears to have two ‘size classes’ rather than an even distribution.
Line 113: How was the water treated and filtered? I would re-word this sentence to be more informative. High light and low turbidity are subjective terms.
Line 116: usually it’s corrugated clear PVC, not metal. If you used metal, why would additional shade cloth be needed – this part is confusing.
Line 117: when was PAR measured? How often?
Line 119: I imagine there was physical mitigation needed to remove algae from the plugs, otherwise, your discussion of the high amount of attention needed to grow microfragments (lines 347-348) doesn’t make sense (if snails and siphoning is all that was needed for 4 months).
General: what was the temperature and pH of the water?
General: remain consistent using past tense (examples: line 217 remain should be remained, line 232 includes should be included, line 283 and 284 ‘is’ should be ‘was’, 298 and 299 ‘have’ should be ‘had’ etc.)
Validity of the findings: Results:
Line 231: separate the number of fragments by species (x ofav and x mcav)
Line 236: ‘larger fragments grew less’, since you are basically doing linear models, I would think it would be more appropriate to say that there was a positive or negative association rather than saying ‘larger fragments grew less than smaller fragments’, or you could say that size was a predictive factor associated with growth. As it states now, I can’t tell what ‘large’ vs ‘small’ is since size is treated as a continuous variable.
Line 255: how many from each species?
Line 257: see same comment for line 236
Line 260: change ‘across’ to ‘between’ (between is appropriate when comparing two things, here two species)
Line 270: I recommend adding/altering the text ‘which was not statistically significant’, nor did we observe…’ The data suggesting there may be a negative trend in growth for Mcav isn’t compelling here so I am not sure alluding to it twice in one sentence is necessary or appropriate.
Line 280: differentially expressed when comparing what?
Line 315 – 318: this sentence keeps tripping me up. I suggest rewording it for easier interpretation.

Discussion:
Line 345: what does ‘processing time’ mean?
Line 347 – 349: there is a lot more to consider when thinking about many small fragments vs fewer larger fragments than just algae mitigation. It is more difficult to maintain the health of large corals within controlled land-based systems (need more space, water flow, electricity etc), there are more issues with pest interactions when there is a lot of skeleton within the colony and once an issue arises the entire colony could be at risk. Also, where are you going to get the biomass to have these large corals? Having many small corals allows for redundancy of genetics within the broodstock, limits the amount of initial biomass necessary, and is easier to maintain for a healthy outplant. By outplanting clusters of small fragments you create the large coral on the reef within a few months to a year, rather than within a land-based system (which costs a lot more to maintain than an outplant on a reef).
Line 370 – 373: Stating that restoration can do more harm than good (and the references listed in this statement that focus on grounding sites, destroyed reefs from quarry excavation, and moving fragments from one reef to another) are examples of transplantation projects that are substantially different than what most restoration organizations do today – particularly the ones on Florida using microfragmentation for restoration. For example, all of Mote’s broodstock was originally acquired through the NOAA Rescue Nursery (corals that were removed from seawalls ahead of construction projects that would have killed them otherwise) or through corals of opportunity, which is defined as a coral detached from the reef that is in a location where it would likely die if left alone (i.e., in sand). New stock is acquired through assisted sexual reproduction events that would not have been successful without human intervention. I think perpetuating falsehoods about restoration causing more harm than good leads to continued misunderstandings of the practices. I recommend lines 370 to 374 get deleted or are rewritten to accurately reflect best practices of today.
Line 408 – 409: here you should also mention that quantifying the microfragments when they are the size of outplants (3cm diameter or more) is another area of attention in future studies.
Line 411: two genes were differentially expressed…in what way? Remind reader if it was up or down in respect to growth in Mcav. Same in line 416.
Line 415: I suggest making this sentence more tempered “The down-regulation of the GBP gene in M. cavernosa fragments exhibiting higher growth suggests further investigations into the potential for immune tradeoffs among faster growing corals is warranted.”
Line 431: change ‘urges’ to ‘suggests’
Line 441 – 445: actually we know that the majority of our Ofav (like 99%) are dominated by Durusdinium at Mote. This could be because our original broodstock were collected from extreme environments (seawalls) or because our land-based system promotes it.

Conclusion:
Consider toning down lines 461 – 463 based on my comments above.
Additional comments: No Comment


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: MECHANISMS AND POTENTIAL IMMUNE TRADEOFFS OF ACCELERATED CORAL GROWTH INDUCED BY MICROFRAGMENTATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: I appreciate the work the authors did to make the points of this manuscript clearer. The figures are improved and the supplemental figures address many of my issues and give the reader more data to consider. I support publication of this new version.
Experimental design: No comment
Validity of the findings: This paper will be a great resource to reef managers who conduct fragmentation and restoration as well as basic scientists interested in immune trade-offs and gene expression patterns related to growth. very nice!
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: MECHANISMS AND POTENTIAL IMMUNE TRADEOFFS OF ACCELERATED CORAL GROWTH INDUCED BY MICROFRAGMENTATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors did a great job addressing all of my initial comments, suggestions, and concerns. My only non-editorial suggestion is that I think identifying if and when this tradeoff between growth and immune parameters subside could be very useful to guide WHEN to move the corals from the 'safety' of a nursery to an outplant site. This could be added as a sentence or two within one of the relevant Discussion paragraphs. Otherwise, I have only editorial comments to suggest at this point. Thanks to them for the significant effort on this paper.

Abstract:
Suggest changing Line 9 to the following: Microfragmentation is the act of cutting corals into small pieces (~1 cm2) to accelerate the growth rates of corals when compared to maintaining larger-sized fragments.

Line 13:  change to "Here we compared..."
Line 14: add comma after 'corals'
Lines 19-20: suggest changing to "Innate immunity enzyme activity assays and gene expression results suggest a potential tradeoff after microfragmentation between growth rate and immune investment.
Line 24: suggest changing to something like "...microfragmentation recovery period that may affect growout survival and disease transmission after outplanting".

Intro
I realize the added text associated with the history of disease influencing the current state of the Florida reef tract was in response to my initial review, but the text sort of over simplifies the situation now. The state of FL reefs now, and much of the Caribbean (as noted) is definitely in part due to disease, but also major bleaching events and hurricanes. I think that integrating a balance into these statements is necessary to be accurate (lines 34 - 37 primarily).

Lines 43 - 46: adding antibiotics to sick coral is not a form of 'restoration', it is generally considered a form of 'intervention' to prevent loss. Separate the intervention from the restoration text within this sentence to clarify and better wrap up this paragraph.

Lines 79-82: this sentence structure is a bit awkward, maybe rethink it

Line 95: change to "The present study..."

Methods:
Line 223: change to "We used..."

Results:
LIne 263: change to "...expansion and as change in buoyant weight, which included..."

Lines 264 - 266: are these two averages and SD related to the 'small' and 'big' frags for each species? Clarify.

Discussion
Line 459: I think you could just use Koch et al. 2021 here too
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: USING CLUSTER AND REST REDISTRIBUTION SET STRUCTURES AS ALTERNATIVES TO RESISTANCE TRAINING PRESCRIPTION METHOD BASED ON VELOCITY LOSS THRESHOLDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The introduction is well organized, but we note the absence of some studies that have already analyzed the possibility of monitoring velocity loss in the set when instruments to measure execution velocity are not available. For example, Gonzalez-Badillo et al. (2017) Velocity Loss as a Variable for Monitoring Resistance Exercise. Int J Sports Med 38: 217-225; Rodriguez-Rosell et al. (2020) Relationship Between Velocity Loss and Repetitions in Reserve in the Bench Press and Back Squat Exercises. J Strength Cond Res 34(9): 2537-2547.
Figures and tables are acceptable. They provide sufficient information according to the results obtained in the study.
Experimental design: According to the authors, the purpose of this study was to examine the effects of CS, RR, and TS on neuromuscular performance and to determine the viability of using CS and RR as alternatives to prescribing VL20% thresholds across multiple BP and SQ sets. However, the study does not appear to have examined "the effect on neuromuscular performance," but only the viability of using CS and RR to control VL20%.

