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: EXHAUFS: EXHAUSTIVE SEARCH-BASED FEATURE SELECTION FOR CLASSIFICATION AND SURVIVAL REGRESSION
Review round: 1
Reviewer: 3

Output:
Basic reporting: Some additional context needed to be provided in the Introduction and the Discussion sections (see Additional comments).
Experimental design: (see Additional comments)
Validity of the findings: no comment in general (see Additional comments)
Additional comments: In the paper, Nersisyan et al. proposed a new feature selection scheme for the identification of gene signatures with a prognostic value. The approach of selection of feature combinations based on an exhaustive search across feature sets of given size seems interesting and demonstrates its power for selecting highly discriminative features.

The authors supplied the paper with all necessary supplemental materials, uploaded their pipeline in the GitHub repository, and provided extensive documentation, which is essential to ensure reproducibility of the paper results as well as its further use.

Overall paper meets the criteria for publication, however, there are several points where clarifications and revisions are needed.

1. The introduction did not to a relevant extent provide a background on the works that were done in this direction. The idea of combining different genes to identify feature sets with higher accuracy has been presented in several research papers and should be discussed in the Introduction section (for example https://ieeexplore.ieee.org/document/6710349, https://doi.org/10.1093/bib/bbaa189, https://www.mdpi.com/2072-6694/13/17/4297, etc.).

2. In the Materials and methods section (line 212-213) it is not clear how what authors mean by transforming FPKM data into microarray units? Did they perform log-transformation of count data or calculated fold-changes?

3. In the “toy” example I suggest authors compare the accuracy of individual features ("perception_vulnerability", "socialSupport_instrumental", and "empowerment_desires") versus constructed classifier containing these three features.

4. In the breast cancer signature authors state that none of the individual genes before preselection did not hit the pre-defines accuracy threshold. However, the authors did not provide the results for the same comparison for genes that passed pre-selection and were used to constrict feature sets. To what extent performance of a single gene is worse compared to their combination?

5. I suggest the authors discuss the possibility of using their approach with only one dataset containing a small number of samples. If dividing the sample into training and validation sets is impossible what other measures and estimates can be used to assess the quality of the selected features.

6. I suggest the authors discuss how to select the one prognostic feature set from many identified ones. For example, in the BRCA example, more than 1000 gene combinations passed the threshold. How to choose one or a few to be used for future comparisons?

7. I also would suggest authors discuss whether signatures identified by ExhauFS perform better or worse compared to the signatures reported previously (not the methods; they did it already in the paper).

8. In Table S1 is not clear what is the total number of patients.

9. Authors report on the SIGLEC15 gene in the discussion, but this gene was mentioned neither in the results section nor in supplementary figures and tables.


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: EXHAUFS: EXHAUSTIVE SEARCH-BASED FEATURE SELECTION FOR CLASSIFICATION AND SURVIVAL REGRESSION
Review round: 2
Reviewer: 1

Output:
Basic reporting: In this revised manuscript, the authors have successfully addressed my previous concerns. I have read through it and now it meets the publication standard of PeerJ. Thus I suggest acceptance of the manuscript for publication.
Experimental design: The Experimental design meets the standard of the journal.
Validity of the findings: No further improvement is needed.
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: EXHAUFS: EXHAUSTIVE SEARCH-BASED FEATURE SELECTION FOR CLASSIFICATION AND SURVIVAL REGRESSION
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have considered the comment and suggestions. The revised publication can be considered for publication in its current state.


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: MACHINE LEARNING DRIVEN BY ENVIRONMENTAL COVARIATES TO ESTIMATE HIGH-RESOLUTION PM2.5 IN DATA-POOR REGIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: In the section of model development,the paper take PM2.5 as response variable, aerosol optical ickness, annual precipitation, annual average temperature,183 wind speed, wind direction, relative humidity, urface temperature, surface pressure, the potential evaporation rate, DEM, normalized difference vegetation index and enhanced vegetation index, population density as  predictor input variables.there are so many predictor input variables ,Are all these predictor variables necessary?For a prediction model, the fewer model parameters, the better the model。Therefore, it is suggested that before inputting these parameters into the random forest, first make a significance test on these parameters and select the variables with significant influence as the input parameters.
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: MACHINE LEARNING DRIVEN BY ENVIRONMENTAL COVARIATES TO ESTIMATE HIGH-RESOLUTION PM2.5 IN DATA-POOR REGIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this study, the authors present the estimation results of PM2.5 concentrations in the Xinjiang province of China. The article is well written, but it should be further improved to meet the standards of publication. Please refer to the specific comments listed below.

