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: GENOMIC CHARACTERIZATION AND PHYLOGENETIC ANALYSIS OF SALMONELLA ENTERICA SEROVAR JAVIANA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is written with very good scientific English. It is structured clearly. The Background the authors provide for the investigation is very thorough with adequate references. The manuscript is self-contained with the results that are relevant to the objectives. The number of the figures included in the manuscript itself is appropriate. I think Table 3 could be included in the supplemental material and table 4 is not needed since the same information is included in the supplemental table 4.

The following edits should be made:
1.	Line 21 and throughout the manuscript: S. Javiana is not correct Salmonella nomenclature. The genus name Salmonella cannot be shortened as “S.” without spelling out the species name enterica. So after first mentioning and spelling out the entire genus-species-serovar name, the following references to it are acceptable moving forward: “S. enterica serovar Javiana”, “Salmonella ser. Javiana or “ser. Javiana”. Please check the Pasteur Institute's guidance at: https://www.pasteur.fr/sites/default/files/veng_0.pdf.
2.	Line 107: delete the repeated “cucumbers”.
3.	Line 165, Supplemental table 1: for each strain, please indicate whether it was part of a known outbreak or considered sporadic.
4.	Line 242: “multiple sub-clusters” instead of “multiple of the clusters”.
5.	Line 243: “potential disease clusters of public health importance” instead of “potential clusters in public health”.
6.	Line 277: comma after “blood”.
7.	Line 303: “of” after the percentage.
8.	Line 360: ‘is in agreement with” instead of “is comparable to”.
9.	Line 369: ‘it” instead of “this”.
Experimental design: Research questions are well defined and fill a gap in the current state of knowledge. AS for the methods I have the following comments:
1.	Lines 163-176: include a brief description (library prep method and sequencing chemistry at a minimum) of the sequencing methods.

2.	Lines 178-198: please clarify what was the reasoning for using the two different SNP discovery methods in this study: the reference-free KSNP and the reference-based CFSAN pipeline? You state that you used the core SNP matrices from the KSNP to evaluate the population structure, not matrices based on all (core and ancillary) SNPs and given that the CFSAN pipeline removes mobile elements from hqSNP calling, the results should be expected to be close the same from the two pipelines. If the reason was that you were concerned the reference-based analysis would bias the population structure then that should be clearly stated over here.

3. Lines 187-192: the choice of the hqSNP reference sequence for each lineage: typically you want the reference sequence to be closely related to the study population, so what exactly do the authors mean when they state that one criterium for the appropriate reference sequence was that is was “not closely related to other isolates”? Also what was the rationale of using internal references for clades I and III but an external reference for clade II?

4. Lines 223-227: what was the reason for only using the 3 reference sequences to identify the virulence factors? Given, how poorly understood the virulence markers currently are in Salmonella, I would determine the virulence factors either in all sequences or at least in a subset of strains from each clade to get an idea how much strain level variation there is within the clade.
Validity of the findings: For the findings and conclusions I have the following comments/suggestions:
1.	Lines 275-284: the sample source being extra-intestinal may be a poor indication of severe disease. The number of hospitalizations and deaths would be better. TN is a FoodNet site, so the disease outcome information should be available for these isolates and should be included in Table 2 and it should be discussed whether severe outcome was overrepresented in any of the clades.

2. Lines 445-448: what was the reasoning to use the clade II reference as a reference when analyzing all isolates together? Because it was closed and the clade I and III references were not? Clade II appears to be the least representative of the overall Javiana population. I would like to see this analysis being performed also using the clade I and III references. As you state on lines 459-462, the use of closed vs. draft reference should not have a drastic impact and sometimes using a draft reference that is more closely related to the study population actually gives better results than using a more distantly related closed reference. In the case of Javiana, the choice of reference may not make much difference because it looks like there may not be that much diversity in the core genome, but the authors should at least attempt to prove that by performing the overall analysis of the 111 sequences using also the clade I and III references.

3.	Lines 475-477: this is an over-statement unless the overall analysis is also performed using the clade I and the clade III references to prove that for Javiana the choice of reference does not make much difference.

4.	Lines 481-483: more importantly, a larger dataset, particularly from other FoodNet sites where the information about disease severity (hospitalizations, death) is known would help shed light on the role of potential virulence genes.
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: GENOMIC CHARACTERIZATION AND PHYLOGENETIC ANALYSIS OF SALMONELLA ENTERICA SEROVAR JAVIANA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting is excellent.
Experimental design: The manuscript is well written and clear. The Introduction is well presented, highlights the relevant aspects of why the research was undertaken, and provides an appropriate, easy to read introduction. It highlights that S. Javiana is a highly-prevalent enteric food pathogen of concern, and discusses its potential as a carrier of antimicrobial resistance. The authors are correct in their assertion that SJ’s population structure is important to study.
	The Materials and Methods are well presented and logical, and they use well-respected bioinformatics tools in the appropriate contexts. On Line 170: I recommend that they make it clear that (ILLUMINACLIP:etc are parameters passed for Trimmomatic to use.
	The Results & Discussion are also clearly presented, and I found all of the Figure, Tables and Supplementary information appropriately tagged and in the appropriate places. The authors present a well supported case that 106 of the 111 SJ genomes that were sequenced fall into three discrete clades. If they were to suggest that and leave it there, I’d have accepted the manuscript pretty quickly without major revisions.
Validity of the findings: I have huge doubts that selecting, sequencing and analysing 110 strains from Tennessee, collected over 18 months, is sufficient to then declare that S. Javiana is polyphyletic. I suggest that the authors provide better context especially with regard to the worldwide distribution of SJ – which is readily available. I would like the authors to put their analyses in the context of many more genomes of SJ, including those found elsewhere in the US as well as those found internationally. These genomes are readily available.
	Furthermore, I’d like to see these analyses presented in the context of a global analysis of Salmonella genomes – most of which are in Enterobase, to see if the branch lengths of their proposed polyphyletic clades bear similar relationships to other polyphyletic serotypes of Salmonella e.g. Newport, Java, etc. Even a simple minimum spanning tree would be sufficient.
	Please perform those requested analyses, and add them to the manuscript, adjusting the conclusions to accommodate the enlarged analysis, OR, rewrite the manuscript removing the assertion that SJ is polyphyletic and replace it with the finding that it has three highly diverse clades.
Additional comments: Other than my above comments, it is a highly polished manuscript and a pleasure to read.


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: GENOMIC CHARACTERIZATION AND PHYLOGENETIC ANALYSIS OF SALMONELLA ENTERICA SEROVAR JAVIANA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: The authors have adequately addressed my concerns in the revision.
Validity of the findings: 1.	Lines 545-560: SNP distances in an epidemiologically verified outbreak that exceed > 25-30 SNPs typically indicate the presence of polyclonal contamination, i.e. multiple different strains causing an outbreak. An outbreak with >200 plus SNPs certainly is a polyclonal one and should be noted here if the authors wish to use this reference. Cluster detection thresholds really don’t apply to polyclonal outbreaks which are challenging to investigate no matter what typing method is used. A vast majority of the Salmonella outbreaks are within 5-20 SNP range, the upper range often seen in zoonotic outbreaks or long lasting outbreaks involving persistent environmental contamination. An article discussing outbreak ecology for enteric organisms is here: Besser, J.M., Carleton, H.A., Trees, E., Stroika, S.G., Hise, K., Wise, M., Gerner-Smidt, P. (2019) Interpretation of Whole-Genome Sequencing for Enteric Disease Surveillance and Outbreak Investigation. Foodborne Path. Dis. 16. Published Online:9 Jul 2019https://doi.org/10.1089/fpd.2019.2636
2.	Lines 574-581: also when put in the global context, the TN strains all belonged to the same eBurst group and clade. So while three clades appeared to be separating in the local analysis containing just the TN strains, from the global context the TN strains are monophyletic which may explain the equal performance of the different references.
Additional comments: 1.	Line 340: add “age’ after “an average”
2.	Line 511: “are common” instead of “is common”
3.	Line 617: “in all of the representative…”
4.	Line 658: clarify that the TN isolates belong to a single global lineage and single eBurst group


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 CRANIAL MORPHOLOGY OF TANYSTROPHEUS HYDROIDES (TANYSTROPHEIDAE, ARCHOSAUROMORPHA) AS REVEALED BY SYNCHROTRON MICROTOMOGRAPHY
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this important contribution, Spiekman et al. describe in exceptional detail the skull and anterior axial morphology of the recently proposed taxon Tanystropheus hydroides. The description is mainly based in 3D modelling of synchrotron tomographic data, and the authors did a superb job in segmenting the individual bones, assembling a reconstructed skull and producing a braincase endocast model. Although the MS has some weaknesses in the present version, the published paper will probably stand out as one of the best anatomical descriptions thus far provided for an early archosauromorph. The manuscript is written in plain and professional English throughout, but some minor language issues detected by me are highlighted in the attached PDF file (but please bear in mind that I’m not a native English speaker myself). The provided background is adequate and well-referenced.
Experimental design: This is an original research. Although a similar study was recently published by the authors (Spiekman et al. 2020), the main goal of the new MS is to provide a detailed anatomical description of T. hydroides, which they do well. The methods are described in sufficient detail and the research is meaningful and relevant, providing valuable information on the anatomy of an enigmatic group of extinct reptiles.
Validity of the findings: Apart from the excellent anatomical description (which is the core of the ms), my main concerns about the work of Spiekman et al. are related to the robustness of their discussion. The anatomical data are employed to corroborate hypotheses that flirt with speculation or that, even if true, lack sound evidence in the context of the present version of the manuscript. This is the case of the putative streptostyly and the argumentation surrounding the aquatic habits of Tanystropheus. Again, I’m not stating that the authors are not correct, but their argumentation may be weak in some points (see below), and would probably benefit from further discussion or different methodological approaches. Most of my suggestions and commentaries are highlighted in the pdf file I’m sending attached to this, but I’ll try to detail some important issues below:

-	Aquatic adaptations of Tanystropheus: The authors, albeit not being so explicit, appear to support the hypothesis of an aquatic Tanystropheus (echoing what was already sustained by Spiekman et al. 2020). Their case, however, is mainly based on the confluent dorsally-positioned nares and the ‘fish-trap’ dentition. Although present in several arguably aquatic animals, confluent/dorsal nares are not an aquatic adaptation per se and are also displayed, among early archosauromorphs, by the fully terrestrial rhynchosaurs. It is plain enough that Tanystropheus fed on marine animals, but its dentition, allied to the overspecialized neck and the overall conservative trunk and limb morphologies, appear to support the ‘shoreline fishing’ habit proposed by Renesto (2005), whereas make unlikely a prevalent aquatic habit for this animal. I should also note that, although lacking biomechanical support, the proposed habit of ‘positioning on the seafloor’ while approaching prey (Spiekman et al. 2020) seems unlikely, as the lack of pachyostosis allied to the lightly-built axial skeleton would hinder a neutral buoyancy to Tanystropheus. New evidence provided by Spiekman et al. (the submitted manuscript) also seem to indicate that Tanystropheus was poorly ‘built’ for a mainly aquatic lifestyle. This include the semicircular canals morphology (pag. 35) and the lack of evidence supporting suction feeding (pag. 36) or salt glands (pag. 37). Again, I’m not stating here that Tanystropheus wasn’t aquatic, and I don’t even have a personal opinion on this matter. I need to say, however that, if the authors choose to argue in favor of a mainly aquatic lifestyle, they should provide enough evidence supporting this hypothesis and discuss the wealth of evidence indicating otherwise (most are summarized by Renesto 2005).

-	Cranial kinesis in Tanystropheus: Spiekman et al. (pag. 36) propose streptostylic intracranial movements in Tanystropheus hydroides based on two osteological correlates, namely a supposedly synovial joint between the head of the quadrate and the squamosal (an otic joint sensu Holliday and Witmer 2008) and a permissive linkage between the quadrate itself and the pterygoid. Cranial kinesis is an uncommon trait among diapsids, and its proposition for extinct reptiles is conditioned to the detection of several musculoskeletal structures (far more than the two correlates proposed by the authors). I’ll quote Holliday and Witmer (2008): “We recognize minimally four criteria that are necessary for inferences of powered cranial kinesis in fossil (and extant) taxa: (1) a synovial basal (basipterygopterygoid) joint; (2) a synovial otic (quadratosquamosal) joint; (3) protractor musculature; and (4) permissive kinematic linkages. Clearly, intracranial synovial joints such as the otic and basal joints are necessary to allow kinetic movement, and protractor muscles would help drive the system. Additionally, the connections between bones (kinematic linkages) must permit and not obstruct movement” (pag. 1074). It is noteworthy that even living taxa presenting all those correlates may not display cranial movements in vivo and that basal and otic synovial joints are apparently plesiomorphic in Diapsida (Holliday and Witmer 2008). As such, the proposed streptostyly in Tanystropheus is far from convincing and I suggest the authors to verify whether more compelling evidence can be drawn from Tanystropheus skull or to simply abandon that hypothesis.

-	I have the feeling that the description, although exceptionally well-written, lacks comparisons with a broader sample of early archosauromorphs/archosauriforms. The comparisons are virtually restricted to timid commentaries about the condition observed in a few taxa, such as Macrocnemus, Prolacerta and Azendohsaurus. I’ve highlighted several passages where further comparisons would be welcome. However, bearing this in mind, the paper would benefit from a thorough revision of the comparative description.

-	I have alternative interpretations of some bones (nasals, postorbitals and postfrontals). All of them are explained in the annotated PDF. Of course, the authors are by far the best persons to judge whether I’m right or not, but I suggest to double check the issues I was able to detect.

-	The paper is stupendously well illustrated, but I still miss detailed photographs of the specimen. CT data can be misleading in the fine detection of sutures, and some structures (e.g. the dentition) would be better illustrated through photographs than through digital models.

-	Please provide a Systematic Paleontology section, including referred material, details on the horizon and locality, as well as an updated diagnosis (I’ve highlighted some potential autapomorphies on the annotated PDF). I know that a Systematic Paleontology was provided by Spiekman et al. (2020), but it is also of great relevance for the present submission.

-	As you have all those great 3D models, it would be nice to provide a table of measurements. That could be useful for future researchers! :¬)

My best wishes or the authors and congrats for the great job!

Felipe L. Pinheiro
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 CRANIAL MORPHOLOGY OF TANYSTROPHEUS HYDROIDES (TANYSTROPHEIDAE, ARCHOSAUROMORPHA) AS REVEALED BY SYNCHROTRON MICROTOMOGRAPHY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The text is clear and very written, with only very minor issues (eg.: line 54 - Tanystropheidae (the clade) is; line 241: the parietals are; line 663 - cannot BE confidently; line 750 - oriented). Also, in the legend of figure 5, change "dental" to "dentary".

The manuscript is structured in a reasonable manner, in which the data presented support very well the statements made. I would only suggest to move measurements of the skull in lines 90-93 to the description section

The figures are also well done and very helpful to better understand the text. My only suggestion is for authors to label some structures in figure 2, especially the frontals, which are mentioned in line 115. The authors must also be careful to call figures in the order of their appearance.

The authors must not forget to update the status of the Miadema et al. and Spiekman et al. references.
Experimental design: The manuscript presents very high quality data. CT scanning, segmentation, and reconstruction of the material are all above standard. The information yielded is very impressive for a mostly compressed material. The anatomical analysis was carried out carefully.
Validity of the findings: The findings significantly increase our knowledge on the anatomy of the enigmatic tanystropheids. It also provides hitherto unknown information on several structures such as the braincase. Both the reconstruction of the skull and the ecomorphological implications of the anatomy described are sufficiently supported.
Additional comments: Dear authors,

Congratulations on the manuscript detailing the cranial morphology of Tanystropheus. The manuscript is very well written and logically organised, with high quality images. The anatomical analysis is very sound and the statements are sufficiently discussed and supported.

I have only three minor suggestions to make.

Lines 279-290: Could this difference in fossa morphology be due to ontogeny? The fossa changes shape as the animals grow in many mammals, just as the sagittal crest becomes taller. Eg: Holbrook 2006 (https://zslpublications.onlinelibrary.wiley.com/doi/abs/10.1017/S0952836902000250).

Lines 634-635: Please re-phrase. Even though the openings have been incorrectly identified, the fenestra ovalis is still framed by opisthotic and prootic.

Lines 117-1180: The authors could include a comment on the seemingly contradictory findings published for the elongation of sauropod necks and reduction of their floccular fossa - Bronzati et al. 2017 (https://doi.org/10.1038/s41598-017-11737-5).


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: TRANSLOCATION OF AN ARCTIC SEASHORE PLANT REVEALS SIGNS OF MALADAPTATION TO ALTERED CLIMATIC CONDITIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript investigates the whether varieties of a perennial herb are locally adapted or exhibit signatures of maladapted due to changing climates. The experiment(s) presented here are designed well, the paper is well written, and the results are interesting, important and generally on point. The number of years that the experimental gardens were maintained was impressive and provide confidence in their conclusions both for the basic science and management aspects of the project. This is the clearest signal of adaptation lag that I have seen in the literature. However, I do have a number of concerns and comments that hopefully will clarify the experiment design, results and associated conclusions for the reader.
Experimental design: Reading through the supplemental methods describing the design of the experiment, I grew a bit concerned about whether this experiment actually measures local adaptation. The plants were given excess fertilizer, had limited contact with the native soil at each site, and were given water on a weekly basis to prevent die off from drought. It seems like what was really measured was how well each variety performed in the different temperature and herbivore regimes of each site. Even so, temperature may have a larger effect than reported as higher temperatures could facilitate more substantial droughts. I fully realize that all field common garden studies have these issues to a certain degree, but I suggest that more of the experimental design be reported in the main text and the issues I raise should be a part of the discussion.

I am a bit confused about why dead plants were replaced at the end of the 2013 growing season with mature plants and how these plants were taken into consideration in the analyses. Please provide more explanation (can be in the supplement if they were not included in any of the analyses). However, I think these were somehow included in the analysis (i.e. Line 260) despite that they did not go through the most severe bottleneck in the experiment (i.e. the first year).