A weak aspect of the design is the determination of the 10RM load. On the one hand, the same repetitions are not performed with the same percentage in both studied exercises (see the articles cited previously). Specifically, it is unlikely that the average number of maximum repetitions at 80% will exceed six repetitions in the squat. Naturally, these repetitions refer to a squat (full squat), not a squat to parallel. In this sense, determining the thigh parallel without intermediate stop may be unreliable. What control has been performed to accept that all repetitions were done with a similar flexion? Moreover, with this type of test, any athlete is being forced to perform one or more tests, up to three, according to the study's authors, of 10RM. The authors should indicate what type of athlete/sport and at what ages it would be appropriate to apply this type of test. With the data from the two uncited articles, it is possible to know the velocity loss in the set with any load without performing any XRM test.

Not all subjects can perform the same repetitions with the same percentage (see articles not cited in the manuscript). This circumstance means that not all subjects will have trained with the same relative intensity if all subjects have reached the 10RM load. Moreover, even if the percentage was the same for all participants, not all will lose the same velocity when doing the same repetitions (see the previous articles), so the fatigue for the same repetitions will be different. In addition, velocity losses for the same repetitions are different depending on the relative intensity at which the training is performed. Therefore, if applied in a workout, the identical rest between repetitions and sets will produce a different effect.

The authors state that “the bar was pressed concentrically immediately after touching the chest without a pause to ensure the most reliable performance.” We assume that the authors are aware that some other study concludes that with the chest stop, the results are more reliable. They should cite this study and try to explain their decision.
Validity of the findings: Lines 277-84. The conclusions that “the study findings suggest that CS and RR are effective at reducing the overall fatigue-included decrease in velocity compared to TS” does not seem relevant. All the “main findings” of the study are logical and known without the study's need. It is common knowledge that the longer the recovery time between repetitions, the less fatigue and velocity loss in the set. If the recovery times between repetitions had been somewhat longer, the velocity loss would have been less. That is, the CS and RR would have shown "greater validity."

The study provides little helpful information, as it sticks to a single load. According to the design, it could not be applied to any other load. Nevertheless, as we have indicated previously, with the data from the two uncited articles, it is possible to know the velocity loss in the set with any load.

The study is justified because losing at most 20% of the velocity in the set may have a better neuromuscular effect than losing more velocity. However, an essential question arises to be solved that may render the study useless. The effect of 20% velocity losses without rest between repetitions may differ from 20% loss with intermediate rests between repetitions or groups of sets. In the studies cited in this manuscript, which serve as the basis for its justification, there was no pause between repetitions.

Lines 327-29 “CS and RR can be used not only do decrease the overall amount of neuromuscular fatigue experienced during resistance training, but also to ensure velocity remains high during training.”
This statement does not mean that CS and RR provide two applications because a lower velocity loss is the same as experiencing less fatigue.

As can be deduced from the above, the statements made in lines 324 25 (the present study shows the viability of both CS and RR to potentially serve as cost-free alternatives to VL20% resistance training prescription) and 383-86 (the beneficial effects of terminating training sets after VL20% demonstrated in prior literature can be achieved by using CS and RR, while avoiding the financial and logistical constraints associated with the use of velocity tracking devices) cannot be applied and, therefore, do not present utility.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: USING CLUSTER AND REST REDISTRIBUTION SET STRUCTURES AS ALTERNATIVES TO RESISTANCE TRAINING PRESCRIPTION METHOD BASED ON VELOCITY LOSS THRESHOLDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: General comments:
Interesting study showing the efficacy of alternative set structures as alternative methods to velocity loss thresholds, which may certainly have utility in team-based sports or large group settings. I commend the authors for their work. To the best of my knowledge, they have performed appropriate statistical analyses with the raw data provided to investigate the purpose of their manuscript. However, I have some requested revisions as addressed below.

Specific comments:
Line 49 – 50:
This sentence may confuse the reader. I would suggest changing ‘above 20% VL’ to read ‘less than 20% VL’.

Line 87:
Please remove ‘or even greater’, as there were no significant differences in muscle strength in this study cited by Pareja-Blanco et al. (DOI: 10.1111/sms.12678).

Line 113:
This sentence may confuse the reader. I would suggest changing ‘above VL20’ to read ‘at less than VL20’.

Line 171:
I think ‘things’ is a typo. Please change to ‘things’ to say ‘thighs’ or appropriate terminology.

Line 209:
This sentence may confuse the reader. I would suggest changing ‘above the 20% threshold’ to read ‘at less than 20% VL threshold’.

Line 317:
Please change ‘VL20%’ to state ‘VL20 – 25%’, since although VL20% may be suggested in the squat from Pareja-Blanco et al. (DOI: 10.1111/sms.12678) and Pareja-Blanco et al. (DOI: 10.1249/MSS.0000000000002295), VL25% may be suggested in the bench press from Pareja-Blanco et al. (DOI: 10.1111/sms.13775), and you investigated both exercises (squat and bench press) in your study.

Line 319:
Please change ‘VL20%’ to state ‘VL20 – 25%’ for the same reason as mentioned in my previous comment.

Line 326:
This sentence may confuse the reader. I would suggest changing ‘above the VL20%’ to read ‘at less than VL20%’.

Line 342:
Please change ‘production’ to ‘generation’ for further clarity.

Line 378 – 381:
Please change the following sentence, “Importantly, RR emerged as the most efficient set structure among the three since it allowed for the lowest VL among the three set structures, and greatest number of repetitions to be performed above VL20% without extending the total training time – which was the case during CS”.
RR is not necessarily 'the most efficient’ set structure because it allowed for the lowest VL and the greatest number of repetitions to be performed at less than 20% VL, as the available evidence does not suggest that VL should be as low as possible for strength nor hypertrophy. Please highlight how both the systematic review and meta-analysis by Jukic et al. (DOI: 10.1007/s40279-020-01423-4) and Davies et al. (DOI: 10.1007/s40279-020-01408-3) found no differences in strength and no differences in hypertrophy when comparing alternative set structures to traditional sets; therefore, alternative set structures do not appear to be superior nor inferior to traditional sets for strength nor hypertrophy. With that being said, you could potentially highlight how RR may be beneficial for team-based sports that involve high velocity movements, desire velocity-oriented profiles, and greater power outputs with lower loads, but it is crucial to highlight that this does not impact strength and hypertrophy when the total repetitions and load are the same between alternative set structures and traditional sets based on the two available SRMAs. Also, Davies et al. found that on average alternative set structures required more total training time than traditional sets; therefore, RR is not necessarily 'the most efficient' based on the broader available evidence. You may want to include that integrating different velocity loss thresholds (and both alternative set structures as well as traditional sets) may be beneficial in a periodized model as both have advantages and disadvantages on the acute responses and favored chronic adaptations. With everything said, I greatly appreciated the novelty of the study, well-written manuscript, and thorough statistical analyses. Awesome work!
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: USING CLUSTER AND REST REDISTRIBUTION SET STRUCTURES AS ALTERNATIVES TO RESISTANCE TRAINING PRESCRIPTION METHOD BASED ON VELOCITY LOSS THRESHOLDS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you for addressing my comments.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPREHENSIVE ANALYSIS OF THE CORRELATIONS OF S100B WITH HYPOXIA RESPONSE AND IMMUNE INFILTRATION IN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English language needs improvement and mistakes corrected. Transfection and overexpression part in the methods as well as bioinformatic analyses and figure legends do are not provide sufficient information on what has been done.
Experimental design: The concept of the manuscript is unclear. The authors show hypoxic up-regulation of S100B expression and invasion in HCC cells and in HCC tumors to tumor cell migration. On the other hand, the subsequent analyses were performed with databases from HCCs and normal livers. As S100B appears to be mainly expressed in immune cells in normal liver (leaving cellular distribution in HCC unclear). It is unclear if hypoxia also up-regulates S100B expression in immune cells, how hypoxia is related to tumor hypoxia. In consequence, does this mean that S100B expression in HCC is due to increased immune cell infiltration in hypoxic HCC and immune cell infiltration is correlated with hypoxia and metastatic properties?
Moreover, this raises the question if the experimental investigations are relevant for second bioinformatics part of the manuscript.
Validity of the findings: The findings appear to be valid, but as the connection between the different parts of the manuscript and the use of the models is not convincing, the conclusions appear questionable.
Additional comments: 1.	Down-regulation of HIF-1alpha and S100B by shRNA expression plasmids in HepG2 cells is not an easy task considering the difficult transfection of these cells and the small efficacy of shRNAs. Neither plasmids are described, how they were generated nor the shRNAs nor is shown how effective the shRNA for S100B was.
2.	Bioinformatic analyses are poorly described.
3.	Figure legends are not instructive concerning the explanation how the data illustrated in the figures were generated.
4.	What is “LIHC”?