1.	Relevant prior literature on spatio-temporal data-driven machine learning methods should be appropriately referenced. The authors only mention spatio-temporal data-driven machine learning in the title, whereas the comparison of different methods for utilizing spatio-temporal information is untouched in the article. The following papers are listed for reference.

[1] Wei, J., Huang, W., Li, Z., Xue, W., Peng, Y., Sun, L., Cribb, M., 2019. Estimating 1–km- resolution pm2.5 concentrations across China using the space-time random forest approach. Rem. Sens. Environ. 231, 111221.
[2] Yang, N., et al.2022. "Geographical and temporal encoding for improving the estimation of PM 2.5 concentrations in China using end-to-end gradient boosting.". Rem. Sens. Environ.

2.	In Figure 1, the legend of the figure is not labeled as English words. In all figures of this article, it is confused for readers that all the high values are marked as blue and the low values are marked as red, which are opposite to the general studies. The authors should explain why they adopted this type of figure legend.
Experimental design: 1.	I thank the authors for providing the raw data. The current raw data only contains the PM2.5 data, whereas the AOD data and the meteorological data of training samples are not presented in the file of raw data. Readers cannot reproduce the experiments via the current data file.

2.	The code of cross-validation could not be found in the source code. Sufficient details of model hyperparameters are not described to replicate the experiments.
Validity of the findings: 1.	The article should clearly define the research question and show how the problems are solved. In lines 21-22, the authors claim that “We hypothesize that combining temporal and spatial information can improve the PM2.5 estimation accuracy because the spatiotemporal data expand the training sample of the model.” I suggest that authors should present the estimating results with and without temporal and spatial information to prove their hypothesis.

2.	In this research, the model is not compared with other results of previous research in this study area. The aerosol emissions could change each year, and different number of training samples also affects the model performance. The authors need to add some experiments and discussions about the model comparison.

3.	Please provide the standard deviations based on the 10-fold results to show whether the proposed model is robust are statistically significant.
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: MACHINE LEARNING DRIVEN BY ENVIRONMENTAL COVARIATES TO ESTIMATE HIGH-RESOLUTION PM2.5 IN DATA-POOR REGIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: This article needs to be polished further with professional English proofreading.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: BIOINFORMATICAL ANALYSIS IDENTIFIES PDLIM3 AS A POTENTIAL BIOMARKER ASSOCIATED WITH IMMUNE INFILTRATION IN PATIENTS WITH ENDOMETRIOSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper by Gan and al, idnetifies PDLIM3 as an interesting gene biomarker of endometriosis. The work is well performed, and convincing. The approach to use three published datasets, screen them for common genes, and building ROC curves with the samples of these articles, as well as from a fourth study is interesting and convincing.
There are I think some flaws in the English such  as line 202 'All DEGs above were performed GO and reactome...' has no meaning. It should be I think something like 'The above list of DEGs was used to evaluate if they were enriched into specific GO or reactome pathways'.
The literature provided is sufficient but I found some other datasets that could be interesting to strengthen again the dataset of DEGs (for instance the dataset GSE12768 from Borghese and coworkers (2008) is almost completely consistent with the DEG list produced by the authors.
About the pictures, the small size of the characters is accessible only when enhancing the size from the images on a computer. There is some space that would allow to use a larger character size.
Experimental design: The design is I think excellent. The flow of the ideas is straightforward. The evaluation of immune cell contents presented in figure 5 is clearly an innovation in the field of endometriosis.
The fact that the gene list identified is largely confirmed by other studies is an extremely strong point. An additional point that could be given is the type of endometriosis that is under study in the datasets presented; are there ovarian (OME), deep infiltrating (DIE) or superficial (SUP) endometriosis. In addition is the fourth verification dataset from the same type of endometriosis than the others? OME was analyzed for PDLIM3 expression at the protein level (figure 4) therefore this question is important.
Validity of the findings: My feeling is that the originality and robustness of these experiment is convincing and robust. The computig of the p-values is state-of the art. The paper is concise and should be of great interest for people working in the field of endometriosis.
Additional comments: An interesting and innovative paper in the field. The statistical analysis is convincing.