As discussed in the methods, results and Figure S4, southern variety plants were larger at planting than northern variety plants. This size is included in the aster models as a highly significant covariate. However, I am not sure whether this covariate’s contribution to fitness is fully accounted for in the likelihood ratio significance tests within the aster model. In a standard ANOVA hypothesis testing framework, type III sum of squares is utilized to control for the variance due to the other factors and covariates; however, this is not necessarily true in the likelihood ratio framework used here. If you disagree, please provide some reference. I think it would be much more convincing to show that there is still a significant variety x site interaction in first year survival or survival/number of flowers using the residuals from a model examining the correlation between size at planting and survival.
Validity of the findings: The clearest issue with this study is the lack of a refresher generation between seed collection and the experimental gardens. The observed differences in fitness could partly or fully be due to differential maternal effects rather than maladaptation. Presumably the temperatures were higher during the collection year for the southern variety compared to the northern variety, and therefore the southern variety may have been more primed to perform in warmer climates – exactly the pattern shown. This is briefly mentioned in the discussion, but it should be brought up in the methods as a caveat and the implications should be better explained in the discussion. An additional point of interest that could be included in the discussion on priming (~Line 498) would be the conditions during the year where seed was collected. What was the mean annual temp that year for each site and how did it relate to the growing season conditions?

In the abstract (L39-41), the conclusion that both populations are maladapted to their home environment. I would respectfully disagree – the southern variety has higher relative fitness in their how environment, and nothing is said about absolute fitness (see point above). Just because a population has higher fitness in a different environment does not mean that it is maladapted. For instance, in Clausen, Keck and Heisey’s seminal work, their high elevation pops performed better at low sites than high sites, but were the only population to survive at high sites. You provide a similar definition on L82. If you want to define maladaptation by home:away criteria, it is should written out explicitly as this is not the norm.
Additional comments: A key property of an adaptation lag is that weather during the experiment is more closely matched to historical climate of the distant variety rather than the native population. The data here perfectly matches this expectation, and I think it would be nice to lay this prediction out with the other prediction of adaptation lag, possibly on Line 136-137 or in the discussion. A related comment, Line 234 has the results that correspond to this prediction, but are in the methods.

I think a management-oriented person may be interested in absolute levels of fitness in this study – that is, did either the southern or the northern variety produce enough offspring to replace themselves. I know seed data was not collected, but any citations or observations in the discussion would be useful information.

Line 123: My personal opinion: The equation here seems unnecessary and breaks the flow of the intro.

Line 150: Any idea how old these perennials can exist in natural populations? Does your design encompass the typically lifespan or the most important part of it? Not necessary to include if unknown, but the reader will be wondering it here.

Line 155: Don’t need the parenthetical phrase as these were defined in the paragraph above.

Line 195: surveyed weekly, monthly, once a year?

Line 208 (and below): please add versions and citations for each package

Line 367: models 40 and 60? Are these defined somewhere.

Line 431: Does the structure of this low level of genetic diversity suggest that there has been a bottleneck or founder event in either population that caused maladaptation? Or are there similarly low levels of diversity in each population?

Figure 1: hard to tell light gray from dark gray points. How about using shapes?

Figure 3: Pretty sure the labels are wrong here as these temps did not match Fig 2 and the most northern garden did not have the highest mean annual temperature. Figure S6 has the same problem.

Figure 5: what do error bars represent (95% CI?, standard error around mean?). Please include in the legend


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: TRANSLOCATION OF AN ARCTIC SEASHORE PLANT REVEALS SIGNS OF MALADAPTATION TO ALTERED CLIMATIC CONDITIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The introduction cites relevant literature, and generally provides the background necessary to understand the experiment.
The grammar is mostly good, but there are a few small errors. I note those that I caught in my minor comments.
The raw data is on github and the metadata is clear. The climate data is not included, as far as I could tell.
The figure captions are very clear and the figures are nice. A few may be unnecessary (see comments below).

1. The quotation of Kawecki and Ebert (line 82) seems misplaced, since it's a definition of a metric of local adaptation, but local adaptation has been discussed earlier in the paragraph too. Maybe provide more context for this quote, or use your own phrasing.
2. line 114: local adaptation is not mutually exclusive with insensitivity to temperature, as populations could be locally adapted to factors other than temperature.
3. line 137: this sentence is missing words, so its meaning is not clear. Regardless, it may be a good idea to be conservative about what you say you can disentangle, as a transplant with two populations will always have many confounding factors.
4. Fig 1c: I don't think you can technically assess local adaptation at the level of subspecies without a comparison to the other subspecies. This panel might be better described as niche limitation of subspecies, with no local adaptation.
5. Fig 2: I don't think you need to show the warmest quarter plot because you never discuss it in the text.
6. I think that Fig 3 could be moved to the supplement, and I don't think the warmest quarter plot is necessary because it is not discussed in the text.
7. Fig 5: The legend text is too small.
7. Fig S2 is unnecessary.
8. The contents of Figs S5 and S6 are not ever discussed in the text, perhaps consider omitting these figures.
9. I had to zoom in quite a bit to read the text on figures S4-S7, but the figures are high enough quality that they are legible once you zoom in.
10. The information in Table S3 would be more clear if it were presented in a plot.
11. The caption of Fig S7 says "raw data" but these are means (e.g. mean size) so that term isn't correct.
Experimental design: Understanding plant responses to climate change is an important current topic, and transplant experiments are a very powerful way of measuring potential responses. This paper contains multiple years of performance data from transplant gardens, and combines a reciprocal transplant with an over-the-edge transplant. These techniques are labor intensive and as a result, they are relatively rare, so this case study is a worthy contribution to the scientific literature.
The methods are generally appropriate, and described clearly with plenty of detail.
1. However, please give more information near line 293 on how these three metrics of local adaptation provide information about habitat quality, variety quality, and fit to environment. What can you learn from each metric of local adaptation that is not revealed by the others?
2. Some of the description of climate conditions in the methods (paragraph beginning 234) might be better placed in the results.
3. line 239: I'm not sure the far southern gardens historic means are relevant, as they were not experienced by plants during the experiment, nor are they the conditions these populations adapted to.
4. line 258: Did you consider trying a quadratic term for temperature? Is it possible that there might be an optimal temperature with declines above and below it? Or are you assuming a priori that all the experimental temperatures are above these optima, and that there will be linear relationships only?
6. line 306: Why did you choose not to include the beyond the edge gardens as "away" sites for both varieties? Explain in the text.
Validity of the findings: 1. Can you evaluate the relative importance of survival vs. flowering on average performance for these varieties? Which life-history stages pose the largest challenge?
2. In the methods, you state that you measured when plants flower. Does their phenology give you any more clues about how temperature is affecting performance?
3. Paragraph beginning line 419: One issue with this experiment is that with just two populations, it is hard to have confidence in the magnitude of the temperature effect relative to other garden effects and intrinsic qualities of the populations. For example, could the results not also be explained by a higher garden quality at Svanvik, and greater load in the northern variety, without an effect of temperature? I don't mean to say that temperature is implausible--I think it's likely important, but it's hard to disentangle its effects from other factors with this design. The two populations used appear to differ by about 1 degree in their historic normal MAT. Is this difference enough to explain the huge difference in performance between the two varieties shown in figure 5b, where the red population's performance does not fall as low as the blue population's maximum unless it experiences a temperature 5.5 degrees warmer than it's historic normal? If you re-made your temperature model, and instead of actual garden temperatures, used the deviation of garden conditions from each population's historic normal (i.e., temperature mismatch), do you still see the southern population outperforming the northern one? Figure 5b makes me think that you would.
4. Section on confounding effects: I think the biggest limitation of your study is that you do not really have replication within varieties. Even though you sample multiple sites for seed collection, your approach doesn't really treat these as independent replicates. Discuss how this limits you ability to isolate temperature from other factors that may differ between northern and southern habitats.
5. line 542: why are sites beyond the range necessary for consideration of maladaptation? couldn't maladaptation also be revealed by within-range transplants?
5. line 454: I don't think the far southern gardens historic means are relevant, as they were not experienced by plants during the experiment, nor are they the conditions these populations adapted to.
6. line 334: I think you mean Fig 4 not Fig 3.
7. line 345: "raw data" isn't appropriate here.
8. line 377: Fig 1a is the map, do you mean a different figure?
9. line 412: Do you mean local adaptation to historic temperatures?
Additional comments: 56: responseS
60: perhaps "tolerance of" rather than "plasticity towards"
99: maintenance instead of maintaining
104: what do you mean by "fixed" here?
136: translocatioN
225: with instead of among
Use of Fig., Fig, and Figure are inconsistent.
Check that figure and table references actually reference the correct figure/table.
Check formatting of references (i.e., sometimes full names are used in main text).


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: TRANSLOCATION OF AN ARCTIC SEASHORE PLANT REVEALS SIGNS OF MALADAPTATION TO ALTERED CLIMATIC CONDITIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Language is basically sound. On line 412 “evidenced” is used as a verb, which is not really appropriate. “shown” or “revealed” will do just fine.