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPREHENSIVE ANALYSIS OF THE CORRELATIONS OF S100B WITH HYPOXIA RESPONSE AND IMMUNE INFILTRATION IN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Very clear and organized reporting.
Experimental design: Experiments and analysis are well designed and executed.
Validity of the findings: Findings are valid, statistically sound and controlled. Conclusions are well stated.
Additional comments: Yan et al. reported that HIF-1α participates in the proliferation, invasion and metastasis of HCC through regulation of the expression of S100B in hypoxia. The study is well designed and analysis and conclusion well performed and written. The paper should be ready for publication after minor language/ presentation suggestions below are addressed:
- Figure 2A: There are two HREs (HRE1 and HRE2), but three promoter sites (-980, -844 and -726). Are the HREs in the three promoter sites? If they are, please label accordingly. Also, what does the two green oval represent? Please clarify in the description. Lastly, the HRE1 sequence (atgct) outlined does not have the consensus sequence of (A/G)CGT(G/C)
- Figure 2B: What is the role of the PGL3-basic? It is not mentioned anywhere in the text or Figure description
- Figure 4A: The blue and orange is not visible unless we zoom in a lot. Is it possible to make the coloring more obvious?
- Figure 5: Please put spaces between 'thatS100BmRNA' and 'normalhumanliver'. Also change 'it majorly expressed' to 'it is majorly expressed'. Please also provide clearer description of the takeaway for Figure 5A and 5B.
- Figure 6B and 10A: What does * and # signify?
- Figure 8: No H-J shown. Also, Figure 8G and 8D are the same.
- Line 14: qRCR. Shouldn't it be qPCR?
- Line 97: please delete the repeated 'Then,'
- Line 227: 'maintenance' can be changed to 'maintain'
- Line 285: 'bounds to' can be changed to 'binds'
- Line 319: 'S100B directly interacts proteins'. Does the author mean 'S100B-interacting proteins'?


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPREHENSIVE ANALYSIS OF THE CORRELATIONS OF S100B WITH HYPOXIA RESPONSE AND IMMUNE INFILTRATION IN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Yan et al. used the Hepatocellular carcinoma (HCC) cell line HepG2 to investigate the role of S100B protein in progression, invasion, and metastasis.
The authors discovered that hypoxia increases S100B expression at both the mRNA and protein levels. They also discovered that HIF-1 binds to HREs in the S100B promoter region and increases its expression. They also discovered a direct association between S100B and HIF-1. In the HepG2 cell line, S100B is also involved in hypoxia-induced cell migration and invasion. The authors discovered that S100B was primarily found in T cells and Kupffer cells in normal liver, indicating a significant correlation with immune response in the liver. They discovered a positive relationship between S100B expression and infiltration levels of immune cells using TIMER analysis. They also reported a positive relationship with immunomodulators. Based on these findings, the authors claim that S100B expression aids in the recruitment and migration of immune cells in the HCC tumor microenvironment. Overall, this is a nice paper with carefully gathered data. It is well written and provides interesting mechanistic insights into how S100B regulates the HCC tumor environment under hypoxic conditions. S100B may also serve as a biomarker and drug target in the treatment of liver cancer. This manuscript, in my opinion, is appropriate for the PeerJ journal.
Experimental design: NA
Validity of the findings: NA
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPREHENSIVE ANALYSIS OF THE CORRELATIONS OF S100B WITH HYPOXIA RESPONSE AND IMMUNE INFILTRATION IN HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is somehow improved, but not all and even my key concerns have not been addressed adequately. The authors show experimentally that hypoxia regulates S100B expression in tumor cells, but S100B appears to enter tumors mainly in the form of immune cells, Thus, the hypoxic regulation of immune cells appears to mediate hypoxia-induced upregulation of S100B in HCC. The authors still did not link their findings in HCC cells with their data base analyses.
Experimental design: The data base and the experimental part are not convincingly linked.
Validity of the findings: The validity of the findings is compromised by the study design
Additional comments: If the authors find a better way how up-reguation of S100B by hypoxia in the cells might be related to the immune cell infiltration and S100B in immune cells, one could re-consider the manuscript. Hypoxic regulation of S100B expression by HIF1alpha, regulating migration is interesting.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPREHENSIVE ANALYSIS OF THE CORRELATIONS OF S100B WITH HYPOXIA RESPONSE AND IMMUNE INFILTRATION IN HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: Reporting is clear and unambiguous
Experimental design: The experimental design is well defined and methods sufficiently described
Validity of the findings: The findings and conclusions are well supported
Additional comments: My concerns have been mostly been addressed in the edited manuscript. There are only a few minor suggestions for change:
- Line 16: please define what NK cell is
- Line 24: please change ChIP-qRCR to ChiP-qPCR


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RELATION BETWEEN LYMPHOCYTE TO MONOCYTE RATIO AND SURVIVAL IN PATIENTS WITH HYPERTROPHIC CARDIOMYOPATHY: A RETROSPECTIVE COHORT STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Generally clearly written article with good language and overall structure.
Abstract
- generally ok

Introduction
- a bit too short. Maybe can highlight why LMR is considered better than current biomarkers or for complementary purposes.

Methods
- study design - not mentioned
- to detail out the sampling method and technique. 499 is universally sampled, any random sampling?
- when was the enrollment started and completed endpoint
- ethics - as informed consent was waived, what other steps were taken to ensure confidentiality and safeguard the data
- stats analysis - how and why did the author decide to split into 3 tertiles. reason to split into 3 and how they decided on the range and cut off points.

Results
- can elaborate further on the non-cardiac deaths and why they should be included in the study
- Tables- maybe can add * for the significant p-value/ values