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: BIOINFORMATICAL ANALYSIS IDENTIFIES PDLIM3 AS A POTENTIAL BIOMARKER ASSOCIATED WITH IMMUNE INFILTRATION IN PATIENTS WITH ENDOMETRIOSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The subject of the manuscript, i.e. endometriosis, is clinically relevant and, as recently defined by Agarwal et al , “Early identification and treatment of endometriosis is essential and facilitated by a shift towards clinical diagnosis instead of relying on surgical diagnosis alone” (Am J Obstet Gynecol 2019;220: 354.e1–12). In addition, endometriosis is now defined as a chronic systemic disease and clinical challenges and novel innovations have been recently reported by Taylor et al. (Lancet 2021; 397: 839–52). On these bases, Introduction and Discussion need to be revised. In particular the major aim of the study “early clinical diagnosis” could not be based on tissue biomarkers such as those described in the present manuscript but on circulating biomarkers and comments on these aspects need to be added in the discussion.
Experimental design: On the basis of the comments in Basic Reporting, it is suggested to clarify that the comparative tissues analyses could help in identifying not only the mechanism of the disease but also potential target/s of therapy. Accordingly, it is necessary to note that pelvic endometriosis, analyzed in the meta-analysis of three datasets (GSE11691, GSE23339, GSE5108) could be different from ovarian endometriotic cysts characterized in the validation dataset (GSE120103). Indeed, the different origin of samples between meta-analysis and validation could account for the no significant different gene expression for six of the seven genes initially identified.
The case material used to validate the expression of PDLIM3 by qRT-PCR and western blot need to be better specified (number of samples and clinical characteristics of patients).
Validity of the findings: My suggestion is to highlight that:
1) The reported data could be considered a revisitation of already described data (GSE11691, GSE23339, GSE5108) that were only compared but not combined.
2) The new evaluation of the meta-analysis dataset with CIBERTSORT, tool not available when the single studies were published, enabled to confirm the strong involvement of inflammation. In this context It could be relevant to analyze also the immune microenvironment of GSE120103 dataset.
Additional comments: Please note that the abbreviation EMT is universally applied to Epithelial-Mesenchymal-Transition. It is strongly suggested to not use the abbreviation EMT for endometriosis .


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: BIOINFORMATICAL ANALYSIS IDENTIFIES PDLIM3 AS A POTENTIAL BIOMARKER ASSOCIATED WITH IMMUNE INFILTRATION IN PATIENTS WITH ENDOMETRIOSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors convincingly responded to my concerns.
I feel nevertheless that the text in the figure is still very small and could be further increased in size.
Experimental design: The design was good from the start. Including the additional dataset that I suggested strengthened the message.
Validity of the findings: Links between PDLIM3 and endometriosis were documented before and this paper is a strong reinforcement to this idea.
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: BIOINFORMATICAL ANALYSIS IDENTIFIES PDLIM3 AS A POTENTIAL BIOMARKER ASSOCIATED WITH IMMUNE INFILTRATION IN PATIENTS WITH ENDOMETRIOSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors included all requested changes for both external reviewers accordingly  no further comments are needed
Experimental design: The authors included all requested changes for both external reviewers accordingly  no further comments are needed
Validity of the findings: YES
Additional comments: No comments