The literature review omits much work on reciprocal transplants and local adaptation from North America, including the classic studies of Clausen, Keck, and Hiesey. Many of these studies have involved Arctic species such as that of Mooney and Billings(1961), McGraw on Dryas octopetala, Chapin on Carex aquatilis, and Bjorkman et al. (2016). The authors should also be aware of our studies of Eriophorum vaginatum in a reciprocal transplant experiment, for which I list some references below. The starred references are particularly relevant

Clausen, J., Keck, D.D., and Hiesey, W.M. 1948. Experimental studies on the nature of species. III. Environmental responses of climatic races of Achillea. Carnegie Institute of Washington, Washington, DC.
Mooney, H.A., and Billings, W.D. 1961. Comparative physiological ecology of arctic and alpine populations of Oxyria digyna. Ecol. Monogr. 31: 1–29.
Bjorkman, A.D., Vellend, M., Frei, E.R., and Henry, G.H.R. 2016. Climate adaptation is not enough: warming does not facilitate success of southern tundra plant populations in the high Arctic. Glob. Chang. Biol. 23: 1540–1551. doi:10.1111/gcb.13417.
Souther, S., and McGraw, J.B. 2011. Evidence of Local Adaptation in the Demographic Response of American Ginseng to Interannual Temperature Variation. Conserv. Biol. 25: 922–931. doi:10.1111/j.1523-1739.2011.01695.x.
McGraw, J.B., and Antonovics, J. 1983. Experimental ecology of Dryas octapetala ecotypes: II. A demographic model of growth, branching, and fecundity. J. Ecol. 71: 899–912. Also see other papers in this series.
Bennington, C.C., and McGraw, J.B. 1995. Natural selection and ecotypic differentiation in Impatiens pallida. Ecol. Monogr. 65: 303–323.
Chapin III, F.S., and Chapin, M.C. 1981. Ecotypic differentiation of growth processes in Carex aquatilis along latitudinal and local gradients. Ecology 62: 1000–1009.
Curasi, S.R., Parker, T. C., Rocha, A.V., Moody, M.L., J. Tang, and N. Fetcher. 2019. Differential responses of ecotypes to climate in a ubiquitous arctic sedge: implications for future ecosystem C cycling. New Phytologist 223: 180-192.
*Schedlbauer, J. L., N. Fetcher, K. Hood, M. L. Moody, and J. Tang. 2018. Effect of growth temperature on photosynthetic capacity and respiration in three ecotypes of Eriophorum vaginatum. Ecology and Evolution 8: 3711-3725 doi: 10.1002/ece3.3939
Parker, T. C., J. Tang, M. B. Clark, M. M. Moody, and N. Fetcher. 2017. Ecotypic differences in the phenology of the tundra species Eriophorum vaginatum reflect sites of origin. Ecology and Evolution 7: 9775-9786. doi: 10.1002/ece3.3445
Chandler, J. L., J. B. McGraw, C. Bennington, G. R. Shaver, M. C. Vavrek, and N. Fetcher. 2015. Tiller population dynamics of reciprocally transplanted Eriophorum vaginatum L. ecotypes in a changing climate. Population Ecology 57:117-126. DOI 10.1007/s10144-014-0459-9
*McGraw, J. B., J. B. Turner, S. Souther, C. C. Bennington, M. C. Vavrek, G. R.Shaver, and N. Fetcher. 2015. Northward displacement of optimal climate conditions for ecotypes of Eriophorum vaginatum L. across a latitudinal gradient in Alaska.  Global Change Biology 21: 3827–3835. doi: 10.1111/gcb.12991
Souther, S., N. Fetcher, Z. Fowler, G. R. Shaver, and J. B. McGraw. 2014. Ecotypic differentiation in photosynthesis and growth of Eriophorum vaginatum along a latitudinal gradient in the Arctic tundra. Botany 92: 551–561 dx.doi.org/10.1139/cjb-2013-0320
*Bennington, C. C., N. Fetcher, M. C. Vavrek, G. R. Shaver, K. J. Cummings, and J. B. McGraw. 2012. Home site advantage in two long-lived arctic plant species: results from two 30-year reciprocal transplant studies. Journal of Ecology 100: 841-851. doi: 10.1111/j.1365-2745.2012.01984.x
Fetcher, N., and G. R. Shaver.  1990. Environmental sensitivity of ecotypes as a potential influence on primary productivity.  American Naturalist 136:126-131.
Shaver, G. R., N. Fetcher, and F. S. Chapin III. 1986. Growth and flowering in Eriophorum vaginatum: Annual and latitudinal variation.  Ecology 67:1524-1535.

    That said, the ms. contains many useful references of which I was unaware, so I hope this has been a helpful exchange of information.

Figures 3 and S5.
Is the legend reversed? It seems that Svanvik should be cold and Tartu should be the warmest site.

Raw data are available. Permits are provided.
Experimental design: The study is a welcome addition to our understanding of plant responses to climate change. Given the wide distribution of Primula nutans, it would be interesting to see if it could be replicated elsewhere in the circumpolar region.

The study was very carefully done, and the analyses seem to be appropriate. I am somewhat concerned about the use of flower number as a component of fitness. Ideally, seed production would be the best measure of fitness, but it was not available in this experiment. Is there any evidence for a correlation between seed production and number of flowers? One can easily envision a scenario where a plant produces many flowers, but they don’t become pollinated or the seeds abort. Perhaps the aster model should be developed for the survival and flowered or not performance measures only and the results compared to those for the complete model.
Validity of the findings: Discussion is appropriate in relation to the issues raised in the introduction. It could be improved by reference to the literature mentioned above as well as other studies. In particular, I would be interested to know whether other studies have reported such a dramatic improvement for southern varieties when moved north. Perhaps more space could be devoted to this topic and less to the confounding effects.
Additional comments: There is no need to cite all of the references listed above. I included them to make the authors more aware of some of the work that has been done elsewhere.


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: TRANSLOCATION OF AN ARCTIC SEASHORE PLANT REVEALS SIGNS OF MALADAPTATION TO ALTERED CLIMATIC CONDITIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have a done an excellent job addressing my previous concerns (I was reviewer 1 for the previous draft). I have a few general comments below that reflect small edits and/or additions.
Experimental design: The authors have added sufficient detail to the methods to meet my previous concerns and do a good job of justifying the decisions that they made.
Validity of the findings: The results justify the conclusions and appropriate caveats are described in the results.
Additional comments: Line 205: I was a bit confused by the wording here - the photoperiod was set to 12hr days?

Line 221 & 223: Probably don't need the italics here. I would save it for your variables below (personal opinion)

Line 414: I am not sure what you mean by "The northern variety, however, did not experience historical temperature conditions in any of the experimental sites". Does this mean that the northern variety has experienced hotter temperatures in its native range  at some point in the past than it experienced in the most southern site? The subsequent comparison with the coldest site makes it confusing.

Line 455: I think it would be useful to have a sentence here that says that the southern plants were no larger at planting than the northern plants. This statement was it the response to reviewers, but would be useful in the manuscript.

Line 490: I don’t actually think your results are in complete concordance with the previous photoperiod study. Your results show that plants that did survive in the southern gardens were able to flower (thus not necessarily needing the cold nights of the northern garden). Making a more nuanced statement here would be useful.

Line 539: I think this section was a useful addition. I also think that it could benefit from one more sentence that provides an explanation for what maladaptation due to drift or stochastic processes would like spatially (i.e. the degree of maladaptation would be random with respect to geographic locality).

Reviewer: Nic Kooyers


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: TRANSLOCATION OF AN ARCTIC SEASHORE PLANT REVEALS SIGNS OF MALADAPTATION TO ALTERED CLIMATIC CONDITIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have responded to reviewers' suggestions and edited the manuscript accordingly. I think that overall the manuscript has improved and is a better presentation of a careful and laborious experiment.

There are still some grammatical and spelling errors that could be corrected, particularly in the revised sections.

The authors should also check their references carefully, e.g., I couldn't find Yien et al. 2019 (line 311 in tracked changes version) in the references list. I didn't check other references, I just happened to notice that one because it sounds interesting and I wanted to look it up!
Experimental design: The methods section is clear and well written.

The aims of the study and the distinct value of within-range vs. beyond-range transplants are highlighted by edits the authors made to the introduction.

I have one follow up from a comment on the previous version (Reviewer 2, comment 2.20). I really appreciate the figure you provided in the response letter plotting performance against temperature deviation from the varieties' historic normals. This figure clearly shows that to an extent, the poor performance of the northern variety is not only explained by temperature (if it were, you would expect both to overlap once temperature deviations were accounted for). This could be stronger genetic load in the northern variety due to drift or expansion, or garden conditions that for some unknown reason favored the southern variety. But very importantly, temperature conditions during the experiment do not fully account for the performance differences between varieties. I think this deserves a mention in the discussion and the figure should perhaps be added to the supplementary materials.
Validity of the findings: I take some issue with the statement "As far as we are aware, this represents the strongest example of climate change induced maladaptation." (lines 823-824) I think you mean that the distance between the southern source and the test site where it performed best is the furthest, but intermediate distances to the north were not sampled, so you don't really know that the Svanvik site is the optimum for that population, it's just the best among those you chose. There are some really excellent, more highly replicated studies of adaptation lag at similar geographic scales (Wilczek et al. 2014, Bontrager and Angert 2019) , so consider being more specific about what aspect of yours is the strongest, should you decide that it's necessary to make that claim at all.
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: TRANSLOCATION OF AN ARCTIC SEASHORE PLANT REVEALS SIGNS OF MALADAPTATION TO ALTERED CLIMATIC CONDITIONS
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: On reading the comments about the water table at the collection sites and visiting some of them via Google Earth, I was struck by the possibility that the plants could be adapted to soils with higher salt content than that encountered in the experimental gardens. Could this have had any effect on the results?
Additional comments: McGraw et al 2015 use the phrase "adaptational lag" instead of adaptation lag.  This phrase is also used in  Aitken SN, Yeaman S, Holliday JA, Wang T, Curtis-McLane S (2008) Adaptation, migration or extirpation: climate change outcomes for tree populations. Evolutionary Applications, 1(1):95–111. https://doi.org/10.1111/j.1752-4571.2007.00013.xI, which is cited in McGraw et al. I suggest that the authors continue to use "adaptational lag" to avoid introducing multiple terms for the same phenomenon.