References
- very few from 2020 reference, none from 2021. if possible to add more, but understandable if it is rare to find studies related to this topic.
-
Experimental design: No comment
Validity of the findings: Study has meaningful outcome and benefits.
Tertile was divided into 3 with stated range. However, it would be beneficial to add reference or source of the cut off points for the ranges.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RELATION BETWEEN LYMPHOCYTE TO MONOCYTE RATIO AND SURVIVAL IN PATIENTS WITH HYPERTROPHIC CARDIOMYOPATHY: A RETROSPECTIVE COHORT STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The writing of the manuscript was clear and easy to understand.
Experimental design: 1. Introduction: The justification of chosen LMR was not clearly stated. The authors need the explain how and why the decision was made to choose LMR as a prognostic factor.
2. Methods: Please add on the inception cohort as the study design was a retrospective cohort. The authors need to state the starting and endpoint clearly (What is the starting point? when is the endpoint, especially for censored cases especially the authors, mention on the end of follow up..when?).
Please calculate the minimum sample size needed for this study.
What sampling methods do the authors use to select the respondents?
Line 93-95: On what basic this LMR was divided into 3? Any citation?
What is the reason choose a time-dependent receiver operating characteristic (ROC) curve?
Validity of the findings: The manuscript explained clearly the statistical analysis and result with various analyses to control all the confounders.
The conclusion is linked with the research objective of the study.
Additional comments: No comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RELATION BETWEEN LYMPHOCYTE TO MONOCYTE RATIO AND SURVIVAL IN PATIENTS WITH HYPERTROPHIC CARDIOMYOPATHY: A RETROSPECTIVE COHORT STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: Thank you for the opportunity to review this manuscript. It’s an interesting study, however, there are a few issues that need to be cleared.  In the introduction, its more beneficial if the author could elaborate more on the relationship between hypertrophic cardiomyopathy (HCM) and lymphocyte to monocyte ratio (LMR). How the lymphocyte to monocyte ratio could affect the hypertrophic cardiomyopathy
Experimental design: For the methodolody – is there any reference age included in this study? Sample size calculation and sampling method were not mention in the methodology. LMR was divided into 3 tertiles; however, there was no reference cited in the methodology.	Kindly mentioned in the method, what is the accrual time, the additional follow uptime, and when is the closure of the study
Validity of the findings: Overall, results were presented in tables and figures. However, few results need explanation. Line 124 - author mentioned WBCC increased across LMR tertiles, however, the result in table 1 showed the contrary. Figure 1- Kaplan Meier demonstrated a decreased cumulative incidence of all-cause mortality?  However the figure showed the contrary. Table 2 is intended for univariate cox regression analysis of all-cause mortality- is it without considering the tertile? Why is tertile not included in the univariate analysis? Table 3, what type of variable is included in model 2. Table 3: adjusted HR for LMR for tertile 2 and 3 were 0.43 and 0.39 respectively – which showed LMR had a protective effect for all-cause of mortality- In the discussion –(Line 193) – lower LMR was significantly associated with adverse outcomes? Kindy explains.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RELATION BETWEEN LYMPHOCYTE TO MONOCYTE RATIO AND SURVIVAL IN PATIENTS WITH HYPERTROPHIC CARDIOMYOPATHY: A RETROSPECTIVE COHORT STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors have made changes according to comments.
Overall well written.
Experimental design: No comment
Validity of the findings: no comment
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RELATION BETWEEN LYMPHOCYTE TO MONOCYTE RATIO AND SURVIVAL IN PATIENTS WITH HYPERTROPHIC CARDIOMYOPATHY: A RETROSPECTIVE COHORT STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors has improved it.
Experimental design: The explanation and correction was done according to the comments
Validity of the findings: Well explained
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE RELATIONSHIP BETWEEN DISTAL TRUNK MORPHOLOGY AND OBJECT GRASPING IN THE AFRICAN SAVANNAH ELEPHANT (LOXODONTA AFRICANA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall the manuscript is well written in its preparation and organization of scientific data. I think the authors could benefit from reading some of the sentences out loud as some of the sentence structure and use of passive versus active voice can shift. Overall the background needs improvement with more sources tying in the similarities and differences between the distal tip of the African elephant versus the tip of Asian elephants as well as other primate species.

The largest improvement in the manuscript would be on the figures. Many of the figures contain inconsistancies and I believe adding some figures/tables labels and schematics could greatly increase the context of this article.

The authors have a clear knowledge and background of the idea of reaching tasks, but I would like to see some more tie in to the unique morphology of the trunk and comparisons to more muscular hydrostats.
Experimental design: Personally the experimental design seemed sufficient towards understanding the author's hypothesis. I would prefer for the authors to include more detailed schematics in the figure to help the readers understand what the actual methodology of this project is. I give more information in the line by line comment, but I would recommend not using each of the elephant's names and completely dismissing the elephant that was used in analysis on only some points. The authors list that the experiments were performed on 6 elephants, however 5 elephants were only used in some of the analysis. In this field your sample size could be indicated to be not-sufficient, but working with elephants is difficult and to me a sample size of 5 is still rigorous for much of your analysis.
Validity of the findings: I would like to see more discussion again to compare what the authors might think in the Asian elephant as the bottom finger on the trunk is composed of cartilidge. The authors also simplified this down to 2D and I'm curious if there is any thought given to how the trunk movement before the tip is changing. The conclusion itself is well stated, but I think targeting a more morphological argument is needed as many of the comparisons given are to animals that have joints that they shift and there is no discussion of joints with the elephant as it is purely a muscular hydrostat.
Additional comments: Line 31 – At the beginning of the abstract it would help if you indicated that you are talking about the hand of a human. There are even descriptors of elephant trunks being called the “hand of the beast” that you could use in your abstract as a transition.
General Abstract – I think it could benefit from discussing more about Muscular Hydrostats in general. Look to many of Bill/William Kier’s publications on discussion of muscular hydrostats as tongues, trunks, and tentacles and you have a clearer background of comparison. Also in the abstract it would help to find a more similar morphology comparison than that of the hand to the reader.
Line 40 – With the new updated African forest elephant species in recent years. Most literature I see cites African bush elephants as (Loxodonta africana)
Line 53 – social interaction, you could discuss as this relates to elephants by citing that elephants often grasp eachother’s trunk’s in a trunk shake type of movement.
General Introduction Comments – Something you should bring up in the introduction is the idea that African elephants which you studied have two fingers at the tip of their trunk, where as Asian elephants only have one. Your method of grasping behaviors is relevant to many of the primate species listed as they also have thumbs which allow them to grip objects in a different way. As African elephants are browsers and Asian elephants are grazers there are different evolutionary reasons why they developed differences at the tip of their trunks.
I recommend adding some of the more recent citations pertaining to elephant trunk reaching movement including: https://www.sciencedirect.com/science/article/pii/S0960982221011337 , https://royalsocietypublishing.org/doi/10.1098/rsif.2021.0215
Line 122 – I would recommend shying away with naming the elephants in the study and instead identifying them by Elephant A, B, C, D, etc. or Elephant 1, 2, 3, 4. This is good to not indicate specific challenges individual elephants have for ethical reasons. I would encourage you to also include a table giving the age, sex, weight, etc. of each of the elephants to allow readers to compare/contrast differences in their gross characteristics.
Line 140 – The paper’s methods could be more useful to future studies if exact descriptors of the quantities were described as well as exact dimensions. Perhaps including a table of all of the exact dimensions of the food items could help with understanding length scale differences.
Line 162 – was any training done with the elephants to perform this task. If so how long did the training last and did you wait until a certain percentage for the elephants to successfully reach.
Line 217 – I’m a bit confused about how the 107 number was found. Did some of the individual elephants reach more than others or was it completely consistent. Especially with you not including the data from N’Dala. In my opinion if you are excluding N’Dala from one part of the analysis it would be best to just exclude those trials all together and not only include that data when applicable.
Line 221 – you indicate that the p-value is significant but what hypothesis did this prove or disprove?
Line 225 – its important to know that just because the p-value is significant does not indicate that they do not differ. It just indicates that the hypothesis test failed. Therefore you cannot conclude that opposite of a failed hypothesis test.
General Figure comments – every one of the figures need scale bars to indicate the size of many of the images. Especially for the experimental design figure 1. A lot of your figures are very small. It is very difficult to read what exactly the plots are saying especially you’re A, B, C.
Figure 4 – you use different graphics of carrots, apple slices etc. on previous graphs, I would recommend adding those to the figure 4 so readers know what each line is. I really think Figure 4 could be visualized better in another form. Each one of your sub-graphs has a different x and y axis in terms of your figures are labeled so some of the differences seem a lot larger than they actually are. You should graph all of them on the same axis with a grasp number from 0-120 and a number of failure from 0 to 6 as those are the entire ranges of your experimental design.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE RELATIONSHIP BETWEEN DISTAL TRUNK MORPHOLOGY AND OBJECT GRASPING IN THE AFRICAN SAVANNAH ELEPHANT (LOXODONTA AFRICANA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Some issues with the clarity of the writing. I have provided more details below - I have attempted to provide examples for how certain sections could be re-written.
Experimental design: Experimental design is clearly laid out.
Validity of the findings: Methodology and statistics appear correct.
Additional comments: This study assesses how elephants will ‘pre-shape’ the distal aspect of their trunks in preparation for picking up an item, such as some food. The authors examine this behavior in six elephants and find that elephants do indeed pre-shape their trunks depending on food size/shape. They go on to suggest that the ability to pre-shape is likely a learned behavior and is based on visual cues (given that a sub-adult and a blind individual exhibit a much lower instance of pre-shaping).
I find the methodology very compelling and the analyses well designed. I can appreciate that large animal biology is difficult to conduct, and there are many logistical challenges associated with getting access to elephants. I feel the results nicely extend the thinking of animals use their appendages for various tasks.
I have a handful of (mostly minor) comments I hope the authors can address. I also offer some help with re-framing certain sentences.