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

Input:
Title: RESPONSIVENESS OF DOMESTICATED GOATS TOWARDS VARIOUS STRESSORS FOLLOWING LONG-TERM COGNITIVE TEST EXPOSURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study investigated if there is a relationship between participation in multiple cognitive tasks to reactions to novelty (environment, object, human) measured at a later stage in tow goat species; dwarf and dietary domesticated goats. Furthermore, beyond solely addressing whether cognitive testing per se is associated with changes in stress responses, the authors aimed at disentangling the effect of testing from effects of confounding factors such as the human contact and the isolation during testing. This is an important topic that benefits the field of animal cognition, as cognitive testing in a variety of captive settings is increasing within the field.
The abstract and introduction fails to transmit one of the important parts of the study, the cognitive test battery and explain what that entailed. Also, it would be necessary to refer to why the chosen tasks are considered “standardized”. The idea that some goats go through a test battery and then after (some delay, that never is specified) are measured on independent traits like response to a novel arena, object and human is very interesting, given the great efforts made by the authors to generate two adequate control groups. However, in its current state the manuscript does not bring forward this point very well. There is no mention of a cognitive test battery in the abstract, the first time the reader “learns” about this is at the end of the introduction. It would be easier for the reader to comprehend the purpose of the article if it is introduced earlier and explained more thoroughly, what tasks, what cognitive capacities are they designed to measure etc. This is nicely listed in a supplementary Table, but still would benefit from some mentioning in the manuscript. Also, the term “long-term cognitive stimulation” needs to be specified and explained, as well as “standardized” object-choice tasks. It seems like the authors tested more than solely object choice tasks, such as associative and reversal learning. Please review the literature or refer to previous work to why you choose to refer to such test battery as “standardized”.

Second, the authors claim that they are testing goats’ stress reactivity in potentially stressful situations. However, there is a large body of scientific work done on novelty response tasks (similar to the ones the authors use in this study), in the animal cognition literature and animal personality literature (few examples; Stamps and Groothuis, 2010; carter et al., 2013; Šlipogor et al., 2021), such as novel object task, open-field test, response to novel human. Such tasks are mostly employed to measure cognitive traits like neophobia, neophilia, explorative behaviors and human orientation (Damerius et al. 2017) and are not referred to as “stress tests”, as for many animals the opportunity to interact with novelty provides an enrichment activity and not necessarily direct stress. Unless there are evidence and data from goats, that already show that goats respond with stress (measured with cortisol, heart rate etc) in such tasks, I do not agree that it is appropriate to refer to these types of tests as stressors since responses to novelty can have multiple different outcomes, both neophobic (avoidance, approach delays etc) and positive explorative (manipulation, physical contact etc), which might be stimulating instead of stressing. Therefore, I advise the terminology to be reflected over and re-phrased in accordance to the published literature. For example, in the abstract the authors refer to cognitive tests as stimulating, but then presume that novelty response tasks on the other hand should be stressors, this is contradictive and needs to be elaborated on. The cognitive tasks may only be stress inducing when the nature of the task requires isolation and thus the responses of the animals are to separation from conspecifics rather than due to the task itself.

Behavioral “Reactivity” sounds like a very laboratory narrowed term, maybe better terminology behavioral “response”. However, if you expect goats to differ from other animals and therefore such tests just measure stress, please describe this with adequate references in the introduction.

Grammar and language:

The manuscript gives a general “sloppy” impression with regards to grammar and language as throughout there are many sentences are too long and would benefit from being shortened and re-phrased. In general, the manuscript does not flow easily for the reader. I suggest a native speaker or additional senior scientist to edit the text.

Passive and active voice is being mixed, for example in the first paragraph of methods section, please commit to one.
Experimental design: In line 199, the authors state “after the cognitive test phase”, please specify and provide the information on HOW LONG AFTER as you claim to test long-term responses.
A novel arena test (NA) is this the same as an open field test? If not, how do they differ? Open field tests are used to measure animal exploration behavior as well as stress responses.

weighing test (WH) – this is not really a test, more a situation also part of the routine lives of these animals. The situation when the heart rate equipment was placed onto the goats would more likely reflect a novel stressful situation rather than the weighing situation.
L206: “the goats had to be moved to a different building for these tests.” It can be possible that such move is a confunding factor contributing to stress rather than the subsequent test itself. This should be elaborated in the discussion.

If I understand correctly the Novel Objects were presented within the Novel Arena, this poses a problem as one cannot rule out the reactions of the arena or novel environment to that from the animals’ response to the novel object. Normally novel object tests are presented in a familiar environment. Also, the Novel Human test, faces exactly the same problem as the human is within the novel arena. Moreover, in the novel human test, was the person someone the goats had never seen before, please clarify. Same question for the weighing test: it says that a human led the goat onto the scale. Was this by the same person for all goats? What were the relationship to that human? such factors may influence responses. Why not use video cameras instead on the sides and subsequently score behavioral responses from the videos, as the two humans can potentially have an influence on the goats’ responses during the weighing situation.