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: GENOME-WIDE IDENTIFICATION AND ANALYSIS OF THE THIOLASE FAMILY IN INSECTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This research focused on thiolase family in insects. Thiolases plays important roles in lipid metabolism but it is poorly understood in insects. This study identifies the genes and provides key information for the future function analysis. The manuscript is generally written well. I have some comments as following:
1、Line 164, it is better to mention somewhere genome quality potentially influences the results, like the number of genes identified and the deduced evolutionary process.
2、Line 178, the author can specify which group have no intron.
3、Line 181, this part reads like introns can help genes respond to enviromental challenges, which is contradicting to the first part of the sentence.
4、Line 182-183, the findings/results cannot support this discussion/opinion yet.
5、Line 204, reference.
6、Line 243,  the author should list protein identifies between target protein and human template for all proteins. But the author only give the information for TFE and SCP2.


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: GENOME-WIDE IDENTIFICATION AND ANALYSIS OF THE THIOLASE FAMILY IN INSECTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The author used clear and unambiguous English, but there are several simple grammatical and typographical errors to be corrected before publication, which are listed below.

Lines 21, 311: “. While, ” => “, while ”
Line 31: Italicize species name.
Line 44: Fig S1A => Fig. S1A
Line 62: belonged => belong
Lines 124–125: Use singular form after “every “
Line 247: We => we
Line 267: contained => containing
Line 273: “Therefore ” => “Therefore, ”
Line 338: delined => declined

There are also several texts and words which should be improved for clarity and better understanding.
Line 90: “gene models” may be “genes”?
Lines 154–155: The sentence, “In total, …”, should be improved into a clearly understandable text.
Line 222: “Except for” here may confuse readers. It may be better to use “In addition to” instead.
Line 224: “seem to be contained” => “contained”
Line 227: largely => much
Line 269: “SCPx by proteolytic cleavage via peroxisomal” => “SCPx via proteolytic cleavage by peroxisomal”
Line 350: and => or

Some figures and table seem to be in insufficient quality:
Fig. 1: The visibility of dots on the nodes is poor.
Figs. 3B and S2A: Scale bars? cannot be read and look strange.
Fig. 5: The resolution of some figures seems to be low. Numbers with dotted surfaces are shown, but hard to recognize.
Fig. 6: (A) The change of the color for expression <400 into white is recommended for clarity. (B) and (C) The legends seem to be wrong texts.
Table 1: Double-byte spaces appear in Iso_Z. nevadensis line and should be removed.

Titles of supplemental figures are missing and should be placed. In addition, Figs S2 and S3 may also need legends for better understanding.
Experimental design: There are several concerns about details of the methods listed below.

Line 91: The URL of SilkDB (http://silkworm.genomics.org.cn) cannot be accessed.
Line 101, 153: The detail of the known sequences should be mentioned, probably by using Table S2.
Line 102: The condition of BLASTP search should be written.
Lines 108–109: The detail of how did the author validate “The validated thiolases” should be clearly written.
Line 123: The author should specify the species used as “three model species” here or in the result texts and explain why selected them.
Line 135: The author should specify the kind of “similarity”. Is it sequence similarity?
Lines 137–138: “Using BmorT2 as the reference…”. I cannot understand where this was used.
Lines 209–210: The method for “In this study, subcellular localization of all the 137 insect thiolase proteins was predicted” was not written.
Validity of the findings: Several sentences in the conclusion seem to be exaggerated or over generalized and should be improved.
Line 349: “randomly and unevenly distributed” has weak evidence derived from small number of samples.
Lines 350–351, 354–355: The results are based on prediction.
Lines 353–354: “3D structures were highly conserved between insects and other vertebrates” was not clearly shown in general.
Additional comments: The author identified thiolase genes in 20 insects and analyzed their features about phylogeny, genes, 3D-structures, expressions of proteins, etc.

Major concerns:
* The analyses seem to be well done, but some of them were not comprehensively done: for example, chromosome distribution and conserved domain analysis.

* The manuscript needs more discussion “based on the results”, especially for the expression profile parts and should be largely improved.

* Some results are discussed only in insects thiolases (e.g., Lines 190–194, 254–255). The author should evaluate the results by comparing them with the known thiolases of the other organisms. Whether the results are insects-specific or not is important for better understanding the thiolases of insects.

* Fragment sequences like only N-terminal parts are treated same as whole genes. Those sequences may be pseudogenes and should be separately treated. In addition, as the author also mentioned in the text, some of the full-length genes lack the catalytic residues important for thiolases and may lose their functions as thiolases. Those incomplete genes should be distinguished with likely genuine genes (e.g., in Table 1).

Minor concerns:
Lines 185–187: I couldn’t understand why the sentence, “It seems that ...”, was derived. Maybe, more explanation is needed here.
Line 251: The values of sequence similarity are better to be shown. This sentence may be better to appear in line 246.
Line 255: The values of 3D structural similarity are better to be shown.
Lines 258–260: “However, …” The meaning of this sentence is hard to understand for me. Maybe more explanation is needed.
Line 264: How did the author judge “this type of thiolase is the most divergent”?
Lines 271–276: The order of sentences may be better to be reordered: known knowledge as first.
Lines 324: “relatively high enriched” seems to be not appropriate phrase for this.
Lines 325–326: “It further supports that insect T2-thiolase genes may be involved in the JH synthesis pathway.” Generalization as “insect T2-thiolase genes“ seems to be unreasonable.


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: GENOME-WIDE IDENTIFICATION AND ANALYSIS OF THE THIOLASE FAMILY IN INSECTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this manuscript, the author (Shou-Min Fang) identified thiolase genes in 20 representative insect genomes using bioinformatics analysis. Insect thiolases were mainly classified into five classes (CT-thiolase, T1-thiolase, T2-thiolase, TFE-thiolase, and SCP2-thiolase). Their intron numbers and exon/intron structures, and predicted subcellular localization are variable.  Homology modeling indicated that insect thiolases share similar 3D structures with mammals and other vertebrates. Transcription analysis suggested that, in B. mori, some thiolases may be involved in steroid metabolism, juvenile hormone and sex pheromone biosynthesis. In general, the manuscript is well written and organized.
Experimental design: The manuscript is pretty well organized and the experiment is carefully designed. Identification of thiolase genes in sequenced insect genomes provide a useful resource for further functional studies.
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: GENOME-WIDE IDENTIFICATION AND ANALYSIS OF THE THIOLASE FAMILY IN INSECTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The author has addressed my concerns well, and I have no further 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: GENOME-WIDE IDENTIFICATION AND ANALYSIS OF THE THIOLASE FAMILY IN INSECTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The author has corrected most of grammatical and typographical errors, and problems of figures and tables, I previously commented, but there are still some are left to be corrected and new ones appeared:

Line 108: have => “that have” or “having”; Thionlase => Thiolase; recogized => recognized
Line 124: “, which” => because
Line 126: genes => gene
Line 236: “Unexceptly” may be “Unexpectedly”?
Line 258: higher => high
Line 304: “head and hemocyte” => “hemocyte and head”
Lines 304–305: “. While, ” => “, while ”
Line 338: “was suggested” => “suggested”
Line 344: “Spodopera littoralis” => “Spodoptera littoralis”
Line 355: higher => high
Line 390: suggests => suggested
The title of Table S2 in the word file is Table S1.
Experimental design: The author has adequately addressed all my comments.
Validity of the findings: The author has adequately addressed all my comments.
Additional comments: The author has addressed most of my comments. However, there are still some concerns that need to be resolved.

Major concerns:
* The author should use all the insects thiolases in the conserved sequence block analysis. In the present manuscript, only 4 of 20 insects were used to derive the conclusion for insects thiolases, which is unreasonable.

* The author seems to regard BmorT2-thiolase as involving JH synthesis pathway only based on weak estimation from gene expression and it still needs further experimental validations similar to those for HarmT2-thiolase by (Zhang et al. 2017).
** Lines 360–361: “Thus, insect T2-thiolases may be involved in the JH synthesis pathway.”: I think it is unreasonable to generalize the JH synthesis about insects from only an example in H. armigera and an estimation in B. mori.
** Lines 365–366: “except for T2-thiolase” is not appropriate, I think.

Minor concerns:
Lines 266–270: Why did the author use only BmorT2 as the reference to calculate the RMSD values here? It is not enough result to support the description: “It was indicated that T1, T2, CT, and AB classes share more similar 3D structures than TFE-thiolase and SCP2-thiolase (type-1)”.

Line 270: “This phenomenon is widespread in both humans and M. tuberculosis” needs references.

Lines 270–274: The sentences, “Generally, T1-, T2-, ... biological unit (Xia et al. 2019)”, seem to mostly describe the general fact and are not appropriate in the Results section. The author should better describe what was derived from the results of Figs. 5G and 5H, or remove this part.