# # #

Abstract
L35 Appendices – do you mean appendages? There are a couple of other places where this appears in the manuscript (i.e., L80, L99).
L46-48 I think there’s a missed opportunity to quickly explain why you only have 3 individuals conforming to your hypotheses about pre-shaping. When I originally saw that only 3/6 elephants pre-shape their trunks I was quite surprised – but you should mention that those that did not were either blind, or sub-adults, indicating that there is likely some learning occurring, and that visual cues are important.

Introduction
L71 ‘when the hand moves forward the object’ -> ‘when the hand moves toward the object’
L105-107 Please re-phrase. I think you’re asking if all elephants exhibit similar trunk grasping shapes for a given item.

Methods
General: You should mention that the blind and sub-adult elephants are used to assess the role of vision and experience respectively in grasping behaviour. This better prepares are reader fop what’s coming.
L164 ‘We captured these prints at every first put of the trunk tip on the box and…’ -> ‘We captured these images when the elephant first approached the box and…’
L166-167 Is this the only definition of a feeding failure? I think this needs a more comprehensive definition. My original thought was maybe a failure occurs when the elephant drops the food item, or some set amount of time elapses as the elephant can’t pick up the item. Just knocking the box seems a little harsh.
L172 ‘print’ -> image
L173 ‘posed’ -> placed
L200 ‘grille’ -> grid
L209 How were these trajectory plots performed. Are these outputs from the trajectory.analysis() R function in geomorph – if yes, some statistics should be reported. For example: magnitude (P.magnitude.diff), angles (P.angle.diff), and shape differences (P.shape.diff). All these describe slightly different aspects of the trajectory that should prove your point. These, and perhaps other outputs depending you your goal, should be reported in the text or in a table.
L209-211 Bit lost here. Please rephrase – I think you’re trying to say the following. ‘These trajectories of the distal shapes over the grasping permitted to see a potential merging…’ -> ‘Trajectories of the distal trunk during grasping often exhibited similar shapes among individuals for the same object.’

Results
L257 ‘the deformation is the most, and the blue parts where the deformation is the less.’ –> ‘the deformation is greatest, and the blue parts are where the deformation is lowest.’
L262 typo
L277 ‘became more thicken.’ -> ‘became thicker.’
L308 ‘The most the object’ -> ‘The more the object’
L319 ‘when Ashanti and Juba mad the’ -> ‘while Ashanti and Juba made the’
L320-322 I don’t understand this sentence. Are you saying that all elephants had a comparable number of failures?
L330 ‘The figure 4 shows’ -> ‘Figure 4 shows’
L346 The figure 5 displays’ -> ‘Figure 5 displays’

Discussion
L366 typo
L387 ‘the preshaping of the hand or paw can be find usually in most of individuals in primates and rats’ -> ‘the preshaping of the hand or paw can usually be found in many primates and rats’
L398 ‘the vision stays primordial’ Please rephrase, I don’t know what this means.
L434 though -> thought

Conclusions
Any thoughts as to why Tana did not preshape the trunk?
L445 grasped -> grasp

Figures
Figure 2 You could also code the point symbols to reflect each food item (i.e., circles for the carrot, squares for the small cube, diamonds for the large cube etc.)
Figure 3 (very nice figure) Was M’Kali never given a large cube? Based on the supplementary trajectory figures it appears M’Kali rarely interacted with the large cube, you should mention so in the methods or results.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE RELATIONSHIP BETWEEN DISTAL TRUNK MORPHOLOGY AND OBJECT GRASPING IN THE AFRICAN SAVANNAH ELEPHANT (LOXODONTA AFRICANA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Overall it appears the authors did an excellent job with the reviews. I really appreciate the re-organizing of the background and it flows really well together with the rest of the paper. I appreciate the author's improvement of the figures.
I have included additional comments on the figures that once made I believe the manuscript is ready for publication.
One item that would still help is by changing the color and size of the scale bar based on the background imagery. So for example in Figure 1C, making the text & scale bar white font. Also updating the thickness of the bar would be beneficial.
I believe Figure 2 should still be improved in terms of making sure each of the axes are scaled the same way. Currently, the CV1 axis is skewed to the left. Making sure the axis positive and negative labels are equivalent will help not bias the appearance of the data for readers.
Figure 3, 4, 5 I would add text labels next to “Apple 1 cm, Carrot 1 cm cubes, etc.” As there is no scale to indicate the number/size of items. This comment is as on M’Kali’s plot there is a small white cube, but on Juba’s plot, there is a small and large white cube. So distinguishing the difference between each of those would help.
Figure 6 would be improved by clarifying the large cube/medium cube/small cube by size. Indicating on the figure each of those would help the readers understand the flow from small to large in the differences.
Experimental design: Including one video of each of the elephants as well as reaches is really beneficial. One thing that could help the authors is by including a single supplemental video file that the authors can then cite in the manuscript in the methods section to help readers have a video to see the experiment live. Also I agree with the authors rebuttal of keeping the names of the elephants as with being more explicit with the number of individuals used for each analysis makes much more sense.
Validity of the findings: The authors made substantial improvements to the discussion and results sections following the reviews provided. There are additional arguments and discussion points made about the uniqueness of this study in terms of muscular hydrostats.
Additional comments: Additionally I would like to apologize to the authors for my delay in getting the review back to you. The authors clearly did quite a bit of work to improve the manuscript and with some additional figure edits I look forward to seeing this scientific work published.
Also I would like to commend the authors on including members of the Zoo where the research was conducted as co-authors. Historically many zoo-personnel are not included as authors and I appreciate you all doing good, collaborative, and well-meaning science.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE RELATIONSHIP BETWEEN DISTAL TRUNK MORPHOLOGY AND OBJECT GRASPING IN THE AFRICAN SAVANNAH ELEPHANT (LOXODONTA AFRICANA)
Review round: 2
Reviewer: 2

Output:
Basic reporting: Writing is much improved, but could do with one more run through.
Experimental design: Experimental design is clearly laid out.
Validity of the findings: Methodology and statistics appear correct.
Additional comments: This study assesses how elephants will ‘pre-shape’ the distal aspect of their trunks in preparation for picking up an item, such as some food. The authors examine this behavior in six elephants and find that elephants do indeed pre-shape their trunks depending on food size/shape. They go on to suggest that the ability to pre-shape is likely a learned behavior and is based on visual cues (given that a sub-adult and a blind individual exhibit a much lower instance of pre-shaping).
	This is the second time I’ve seen this paper and some clear improvements have been made. I’m glad to see that the authors took the time to respond to my comments in the rebuttal letter – I agree on the CVA plot, the current version is superior.
I have no major comments and just a handful of minor comments that I think the authors should be able to easily incorporate. I would however ask the authors to have another person check the English again – it’s much improved, but there are some sentences that may benefit from a quick edit.