For all tests, in the text there are no mentions of the exact behavioral measurements used in the analyses. L297: you state that you used 9 behavioral measures, even if you present them in a Table, it would be of great importance to list them in the text too.
Validity of the findings: It would be nice with any findings on the relationship between the outcomes of the test battery and the “Stress tests”. Where goats that perform better on cognitive tasks less responsive during the “stress tests”? This was a question that one expected from the introduction but is not addressed in the result section at all.

L382: This would be easier to read if it is shortened and would refer to the two hypotheses - present them clearly in the end of the introduction and in result section briefly, but then no need for a long repetitive section in the discussion.

One possibility to why there was a lack of connection between the cognitive testing and the following stress tests, is the fact that with the current experimental design it is impossible to disentangle interest in objects from isolation stress. One would need to measure interest in novel object in a familiar, every day environment to ensure that such response does not intervein with the stress of being separated into a novel arena. The authors briefly “touches” this topic L445-448, but would have to elaborate on their findings more clearly before interpreting the results.

L462-470: In this section the authors nicely discuss potential explanations of why different groups of goats responded differently in their tests and acknowledge the variation in rearing backgrounds as well as upbringing experience. It would be nice to explore if these rearing influences had an effect on the outcomes of the cognitive test battery as well as the novelty response tests.

L469: phenotypic variation? but what you mean is rearing and background history.
Concluding comment may represent a line of misinterpretations: “We found that long-term experience with standardized cognitive testing per se does not reduce stress reactivity of goats in subsequent stress tests.” This is an overstatement given the lack of definitions in the terminology in the first place and given that the stress response was not properly disentangled from isolation response.
Additional comments: Detailed comments:
ABSTRACT
L23: Very long and complicated introductory sentence.
L41: This sentence is a bit out of place and not clear on how it connects to the rest that has been said in the abstract.
INTRODUCTION
L49: purposes instead of aim?
L51: one space too much
L52: I am not sure what the word learning at the first part of this sentence adds, it seems a bit unexplained and should therefore better be left out or one should explain the context of it here. I think the authors are trying to say something like: Because cognitive tests can potentially cause the participating animal stress, as provided tasks often requires successful learning by the animal before it experience positive emotions (Langbein, Nürnberg & Manteuffel, 2004). I would also take out “initial stages”, as there are also cognitive tests that demand attention and learning for a prolonged time.
L56: “execution”? maybe better completion, participation?
L57: “were”, maybe better has been?
L59: “a call-feeding station”. The reader does not know what this is, please elaborate and explain.
L60: “that the animals were” better that those individuals… or those subjects were… compared to … conspecifics.
L61: Who are they? Specify please.
L65-69: too long sentence, please split and clarify.
L67: “be isolated” from their group of conspecifics? Please specify.
L73: in which species? It would be nice if the reader was given that information.
L92: bind the paragraph to previous with some wording like: “Moreover”, “Furthermore” etc.
L96: “was” replace with “has been”
L103: “extensive husbandry conditions”, in what way? Please clarify and explain.
L110: “were” replace with “has been”
L111: “highly developed cognitive capacities” – like what capacities? Please explain rather than just make a blunt statement.
L114: “by means”, replace with “using”
L116-123: This section is rather suited for the methods part; it makes the introduction harder to grasp.
METHODS
L131-132: No need to replicate the word “Animals” in both headings. Delete “Animals” from L131.
L133: Delete “namely” and replace with simply “;”
L138: “few exceptions”. Exceptions always has to be mentioned and clarified.
L138: “aim”? replace with “purpose”
L139-140: “The potential milk yield of Nigerian dwarf goats does likely not exceed 0.3 kg/day (Akinsoyinu, Mba & Olubajo, 1977)” Unclear wording, do you mean: generally does not exceed?
L140-141: Wrong grammar, please rephrase this sentence.
L146: “and artificially raised” please explain what this means.
L183: do you mean habituation process of the goats or habituation tests? Please specify.
L189-190: The sentence does not flow, please correct grammar.
L190: “administered”? what does this complex word add? Why not simply individuals of ISO treatment
L225: This sentence makes no sense, needs elaboration.
L282: As you exclude these animals with the definition of them being too stressed out during the cognitive test battery, please give the reasons to why you believe they were stressed out.