Lines 303, 307: “In total, 5 out of the 6 thiolase genes presented expression evidence.”, “Four thiolase genes presented expression signals in the silkworm PGs.”: Figs. 6B and 6C do not show the results of all the 6 thiolase genes. The author should show all the results in the figure.

Lines 337–339: “It” in the sentence, “It presented expressions ...”, indicates SCPx gene, but Fig.6B shows the expression result of SCP2 thiolase gene. It is better to fill the gap between SCPx gene, SCP2 thiolase gene/protein, and SCP2 protein just before here for easy understanding.

Lines 348–350: The two sentences may be better to be combined into one sentence.

Lines 376–377: The author wrote that “Simultaneously, BmorTFE and BmorT2 showed similar expression patterns as BmorSCP2 (type-1).”, but BmorTFE and BmorT2 seem to show different expression patterns from BmorSCP2 (type-1) in Fig. 6C for me.

Lines 31–33, 390–391: “may be involved” or “might be involved” is appropriate instead of “were involved” because of the weak evidences.


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: GENOME-WIDE IDENTIFICATION AND ANALYSIS OF THE THIOLASE FAMILY IN INSECTS
Review round: 3
Reviewer: 1

Output:
Basic reporting: There are two new errors appeared in this revision:

Line 249: “in in”=> “in”
Line 272: structrue => structure
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The author has addressed all my comments.
Below is just a recommendation for easy understanding.

The omission of data with no signals in Figs. 6B and 6C is better to be explicitly written in the main manuscript and/or the figure legend.


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: RESPONSE OF BOLIVIAN GRAY TITI MONKEYS (PLECTUROCEBUS DONACOPHILUS) TO AN ANTHROPOGENIC NOISE GRADIENT: BEHAVIORAL AND HORMONAL CORRELATES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors have done an excellent job with the writing, literature review, and presentation of data.
Experimental design: The researchers have provided a very strong descriptive basis for their study (including identification of individual animals; mapping of home ranges; and measurements of noise pollution), but the experimental (mannequin) and physiological (cortisol) outcome measures are hindered by small samples and too many confounding issues.
Validity of the findings: My main concerns are with the cortisol data.  There are a number of factors which may have contributed to the lack of findings here.

1)	Time of day.  Cortisol varies systematically with time of day.  I see no mention of what time of day the samples were collected or any examination of its effects.  The possible circadian effects are mentioned in the discussion, why aren’t we given more information on the actual samples? A previous reviewer also asked about time since exposure to the mannequin – this information is also possibly pertinent, and missing from the manuscript.
2)	Potential pregnancy.  Cortisol is higher in pregnant females.  Since three groups had weaned infants, what was the possibility that the female in each of these groups was pregnant?  If the time of year makes this unlikely, fine.  But if the assay was not detecting pregnant females, then it adds further doubt to how well it was working.
3)	Low levels.  It is very difficult to evaluate why the measured cortisol levels are so low here, but it is worrying and the authors don’t really have an explanation.
4)	The validation is also puzzling.  Essentially, they did not validate for parallelism, and the 4 samples that they were using for the validation had “identical” values.   This seems odd and unlikely.
5)	The authors themselves point out the numerous confounds that may have existed and that were not incorporated into their analyses.

Altogether, I expect that the cortisol measures are just too messy to be reliable, or that something happened to affect the samples.

I also wondered to what extent the authors are trying to assert that the titis viewed the mannequin as a human – which seems to be what they’re suggesting in lines 117-119.   How do you know that they weren’t just reacting to a giant novel object?  How do these reactions compare to the reactions to an actual human?
Additional comments: Line 401 and Table 3: Should be “Tail-twining” not “tail-twinning”


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: RESPONSE OF BOLIVIAN GRAY TITI MONKEYS (PLECTUROCEBUS DONACOPHILUS) TO AN ANTHROPOGENIC NOISE GRADIENT: BEHAVIORAL AND HORMONAL CORRELATES
Review round: 1
Reviewer: 2

Output:
Basic reporting: L36: Where are the predictions for the effect of noise on group movement and behaviour?
L53: This sentence is too long, try to break it up a little so its easier to follow.
L58: I’m not sure that light and noise pollution comes under the bracket of human-wildlife conflict. A human-wildlife conflict normally occurs when an animal poses a threat to humans leading to persecution of the animal and a conflict about the best way to resolve the issue. I would remove this sentence to think of an alternative framework that this paper fits within.
L66: “which is considered as one of the most pervasive acoustic perturbations on Earth” - what specifically is considered this? The layout of this sentence is slightly confusing.
L79: I don’t think that increased vigilance is a way to stay away from the source of stress - maybe rephrase that
L89: “field studies of Neotropical primates are only beginning to emerge in few taxa” - is this in general or in terms of noise pollution? You actually cite a lot of papers on this topic so can you argue that this is an emerging research topic?
L101: I’m not sure this sentence on edge effects is really relevant here.
L117: “In addition, to assess whether the monkeys” - should have an apostrophe after monkeys.
L140: Maybe provide more description of how you subjectively subdivided the study area.
L190: You could specify that you have three recorders at the highway to clarify how you have 17 recording sites.
L206: The RMS index is very well explained and would be clear to non-experts what this index represents and how you would interpret that data. I think it would be good to give a similar explanation of the M index.
Line 229: Not sure about the use of the word “his”.
L542: Sentence beginning “Our findings concur with previous studies of black-fronted titis” - this sentence doesn’t really add anything or explain what finding your study supports. You state that call B is produced in a variety of situations but fail to give any examples.
L565: Should say “We think it is unlikely”.

Figure 1A: I find it difficult to see where the park is located on the map. Maybe zoom out slightly and include an outline of the park.
Figure 2A, B and C: Why are these graphs reported for the RMS index and not the M index?
L320: What do you mean by extreme values? Maybe explain a little more why you are including certain things in your model.
Experimental design: L171: Why did you orient the sound level meter towards the highway and towards Campo Verde?
L172: How did you sample all of the stations on a grid 5 times a day? Did you have 9 people?
L257: How did you decide on the 1m and 1.5m to 7m cut-offs for loose and close associations?
For the statistical analysis, I would recommend mixed models with individual values instead of averaging across all of your variables e.g. averaging across cortisol values and noise values.

You should describe the different call types in the methods, how you distinguish them and what they represent.
Validity of the findings: Could there be an effect of habituation by the observer in the more remote groups?

Could the structure of the forest or physical disturbance explain these patterns instead of noise pollution?

L576: I would round this paragraph off by referring back to your study. What does your research have to contribute to this conversation?
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: RESPONSE OF BOLIVIAN GRAY TITI MONKEYS (PLECTUROCEBUS DONACOPHILUS) TO AN ANTHROPOGENIC NOISE GRADIENT: BEHAVIORAL AND HORMONAL CORRELATES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors did a good job improving the paper but it still needs some adjustments. I made some suggestions on how to improve the abstract and the discussion directly in the word file.
Experimental design: No problems regarding the experimental design.
Validity of the findings: Conclusions are well stated, linked to original research question.
Additional comments: The authors did a good job improving the paper but it still needs some adjustments. I have some suggestions on how to improve the abstract and the discussion and you can find them directly in the word file.


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: RESPONSE OF BOLIVIAN GRAY TITI MONKEYS (PLECTUROCEBUS DONACOPHILUS) TO AN ANTHROPOGENIC NOISE GRADIENT: BEHAVIORAL AND HORMONAL CORRELATES
Review round: 2
Reviewer: 1