Minor Comments
L69 Typo? macacas -> macaques
L180 An example of grasping film can be found in the supplementary data – I couldn't see this among our files but I trust the final version will include this video.
L198 These landmarks formed the trunk distal parts contours, and then this 2D sliding depicted the different distal shapes. – I'm not really sure what you're trying to say here. Perhaps that the semi-landmarks defined the distal borders of the trunk tip?
L203 The GPA serves to remove the effects of translation, rotation, and scaling from your shape data – I'm not sure why you're talking about correlation here.
L236 typo? proscruste space -> Procrustes space


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: POLYMORPHISM IN A NEOTROPICAL TOAD SPECIES: ONTOGENETIC, POPULATIONAL AND GEOGRAPHIC APPROACHES TO CHROMATIC VARIATION IN PROCERATOPHRYS CRISTICEPS (MÜLLER, 1883) (AMPHIBIA, ANURA, ODONTOPHRYNIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: Review: Proceratophrys biogeography and taxonomy

This paper focuses on the taxonomy and biogeography of the genus Proceratophrys in Brazil. On the positive side, the authors appear to have evaluated a variety of different morphological and chromatic characteristics for a large number of locations and populations, and they present some convincing arguments that previous researchers may have designated species without sufficient rigor or justification. On the negative side, this paper is very poorly written. The paper is riddled with what appear to be inappropriate conclusions, awkward and garbled sentence structures, and simply incorrect grammar. I do not have the time or inclination to identify or fix grammatical errors – I suggest the authors get help to identify and correct these errors, because they make it very difficult to evaluate the quality of the science being presented. Of greater concern is that the authors make many statements that seem to be entirely unsupported by any evidence or reference, and which appear unconnected to their main arguments. I have tried to identify these issues in the manuscript (see comments on specific parts, below), because it is imperative that these be corrected (or removed) for this work to be of publishable quality. In general, I recommend that the authors focus on their main argument (that the levels of variation they have identified have important implications for Proceratophrys taxonomy), and try to avoid getting side-tracked and making unsupported claims.

L47: “Understanding how evolutionary mechanisms operate on populations through morphological variability in individuals has been the main objective of the most different studies since the times of Darwin (Futuyma 1987; Huxley 1940).” This sentence does not make sense as written and needs to be modified. Did you mean “most studies”?
L91: “chromatic polymorphism provides these species with a varying amplitude which enables them to occupy, adapt and reproduce for generations in determined environments” It is not clear what is meant by “varying amplitude”. Please explain what you mean or remove this phrasing. Also, it is not clear what is meant by “determined environments”.
L96: “The adaptive importance of polymorphism for organisms, including P. cristiceps, lies in the improvement of the reproductive and survival capacities of individuals in response to stress caused by the environment or predators (Ridley 2004).” This is a very general statement about polymorphism that has only been validated in a limited number of species. Is there evidence that this is the case in P. cristiceps? If so, reference it, if not, you cannot use a general reference (a textbook) to claim evidence concerning what is going on in P. cristiceps (unless Ridley specifically discusses P. cristiceps, which I don’t think he does). In any case, if there is such evidence for P. cristiceps, you should cite the original references.
L99: “Regardless of the degree of changes, a limiting factor for survival is seen in the ability for morphophysiological modification, whether intraspecific or interspecific (Ricklefs 2008).” I do not understand what the authors are trying to say here – please rephrase and rewrite for clarity.
L101: “In this case, the survival and adaptation of organisms depends on the maintenance of a number of individuals in these populations in such a way that the evolutionary mechanisms can act on them, and thus give rise to what we call biodiversity” Again, it is not entirely clear what the authors are trying to get at here – rephrase.
L. 124: “Specimens Maturity” is awkward and grammatically incorrect – rephrase.
L. 134: A Chi Square test should only be used for comparisons of categorical data – it is not clear if that is what the authors did.
L. 139: “whose chromatic patterns were statistically functional with respect to the analysed frequencies.” I have no idea what this means – rewrite or rephrase.
L. 183: Change to “enabling us to rexamine…”
L. 192: “(not intercross)” – not clear what this means – explain or rewrite.
L. 193: “we decided to find presumably intercrossing sets aiming to mitigate methodological eventualities, or the “demes” so to speak” Again, I have no idea what the authors are trying to say here.
L. 245: “We used two software programmes with the goal of mitigating the possible effects caused by PM(g) and PB(g) in the BAM diagram (biotic, abiotic, and movements) of probabilities (errors of omission and commission) for species with restricted vagility”.  I am not an expert on these methods, but I don’t think they are explained very well here. I recommend the authors take abit more space to explain these methods in more detail, making sure to explain any field-specific jargon.
L. 333: “Furthermore, we observed a proportional expression of the six phenotypes for each relative frequency of P. cristiceps (14:43:6:9:8:20), which was also maintained internally among the samples and localities”.  It is not entirely clear to me what this means – I think further explanation is in order.
L. 401: “being exclusively distributed in the Caatinga and transition phyto physiognomies according to their area of habitat suitability.” Again, it is not clear to me what this means – please explain or rephrase.
L. 421: “When dealing with these probable varieties, it is necessary to identify categories which can be in themselves equivalent in an experimentally comparable way.” Not clear what this means?
L. 477: “random events involving skull conformation to food resources” Not clear what this means.
L. 485: “Furthermore, there are underlying factual (and experimental) requirements to explain the morphologic divergence and the alleged taxonomic diversity (Van Holstein & Foley 2020).” Not clear what is meant here – I think further explanation is in order.
L. 546: “but which less explain the proposed species,” Rephrase.
L. 566: Furthermore, they are not even tested under an experimental model of diversification dynamics (Ajmal Ali et al. 2014; Annibale et al. 2020; Schindel & Miller 2005; Van Holstein & Foley 2020)”  I think this requires further detail and explanation.
L. 569: “and why not for the P. biggibosa, P. boei and P. appendiculata groups whose taxonomic history involves controversies and the use of equally variable phenetic traits? Where evidence of pre- or post-zygotic barriers or their biogeography continue to be elusive?” This sentence is garbled and it is not clear what the authors are trying to say. Reference is made to past controversies without explaining them.
Experimental design: The experimental design appears generally appropriate, but I would have liked more detail on how the Chi Square tests were carried out.
Validity of the findings: L. 428: “The variations observed in Proceratophrys cristiceps at the chromatic level indicated selective value in the studied species. This fact is reflected in the differential frequencies of the chromotypes that seem to signal some advantage of individual survival. The distribution of animals in the soil found herein suggests having a predominant role in the bias of chromotypic frequencies throughout post-larval development, i.e. it indicates a frequency-dependent selection (Bond 2007).”  In my opinion, this paragraph is entirely speculative and should be removed or replaced with something much less assertive. As far as I can tell, the authors present no evidence that these different morphs are adaptive, other than a few pictures where some of the color morphs appear to blend into the background. This is not good evidence. Referencing a general review paper on predation and polymorphism by Bond also does not provide evidence. It is possible that these colors are an adaptive form of camouflage, but you need to carry out carefully controlled experiments to demonstrate that these color patterns are adaptive, not simply show a few pictures. The authors claim that the frequency distributions they observed somehow support the hypothesis of camouflage and frequency-dependence, but it is not at all clear how or why they came to this conclusion, and I did not see any valid test of distributions in the context of predictions of the camouflage hypothesis.

L. 450: “Therefore, we deduce that it may be a chromatic polymorphism with a strong genetic basis”. As far as I can tell, you do not have solid evidence for a genetic basis. You would need to do common garden experiments to demonstrate this, and those are not described in this paper.
Additional comments: Finally, the authors have put a number of the figures in the discussion. It is not clear why that is the case – typically figures are placed in the results. Personally, I find this approach confusing.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: POLYMORPHISM IN A NEOTROPICAL TOAD SPECIES: ONTOGENETIC, POPULATIONAL AND GEOGRAPHIC APPROACHES TO CHROMATIC VARIATION IN PROCERATOPHRYS CRISTICEPS (MÜLLER, 1883) (AMPHIBIA, ANURA, ODONTOPHRYNIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: In general, this is a quite well-written article. I am not a native English speaker so I cannot fully assess the quality of the English but I think it's correct for the most part. However, some sentences are in my opinion unnecessarily wordy and thus difficult to read. There are also some typographic errors and some weirdly used words. Wherever I could, I suggested corrections (see the attached annotated PDF) but this is by no means an exhaustive check. Anyway, the text needs to be carefully checked again.
Experimental design: I believe that the methods used to tackle the problem of colour variability in Proceratophrys cristiceps are appropriate (although I am not familiar with some of the used methods such as ecological niche modelling). However, in a few cases, I would like some additional details which could help the reader to better understand what was done by the authors.