Suggested literature (just a few)
Stamps, J., & Groothuis, T. G. (2010). The development of animal personality: relevance, concepts and perspectives. Biological Reviews, 85(2), 301-325.
Carter, A. J., Feeney, W. E., Marshall, H. H., Cowlishaw, G., & Heinsohn, R. (2013). Animal personality: what are behavioural ecologists measuring?. Biological Reviews, 88(2), 465-475.
Šlipogor, V., Massen, J. J., Schiel, N., Souto, A., & Bugnyar, T. (2021). Temporal consistency and ecological validity of personality structure in common marmosets (Callithrix jacchus): A unifying field and laboratory approach. American journal of primatology, 83(2), e23229.
Damerius, L. A., Forss, S. I., Kosonen, Z. K., Willems, E. P., Burkart, J. M., Call, J., ... & van Schaik, C. P. (2017). Orientation toward humans predicts cognitive performance in orang-utans. Scientific Reports, 7(1), 1-12.


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: RESPONSIVENESS OF DOMESTICATED GOATS TOWARDS VARIOUS STRESSORS FOLLOWING LONG-TERM COGNITIVE TEST EXPOSURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: (Comments listed in order of importance)

•	Line 343 onwards: Linear mixed-effect models section – it is more conventional to report the test statistic etc., not just the p-values
•	Lines 30-33: Especially for the POS treatment and somewhat for the COG treatment I feel you should either change the treatment names or describe them in a way that it is obvious where they derive from. In respect to the POS treatment, this is better described at the end of the introduction (Line 117), but the link between the COG treatment name and description becomes more unclear (Lines 115-116). This will enhance memorability to readers as to what treatments involved further on in the text.
•	The term selection lines is used throughout (e.g. lines 35, 133), but I feel this it is used inappropriately as authors were not just comparing between two distinct breeds as they used three different dairy goat breeds and cross-breeds.
•	Supplementary material: Table s2 onwards: explain the shortened terms used in the description above, i.e. est. = effect size?, s.e. = standard error etc.
Experimental design: (Comments listed in order of importance)

•	How long did it take for the goats to clear these treatment trials and what was the average duration between completion of the COG condition and the start of the experimental trials? This is important information and should be mentioned in the methodology section, but I cannot find this written anywhere
•	What was the average time interval between the stress-tests?
•	Was it always the same person conducting the battery of tests for each goat? This could at least have important implications for the novel human test
•	As human associated rewarding is a major factor in the experimental design, I feel  what other forms of human contact did the goats receive before onset of tests (e.g. husbandry routines etc.) should be explained in the methodology section
•	Line 171 onwards: Treatment groups section: it would be very useful for readers to have the tests briefly described in a bit more detail to better understand them without having to be referred to another document. This supplementary document would be useful to keep in, but I would look for a bit more detail of what tests are involved and how regularly they were exposed to testing
•	Supplementary material: Did any goats fail to reach the success criteria for these cognitive tests? If so, what happened to such goats?
•	Supplementary Materials: Table S1 – make the description of the visual discrimination task clear, for example by explaining only one of the cups was baited
•	Line 198: did you consider order effects/ randomisation of test conditions? Why did you choose to do the tests in a standardised order?
.	Supplementary materials: it would be useful to have a photograph or diagram of each test paradigm used
•	The authors used three breeds of dairy goat and some mixed breeds. Did you look for the effect of breed on results, rather than just splitting comparisons between dairy breeds vs pygmy goats?
•	The authors mentioned using an external microphone to look at acoustic responses. Did you investigate the effect of stress tests on acoustic parameters relative to treatment condition?
Validity of the findings: Comprehensive information has been provided regarding subjects, the raw data and even R scripts used in analysis.