Output:
Basic reporting: All good here
Experimental design: Experimental design is solid
Validity of the findings: I still have some concerns over the fecal cortisol analysis; however, the authors do a good job in the discussion of talking about the limitations.
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: FREEZE-LIKE RESPONSES TO PAIN IN HUMANS AND ITS MODULATION BY SOCIAL CONTEXT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written and mostly easy to follow. There are some minor comments in the general comments section to assist with the readability. The main focus is on the reporting of the participant numbers and ensuring this is accurately described considering the high drop out number.
Experimental design: The experiment is well considered, the question is well articulated and matches with the methods, reporting and discussion.
Validity of the findings: Negative results are well accepted. There are some minor comments below regarding the interpretation and not overstating the findings.
Additional comments: Methods
1.	Please explain why a medium effect size was used in the power calculation.
2.	Line 139 make clear that the electrocutaneous stimulus was designed to be painful
3.	Line 149 rephrase “receive stimuli of maximally this amplitude” to explain more clearly the effect of the amplitude on what was experienced by the participants.
4.	Line 165 refers to table 1 and I was expected to see the preparation and calibration phrase included in the table. Please rephrase to explain more clearly that table 1 only refers to the acquisition and generalisation phase. Perhaps make clear the distinction between the preparation and calibration and that the experimental phase contains the acquisition and generalisation phase and this is how it is explained further down in the manuscript.
5.	Line 168 the phrase absence of any findings indicates that no data was gathered, rephrase to explain more clearly that there was no data was not meaningful due to the high drop out rate. Very good that it is still reported in the supplementary material.
6.	Line 183 refers to the task instructions please include these as a supplementary material or indicate that the instructions are available from the authors for replication.
7.	Line 186 experimental phase: could the authors consider a more clear table or figure to visually guide the reader through the experiment. Table 1 attempts to do this however I feel it could be more clearly explained.
8.	Line 189 it was not clear how it was decided which trials would have the auditory cue and of those trials with the auditory cue which would have the electrocutaneous stimulus.
9.	Line 264 the reference to table 1 is confusing here, as to how table 1 would help the reader understand the heart rate recordings.
10.	Statistical analysis: : Line 272 when describing the ANOVAs please explain the block references i.e. A1, A2, A3 and what these refer to. The references to A1, A2 and A3 are not included previously in the manuscript and it is not clear to the reader.
Results
11.	Line 287 with regards to the participants who did not finish the experiment, the methods section states that 39 volunteered and that 7 participants stopped their participation and that 36 completed the full experiment. Please clarify and alter the text to explain how 36 participants still complete the full experiment when 7 dropped out from a starting number of 39.
12.	In addition to clarify the point above, given the high dropout rate 7/39 (18% dropout) please describe the characteristics of the participants who dropped out and if they differed to the participants who stayed in on any of the outcome measures that were assessed. i.e. anxiety, gender.
13.	Please also clarify the number of participants who were included in the final analysis. The numbers in the results indicate that 39 participants were included.
14.	Line 294 with regards to reporting of the t tests and interpretation of the p values, please define in the methods section how you planned to interpret the results. You describe a “slight trend” regarding valence, and that for control you describe a significant decrease. The reader would assume that you set the significance level at 0.5 but please make this explicit. In addition, to reporting the p values please also include the mean difference of the pre and post scores where possible, unless the scientific editor please this is not necessary.
15.	Line 339 it is stated that a planned comparison was used, however there is no further explanation for this. As a reader I was unclear of the reference that was used and why this was included. In the referenced article (27) were similar comparisons done? Please explain this.
16.	For Hypothesis 2 in the results section please include how many participants were included in this analysis for the low and high anxiety groups.
17.	Furthermore for hypothesis 2 with the reporting I feel it would be important to include the mean between group difference and if the large SD are not explained further on they I would ask that the large SD are explained somewhere in the text. It is hard to appreciate that difference in the between groups scores when the SD are so large.
18.	Regarding figure 2 and 3, I appreciate that this is a common graph for this style of research however I would encourage the authors in the future to consider presenting the results in graphical form in such a way that allows the lower SD to also be presented and not just the upper level. Given the results in this paper I do not feel it is necessary to change the graphs but would encourage this be considered for future manuscripts.
19.	Hypothesis three- my comments above regarding reporting the between group means and SD is also appropriate for hypothesis 3.
Discussion
20.	Line 375 between “relate” and “the” it seems a word is missing
21.	Nice interpretation of the results, link to previous research and possible explanations for the results.
22.	Line 395 I would like the authors to consider rephrasing the term partial support. The description in the next line that the effect was specific would be a more accurate way to begin this paragraph and ensure the reader is not mislead.
23.	I applaud the authors for their interpretation of the results in relation to hypothesis 2 in particular line 403 to 405
24.	Would the authors please provide a possible explanation for the overall decline in heart rate across blocks for highly anxious individuals.
25.	Line 417 replace colon with full stop.
26.	Line 426- the extrapolation to clinical relevance is a large jump. I would prefer the authors to indicate that the findings “may” be clinically relevant not that the finding is clinically relevant.
27.	Line 454 states that all participants were female however in the participants section it states that of the 39 participants 31 were female. Please correct this.
28.	I would be interested for the authors to present their views on where this line of research could go in the future and what questions future studies should evaluate based on the findings of this research.
Conclusion:
29.	Line 463 I would ask the authors to tone down the expression that bradycardia was especially pronounced and while you then state this was only in the anxious participants I still feel this overstates the findings. Please reword this statement.
30 I would encourage the authors in the future to predefine their analysis plan using an open access repository to make it clear what was decided prior to the analysis being conducted.


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: FREEZE-LIKE RESPONSES TO PAIN IN HUMANS AND ITS MODULATION BY SOCIAL CONTEXT
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	Your introduction needs more detail to provide a better justification for your project. I suggest that you improve the following elements:

• Which behavior changes are correlated with the transition from acute to chronic pain? From a psychological view, what are the differences between acute and chronic pain?
•More explanation on how a maladaptive responses can be linked to the social context should be added this section.
•Providing more quantitative comparison of the chronic pain prevalence and severity with adults living in different social environment would be relevant.
•The introduction would be strengthened by adding brief mention of other freezing indicators (Respiration, increased muscle tonus, reduced vocalization) and their related physiological explanations.

2.	Figures may not reproduce well. I recommend that the authors darken the colors and thicken X and Y axes
Experimental design: 1. A clear link between the proposed objectives, expected outcomes, and justification for the study have not been well established. It appears that the main thrust for the study is that no one has investigated these variables in patient with pain. It is not clear how these expected results, by using an electrocutaneous-induced acute pain model on healthy individual, will help inform future pain management protocols nor how the social context plays roles in chronic pain development and maintenance. Please explain why you used this study population and not chronic pain population.

2. Aims (Hypothesis) 1 and 2 propose to examine the impacts of threatening social context on specific outcomes in relation to anxious level. However, anxiety level was not an eligibility criteria. Did you have de same number of low anxious participants than the high anxious participants?

3. Since physical activity levels, sleep hygiene, energetic drinks have effects on postural stability, I would recommend to record this data of each participant.

4.The sample size calculation show adequate power, but I was unable to find the standard deviation of which parameter was using in this calculation.

5. Your stabilimetric force platform is not the most appropriate tool for obtaining COP derivation parameters (ellipse area of COP and the mean COP velocity in the anteroposterior (A/P) and mediolateral (M/L) directions). Only these COP parameters are sensitive and reliable postural control measures as published by Oliveira et al. 2019 (M.R. Oliveira et al. / Journal of Bodywork & Movement Therapies 23 (2019) 594e597). Please explain why you used this method. What are the psychometric properties of your measurement tool?

6. Please provide a complete electrocutaneous stimulation parameters as published by Geng et al (Geng et al. Journal of NeuroEngineering and Rehabilitation 2011).

7. Please add an explanation and references on your auditory cue parameters.

8. The social context method needs more details. Please explain how bias and variance were calculated. Why did you choose this method? Was it the most appropriate method for this issue (Virtual environments, dynamic or static face stimuli, photo-realistic faces, videos)?
Validity of the findings: 1. How many participants were included in the groups «low anxious participant»  and «high anxious participant»? Please explain de validity of the statistical analysis.

2. What were the sample characteristics (social environment, age, education level, physical activity level, initial COP values, anxiety, BMI)?

3. You need at least 36 patients to validate your statistical analysis. 39 participants were recruited, but 7 patients have dropped out before the end. 39 -7 = 32 and dropout rate of 18%. You wrote «A total of 36 (93.21%) completed the full experiment», is it a mistake? If you have 32 participants, please explain de validity of the statistical analysis. How dropout patients were handled in your statistical analysis?

4.  Hodges et al. (P.W. Hodges, K. Tucker / PAIN  152 (2011) S90–S98 ) have shown how patients moving differently in pain. Explain how your sample population or pain model may induce a limitation in your results.
Additional comments: This study tests the proposal that pain stimuli can induce a freezing response in different social contexts. It finds correlations between pain anticipation, social context, body sway, anxiety and face expressions. This is an interesting study. However, in my opinion, this manuscript has a number of general and specific problems (major), ranging from basic grammar and readability, experimental design, statistical analysis, and validity of the findings. In several instances I also suggested to cite more relevant and recent literature. I do believe there is valuable data to be published from this research, but as it stands the manuscript is weak and unrefined. This is why I suggest a withdraw for the manuscript, as I believe the changes that need to be made to this manuscript are many and would be difficult for the authors to achieve in the time frame allowed. I hope that the following critiques will aid the authors in the refining of the manuscript to allow for re-submission in the future.


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: FREEZE-LIKE RESPONSES TO PAIN IN HUMANS AND ITS MODULATION BY SOCIAL CONTEXT
Review round: 2
Reviewer: 1

Output:
Basic reporting: LINE 236: The sentence with maximally in it does still not make sense: I think it would be better written: "After selecting the pain stimulus the participant was informed that (s)he would repeatedly receive stimuli of the maximum calibrated intensity during the remainder of the experiment."
Experimental design: I am now happy with the reporting of the results. Initially I had hoped for the mean differences, however on reflection on the nature of this research I think the information that the authors have provided is appropriate. It makes the findings much more transparent.
Validity of the findings: I am happy with the way the authors have interpreted the findings now.
Additional comments: Well done on the changes to the paper. I appreciate they were significant and time consuming changes. I hope you feel they improved the paper. From the perspective of the reader the paper is now much easier to follow and it is clear what happened with the participants along the way.