Lines 117-122. I think it would be beneficial to indicate - in the case of preserved specimens - how were they stored (e.g. frozen, stored in alcohol, formalin etc.). I do not see this information either in the main text or in the supplemental data. Also, if they were stored in alcohol, wouldn't this affect the results as the alcohol dehydrates the specimen and may change its colouration, especially after long storage?

Lines 124-142. I am not familiar with this group of amphibians so I have a question here. Is there a possibility to confuse P. cristiceps with another species? A cryptic congener maybe? If so, it would be good to indicate how the specimens were classified as P. cristiceps. An identification key or molecular analysis? If there are no other similar species, you can disregard this point.

Lines 265-274. Unfortunately, I do not understand how you achieved this. Please describe your procedure in more detail.
Validity of the findings: In my opinion, the main findings are well supported. However, please see the questions posed in the "Experimental design" section.
Additional comments: The manuscript authored by Kleber Silva Vieira, Erivanna Karlene Santos Oliveira, Washington Luiz Silva Vieira and Rômulo Romeu Nóbrega Alves aims to describe the variation in colouration and morphometrics in a South American amphibian Proceratophrys cristiceps. It is an important task because in most species the intraspecific variation is usually insufficiently known. This is quite unfortunate because, without good knowledge on intraspecific variation, it is difficult to correctly delimit the species and in consequence to use correct names which, of course, further hinders studies in many other aspects of biology. This contribution is thus needed and will be welcomed by other researchers studying the morphology of South American amphibians. To sum up, in my opinion the article will be acceptable after moderate revisions.

Some further comments:
Lines 1-3. I suggest changing the title to "Polymorphism in a Neotropical toad species: An ontogenetic, population and geographical approach to chromatic variation in Proceratophrys cristiceps (Müller, 1883) (Amphibia, Anura, Odontophrynidae)". I know that there are two brackets just next to each other and that does not look very aesthetically but I think that in this context it is justified. I also think that "Neotropical" should be capitalised (names of biogeographic realms are capitalised in English if I'm not mistaken).

Lines 173-174. "For example, morphometric variation in animal research can be identified as either sexual dimorphism or originated from ontogeny (allometry)" - That is quite confusing. The intersexual morphological differences can also result from allometry. I suggest removing the word "allometry", it is not needed in this sentence.

Line 178. Of course, the authors will make the final decision about this, but I suggest putting Figure S2 into the main article. What measurements were taken is very important information and in my opinion, it is better to give it in the main text rather than "hide" it in a supplement.

Line 470" "However, because they are anuran amphibians..." - This doesn't sound particularly well. Please, try rewriting. Maybe something more like this: "However, because there is no clear concordance with the Bergmann's rule in anurans..."

Line 477. "random events involving skull conformation to food resources" - I am not sure whether I understand this correctly but to me, the "skull conformation to food resources" is a result of the selective pressure related to the adaptation to certain diets and thus not a random event but is of presumably strictly adaptational nature. Please reword or indicate what events do you have in mind.

Lines 493. "Fig. 9 A" - to be honest, I don't think this figure is necessary in the main text. I would move it to the supplement but of course, the authors will make a final decision about that.

Lines 514-524. "Figure 10" - the description of this figure is too vague. There is a good chance that the reader will not understand this figure unless it is more clearly described (same as my comment about the lines 265-274).

Lines 526-535. "...our observations indicated that the nodules (including warts and tubercles) were extremely variable in terms of number, shape and distribution, either isolated or regionally, in the same individual or between specimens" - you do not give any specific data about that (or am I missing something?), nor cite previous works that support this claim.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: POLYMORPHISM IN A NEOTROPICAL TOAD SPECIES: ONTOGENETIC, POPULATIONAL AND GEOGRAPHIC APPROACHES TO CHROMATIC VARIATION IN PROCERATOPHRYS CRISTICEPS (MÜLLER, 1883) (AMPHIBIA, ANURA, ODONTOPHRYNIDAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: I am satisfied that the authors have made a sincere effort to improve the writing in this document. There are still some awkward constructions and grammatical errors here and there. I leave it to the editor to decide whether this demands further revision.
Experimental design: I am satisfied with the revisions.
Validity of the findings: I am satisfied with the revisions.
Additional comments: NA


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RESEARCH PROGRESS OF ALDEHYDE OXIDASES IN PLANTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript summarizes recent studies about AO in plants considering genes and enzymes involved in synthesis and function that is in the scope of the Journal. Although authors made a substantial revision of the bibliography, considering the number of recent investigations of AO in specific plants, authors need to integrate the information and include a discussion to establish further investigations or points of view. In addition, authors must use figures and diagrams to support the information in text and point out the missing information or opportunity areas for new research. All of these suggestions could help to increase the audience of the manuscript.
Experimental design: The authors declare the use of Web of Science (Clarivate Analytics). Keywords such as “aldehyde oxidase,” “abscisic acid,” “indoleacetic acid,” and “stress. Considering the broad range objective of the manuscript, other keywords should be included to search among plants, mechanism, regulation, inhibition, etc. that helps to integrate the information in the manuscript.
The sources are adequately cited, but the organization of the review needs to be corrected in some paragraphs. Example: 66-68 indicates that details in AO in IAA production will be discussed later, but is not clear in the manuscript. There is a lack of paragraph connection in lines 104-105. It should be clear when authors make their personal interpretation or discussion (Example line 125).
Validity of the findings: The introduction requires some structure corrections to better meet the goal of the manuscript. The authors dedicate an important part of the introduction to the zeaxanthin epoxidase enzyme function and precursors, which miss the idea covered in each subject of the manuscript. Also, the conclusion should include unresolved questions or further directions. The authors only dedicate three lines to the conclusion.
Additional comments: The abstract should be improved in the specific points of view and discussion of the review.
In line, 38 authors indicate that ABA genes are identified in various plants but only describe enzymes characteristics and not gene regulation.
I suggest the use of a figure or pathway diagram to better support the idea described in lines 40 to 46.
It is not clear if there is a lack of information regarding AOs or the existing information is not clear enough to support the two reasons described in the 50-54 lines.
Line 68 - It is not clear which pathway the authors made reference.
Line 75-77 avoid independent and short ideas.
Line 91-93- ¿Incomplete or missing idea?
Line 95-97 is not necessary to explain.
104 – 105 lack of paragraph connection – miss the main idea – substrate specificity, structure, and expression?
125-125 It is not clear if the sentence is an author idea or hypothesis
126-127 The website has many tools, and it is not clear how the authors made the hypoxia prediction.
Line 128 - In contrast with other AO described in the manuscript the information is scarce.
The table included does not support the manuscript. I suggest a figure or pathway diagram
The manuscript has a good structure, but I found some writing issues related to determiner use (a/an/the/this, etc.), comma misuse within clauses, passive voice misuse. I suggest a revision to improve clarity.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RESEARCH PROGRESS OF ALDEHYDE OXIDASES IN PLANTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors need to pay attentions to the English language and use grammatically correct sentences, especially in lines 50-56 require both spelling and grammar checks.
The review article has referenced a significant number of articles studying the biosynthesis of ABA/IAA involving aldehyde oxidases. The basic properties of AO’s catalysis and physiological functions are clearly presented allowing general audience to comprehend before detailed discussion of each AO isoform in plants. The authors can strengthen their arguments about how this review article will impact the indicated fields in lines 50-56 of introduction.
A figure depicting the basic biochemical or physiological processes of AOs will help readers with different background appreciate this work better and easier. Additionally, a table summarizing the differences and similarities among AO isoforms will serve the same purpose.

Yes, the review has broad and cross-disciplinary interest and within the scope of the journal.
No, the field hasn’t been reviewed lately.
Yes, Introduction adequately introduce the subject. The motivation of this review could be strengthened as mentioned above.
Experimental design: Yes, the content is within the Aims and Scope of the journal.
Yes, the Survey Methodology is consistent with a comprehensive, unbiased coverage of the subject.
Yes, sources are adequately cited and appropriate.
Yes, the review is organized logically into coherent paragraphs/subsections
Validity of the findings: In the last session of the article extends beyond the well-characterized functions of AO and four AO isoforms, so the session shouldn’t be named as ‘Summary’. On the other hand, the benefits of this work stated in lines 50-56 are not only summarizing the current advances of the discussed field but also providing useful information to agriculture, whereas this session falls short of intended discussion and implications. Alternatively, a future direction of AO research may be discussed in the ‘summary’ session.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RESEARCH PROGRESS OF ALDEHYDE OXIDASES IN PLANTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: (1) In the basic information, the biosynthetic processes of IAA and ABA are explained. However, it is difficult to understand only the text description. Please illustrate the biosynthetic process including chemical structures and clearly show the pathways in which AO is involved.

(2) In addition, the biochemical and physiological functions of AO1 to AO4 are summarized, but it would be easier to understand if the localization, characteristic substrates, and physiological functions in plant are summarized in a table.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In the introduction, "GA20-oxidase" is explained, but please add more details about its potential as a target for gene regulation in agriculture.

In the biochemical and physiological function of AO1, it is stated that AO1 may be involved in the hypoxic response because it is induced by hypoxic stress. Please add more explanation about the physiological significance of hypoxic response in plants.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RESEARCH PROGRESS OF ALDEHYDE OXIDASES IN PLANTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript summarizes recent studies about AO in plants considering genes and enzymes involved in synthesis and function that is in the journal's scope. The authors made substantial modifications to improve the manuscript structure, adding references and figures to integrate the information. Also made all the modifications suggested in the review document.
Experimental design: No comments
Validity of the findings: The authors added more information in the introduction section that makes it easier to understand the manuscript goal and unanswered questions.
Additional comments: The authors improved the abstract, including specific points of view and discussion of the review, and corrected it according to all the suggestions.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RESEARCH PROGRESS OF ALDEHYDE OXIDASES IN PLANTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RESEARCH PROGRESS OF ALDEHYDE OXIDASES IN PLANTS
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DISTURBANCE HAS LASTING EFFECTS ON FUNCTIONAL TRAITS AND DIVERSITY OF GRASSLAND PLANT COMMUNITIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is written clearly, has adequate structure and a clear rationale. Literature references are adequate, but the number of figures can be reduced by joining figures in a single plate. Furthermore,  figures may be improved by adding p-values (actually this information is placed in tables 2 and 3, and this alteration enables the exclusion of the mentioned tables)
Experimental design: Major flaws in experimental design must be addressed in future versions:
1- No data about abiotic changes in disturbed/undisturbed plots (soils, microclimate, ..) are presented, although the introduction highlights the context between abiotic changes due to gaps/disturbances and its consequences on species composition (e.g., l. 63, l. 77)

2- What about intraspecific trait variation in response to disturbed/undisturbed plots? Plant species differ regarding functional traits, but phenotypic variation along environmental gradients (disturbances) are common phenomens and may affect CWMs of functional traits in disturbed and undisturbed plots.

3- Modelling: 12 CWM traits values or functional diversity values are modelled in function of disturbance regimes using plot pair as a random effect - may overfitting be an issue here?

4- small plot size (50 x 50 cm): I am not familiar with the specific vegetation analyzed in this study, but
 I wonder, if the plot size is sufficient to sample diversity of this habitat type. Some statistics on that (species-area relationships etc) would be highly welcome to show if minimum area for phytosociological surveys is sampled.
Validity of the findings: The study is really interesting, but small sample area (3m2 only) and comments on statistics (previous section) make me wonder about the validity of the presented findings. At minimum, supporting statistics should be presented as outlined above.
Additional comments: No comments.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DISTURBANCE HAS LASTING EFFECTS ON FUNCTIONAL TRAITS AND DIVERSITY OF GRASSLAND PLANT COMMUNITIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Smith et al report the findings from an interesting study comparing the trait-space occupied by established and post-disturbance grassland vegetation.  The findings are novel and I look forward to seeing this in it's final form.  My biggest concern is the language surrounding the disturbance treatments, I typically think of grassland disturbance as one that removes/alters above ground cover, but does not result in terminating the plants.  This was not the case, I didn't realize that the vegetation was completely terminated in the treatment plots until I was into the methods.  This doesn't necessarily take away from the novelty of the study, but it does need to be clear that this is a study comparing established vegetation with what is found two-years into the recovery/recolonization from a bare soil condition- which is very different from a grazing or fire type disturbance that I would typically associate with grasslands. To me this is a matter of the temporal age of the stands being compared - tracking the recovery of similarly aged plants is different than comparing established and recolonized communities.

Specific comments:
L38 - "vegetation removed" to "plants terminated"- big difference here.  I would interpret the original wording to mean that the plants were still alive and they tracked the regrowth.  Instead all plants were terminated and they tracked recolonization

L46 - functional repeats
L53 - Again taking issue with the disturbance wording - the functional character of recolonized vs established communities is different.
L152-3.  Wouldn't successive herbicide applications also terminate plants germinating from the seed back at that time? Yes the seed bank and seed rain is still there, but anything colonizing it during that two year treatment window was terminated?
L155- change to post-treatment
L179- curious about the outliers that were removed
L185-187 - There is no before/after disturbance data presented.  There is a comparison between recolonized and established communities
L196 - What distributions were used for the LMM's for each response variable?
L234 - There is no test of a legacy of disturbance here.  Again - recolonized vs established communities
L282 - I'd hardly say this is a study of lasting impacts - two years of recovery is still very early in the successional trajectories of grasslands
L289- Couldn't agree more - but change to functional trait shifts resulting from different successional stages - or something to that effect
Tables - Fix functional richness definition
Experimental design: Solid aside from previous comments re: wording surrounding the treatments and interpretation
Validity of the findings: See above
Additional comments: See above


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DISTURBANCE HAS LASTING EFFECTS ON FUNCTIONAL TRAITS AND DIVERSITY OF GRASSLAND PLANT COMMUNITIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No additional comments.
Experimental design: During previous round, I mentioned four concerns related to methodology/experimental design:
1- no data about environmental changes in disturbed plots: authors revised manuscript accordingly
2- intraspecific trait variation was not considered: authors revised accordingly, stating that the study as a 'first-level test', but failed on adding comments regarding follow-up research
3- overfitting, as 12 observations were modelled by a fixed and a random effect: authors do not provide additional tests
4- small sample area: Authors do not provide species accumulation curves or species-area relationship to address my concerns. In their response, authors cite two studies that attest appropriate plot size - one is about soil seed banks (and not community composition), for the other one, reference data are lacking.
Validity of the findings: As outlined during the previous round, I am not convinced about the validity of the presented findings, and, as detailed, little efforts have been done to improve the manuscript.
Additional comments: No comments.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DISTURBANCE HAS LASTING EFFECTS ON FUNCTIONAL TRAITS AND DIVERSITY OF GRASSLAND PLANT COMMUNITIES
Review round: 3
Reviewer: 1

Output:
Basic reporting: OK
Experimental design: Ok
Validity of the findings: OK
Additional comments: Sorry for delay, but I was in holiday!