Regarding the final comment in my previous review about predefining the analysis plan, I appreciate that the authors understand the importance of this and acknowledge that this was not common practice when the study was designed. I feel that to help encourage others researchers that the authors should still make a comment in the discussion that not having a predefined and publicly available analysis plan is in fact a limitation and that future studies. A reference to a paper like the ones below would be appropriate:

Lee H, Lamb SE, Bagg MK, Toomey E, Cashin AG, Moseley GL. Reproducible and replicable pain research: a critical review. PAIN. Published online April 2018:1. doi:10/gf9dbc

Nosek BA, Alter G, Banks GC, et al. Promoting an open research culture. Science. 2015;348(6242):1422-1425. doi:10/gcpzwn


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: HIGH-DOSE TESTOSTERONE SUPPLEMENTATION DISTURBS LIVER PRO-OXIDANT/ANTIOXIDANT BALANCE AND FUNCTION IN ADOLESCENT MALE WISTAR RATS UNDERGOING MODERATE-INTENSITY ENDURANCE TRAINING
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	The manuscript is clearly written and is comprehensible. The introduction is sufficient to orient towards the aims and objectives of the work.
Experimental design: 1.	The research question is well-stated and the methods are sufficiently elaborated.
2.	Proper statistics have been applied.
Validity of the findings: 1.	Is it known what could be a possible trigger for oxidative stress in liver after high testosterone supplementation?
2.	Given the known role of Androgen receptor signaling in regulating lipid and glucose homeostasis through liver cells, the authors should discuss the effects of supraphysiological levels of testosterone in the light of their results. What about the insulin sensitivity at high testosterone supplementation? It may also pose detrimental effects.
3.	Testosterone can have effects on multiple signaling pathways and organs, but since this paper is more focused on oxidative stress and liver injury, authors should check how Nrf2 signaling (which is a master regulator of antioxidant defenses) is affected. We see that high dose TE leads to increased lipid oxidation (TBARS) together with decrease in antioxidant defenses like CAT, it would be interesting to know if this is due to down-modulation of cellular defenses or exhaustion of resources.
Additional comments: 1.	The discussion is too elaborate, cutting it short wherever possible would make the manuscript more coherent.
2.	Line 211 replace ‘that in’ with than.


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: HIGH-DOSE TESTOSTERONE SUPPLEMENTATION DISTURBS LIVER PRO-OXIDANT/ANTIOXIDANT BALANCE AND FUNCTION IN ADOLESCENT MALE WISTAR RATS UNDERGOING MODERATE-INTENSITY ENDURANCE TRAINING
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language used in the manuscript is clear, mainly in the discussion.

Your introduction is very good, however it is necessary to insert the article reference at lines 62-63 and 115-116 and remove website. I also suggest that you improve the description at lines 62-63 and 115-116 to provide trustworthiness for your study (specifically, you should insert the websites in supplementary data)
Experimental design: If the testosterone dilution at line 135 are included in another study, please cite him in this section.

Considering the methods section, experimental design, a suggestion is to group information from line 159-161 with line 146 to the information not to get lost. In the same section, the authors have data of food or water consumption? If do not, a suggestion to the authors is to use in next studies the control of water and food consumption weekly or, if it is possible, daily. This data control can help to better understand the BW of groups EndTr, mainly with the use of testosterone protocol. For observation, another suggestion to next studies is to change bedding three times a week.
Validity of the findings: The manuscript “High-dose testosterone supplementation disturbs liver pro-oxidant/antioxidant balance and function in adolescent male Wistar rats undergoing moderate intensity endurance training (#46526)” presents a very important data about the use of testosterone in the adolescent phase. This data can contribute to evidence the precautions about the use of testosterone.
Additional comments: To value your paper, another suggestion is to create a figure that explains your results and summarizes the discussion theory, if it is possible, insert in the article or supplementary data.


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 AND IDENTIFICATION OF KEY GENES AND SIGNALING PATHWAYS IN THE OCCURRENCE AND METASTASIS OF CUTANEOUS MELANOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: (1) The language in this manuscript is ambiguous, and the conclusion is exaggerated. Please see Major Q1.
(2) Picture quality is basically no problem..
(3) No raw data is provided.
(4) Literature well referenced.
Experimental design: (1) The experimental method has some problems and needs to be redesigned. Please see Major Q2 and Q3.
(2) There is no problem with the statistical method in this manuscript.
Validity of the findings: (1) The findings of this study need further experiments. Please see Major Q1 to Q3.
Additional comments: Melanoma is a malignant tumor of melanocytes, and the incidence has increased faster than any other cancer over the past half century. Most primary cutaneous melanoma can be cured by local excision, but metastatic cutaneous melanoma has a poor prognosis. Therefore, the research on the mechanism of cutaneous melanoma metastasis will be beneficial to diagnose the metastatic melanoma early, improve the treatment, and prolong the survival of patients. In this study, Dai Hangying and colleagues identified the key genes and molecular pathways involved in the occurrence and metastasis of cutaneous melanoma. The study provides the conclusion, which showed that some DEGs were found from normal skin, primary melanoma and metastatic melanoma samples. These hub genes may play an important role in the generation, invasion, recurrence or death of CM, providing new ideas and targets for the diagnosis and treatment of metastatic cutaneous melanoma. There are some problems in this study, which require the author to revise the research design, and there are some major concerns that need to be addressed.

Major concerns:
1. “Validity of the findings” The title of this manuscript is that “Comprehensive analysis and identification of key genes and signaling pathways in cutaneous melanoma metastasis”. However, from the analysis results and conclusions of the manuscript, the author did not identify convincing analysis, but only found a bunch of differentially expressed genes, analyzed the potential functions and pathways, and found a dozen genes related to prognosis. The correlation analysis used in this study can only indicate the possibility of an important role and should not be exaggerated. The author had to make a careful revision of the language.
2. “Validity of the findings” The authors used multiple databases to analyze differentially expressed genes between primary melanoma and metastatic melanoma and identified these genes as the key genes associated with melanoma metastasis. However, the process of metastasis is complex, and the genes specifically expressed in the metastasis foci may be a passive process for the cancer cells to adapt to the microenvironment of the metastasis foci, rather than "drivers". Therefore, there is a big deviation between the subject design idea and the research objective in this paper, and the results and conclusions cannot support each other. I would suggest that the authors reformulate the purpose of the study and redesign the analysis process according to the purpose. The key is not to make the purpose too large, but to focus on a specific clinical problem or tumor biology problem.
3. “Results – Validation of hub genes and survival curve analysis” Line 201-208. When using TCGA database for verification, the author firstly verified the expression differences of selected genes in primary foci (n = 104) and metastatic foci (n = 368), and then analyzed the relationship between related genes and survival prognosis of patients, proving the importance of related genes. However, as can be seen from the survival curve in Figure 8, the author did not simply analyze the relationship between the expression of relevant genes in the primary site and the survival prognosis of patients, but included all the data of the primary site and the metastatic site into the analysis (n = 250+), which was obviously a logical problem. The prognosis of patients with metastatic foci is certainly worse than that of patients with primary foci alone. The analyzed genes have been found to be abnormally expressed in metastatic foci. If the samples of all patients are integrated and analyzed, the correlation between these genes and prognosis is inevitable and has no real diagnostic value. Therefore, the authors analyzed the survival or prognosis of related genes in patients with primary and metastatic foci respectively.


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 AND IDENTIFICATION OF KEY GENES AND SIGNALING PATHWAYS IN THE OCCURRENCE AND METASTASIS OF CUTANEOUS MELANOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors provide a detailed bioinformatics analysis of the mechanisms involved in cutaneous melanoma progression. I believe that some aspects of their analyses are over-represented, while some other ones are not. I would focus mainly on the comparison between PM and MM.
I beleive that the English language should be improved. Please check that the correct tense for all verbs is used .
Experimental design: My major concern regarding the experimental design is whether the comparison between melanoma (primary or not) and normal skin, in order to identify Differentially Expressed Genes, is valid. I mean that the comparison between cancer and normal melanocytes could be much more meaningful.Could the authors check also this comparison at least at the datasets that contain samples of normal melanocytes or nevi?
I would suggest that the authors include a table describing the datasets that they have analysed, presenting details concerning the number of normal skin, primary or metastatic samples per analysis. The number of differentiated genes could also be indicated.
Validity of the findings: I believe that the presented bioinformatics analyses are robust and statistically sound.
In my opinion there are many tables concerning the GO and Kegg-based analyses. I would suggest to keep those concerning the PM versus MM DEGs and present the others as supplementary files.
Regarding figure 7 I couldn’t understand why the y axis is indicated in Transcripts per million and why the category normal is presented with n=1.
Additional comments: Please correct lines 271-272


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 AND IDENTIFICATION OF KEY GENES AND SIGNALING PATHWAYS IN THE OCCURRENCE AND METASTASIS OF CUTANEOUS MELANOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: - The use of English is verbose and not clear. There also contains grammatical errors and typos. The authors should re-check and revise carefully.
- Literature reviews are weak. The authors should add more literature references about some related works.
- Some abbreviations need to be defined at the first use.
Experimental design: - Research design is a big concern. Why did the authors perform the analysis among three groups? If the problem aims to address cutaneous melanoma metastasis, why did the authors not compare between metastasis and non-metastasis only? Or if the authors compared three groups, why did the authors separate into three binary problems? Is it possible if the authors perform the analysis on all three groups together?
- Methods have not been explained well and it is not easy to replicate. Also, it is important that the authors could release source codes for analysis.
- GO database and analysis has been used in previous works related to biomedical such as PMID: 31277574 and PMID: 31921391. Therefore, the authors should provide some references in this description.
Validity of the findings: - The choice of p-value and cut-off value is a question. Why did sometimes the authors select significant level of 0.01, 0.02, or 0.05 etc? At least, these values must be consistent.
- There are many works that have addressed the same question such as PMID: 31173190, PMID: 31937175, or PMID: 32547879. Thus what are differences between this study and the others? The authors should discuss and compare with some related works.
Additional comments: No comment