However, in order to make the behavioural data set more robust, I was wondering whether you had considered using a secondary coder other than just for the weighing test?
Additional comments: N/A


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: RESPONSIVENESS OF DOMESTICATED GOATS TOWARDS VARIOUS STRESSORS FOLLOWING LONG-TERM COGNITIVE TEST EXPOSURE
Review round: 1
Reviewer: 3

Output:
Basic reporting: This study how long-term experience with cognitive test per se does not reduce stress reactivity of goats in subsequent stress tests. I believe that this study is essential in order to absolutely validate any results obtained in experiments carried out with domestic species (cognition, behaviour, etc). On the other hand, the study provides relevant information that needs to be further studied to ensure proper management of livestock.
The language as well and the English are at highest levels. The manuscripts flow easily its informative and contains all the background information needed. All data, tables, figures and raw data included in the supplementary material are satisfactory well ordered and informative. The objective of the study is sound and the hypotheses too.
The information contained in lines 100-108 regarding the two breeds of goats perhaps could be changed to the discussion, since its mainly the justification of using them and why is important. Nevertheless, if the paragraph maintains its current location the quality of the manuscript remains intact.
Overall, I find the manuscript very pleasant to read, informative and comprehensive and I have no punctual comments.
Experimental design: I believe that the topic of this manuscript is highly relevant, that it is original and fall in the scope of the journal. The hypotheses are clear and sound. I believe that the experimental design is indeed answering all the research questions formulated by the authors. In my opinion, the experimental design is complex, however, the authors found a way to address all the questions and answer them including all possibilities. The methods are well described and easily reproducible.

I believe that this study addresses a very important subject which is usually just “solved” by a period of “habituation” before running cognitive or behavioural tests. However, it is imperative to know if the long-term cognitive test exposure per se has a positive impact, backing it up with physiological (cardiac frequency) and behavioural assessment.

The experiments are clear the justification of the different groups investigated is well explained.
I have no punctual comments about the experimental design or the methods used due to the clarity of the manuscript.
Validity of the findings: In my opinion, the results of the study are absolutely valid, mainly for the good and neat experimental design. Many researchers still reject studies with negative results. I find these negative results very important and necessary in order to design further research. The fact that cognitive testing per se, usually believed to decrease the stress in subjects exposed to further tests, and that the reward-associated human-animal interaction having a positive impact in the subject’s stress, are not supported by the results obtained is very imperative. I believe that the findings of this study highlight the big gap of knowledge that is still missing and the clear necessity that more researching study cognitive testing, habituation, human-animal interaction is greatly needed. Additionally, it points out how other studies might obtain results that lacks validity.

The results are very well discussed, the statistical analysis well done and the authors have provided alternative justification of the results obtained. The study highlights how important is to keep investigating these subjects that are essential for all researchers studying, cognition, behaviour and animal welfare.

I have no punctual comments since the I find the discussion and conclusion of the results absolutely valid. I must add that the manuscript is interesting and very pleasant to read.
Additional comments: Dear authors

I found your manuscript interesting and very pleasant to read. Overall, it is clear, the background information provided covers well the topic and the experimental design is absolutely sound. Your data figures and statistical analyses are satisfactory and, in my opinion, validate your findings. I would encourage you to keep designing studies to learn more about the stress experienced by livestock in general. The use of two breeds is also highly commendable and increase the validity of your results. The discussion is compelling and the possible explanations you provide are sound and well backed up with great references. I honestly did not find any punctual comments to make, the manuscript is excellent as it is.


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: RESPONSIVENESS OF DOMESTICATED GOATS TOWARDS VARIOUS STRESSORS FOLLOWING LONG-TERM COGNITIVE TEST EXPOSURE
Review round: 2
Reviewer: 1

Output:
Basic reporting: I believe the authors have made necessary efforts to improve the points raised during first round of revisions.
Experimental design: No additional comments. I am satisfied with the clarifications made in the new version and all my previous raised issues were properly addressed.
Validity of the findings: Sufficient explanations have been added to improve the manuscript. However, as the authors state "the aim of the current study was to assess the impact of cognitive testing on subsequent performance in conceptually different cognitive test". I would advice the authors to be careful using the word cognitive testing if what they refer to is the perception of novel humans/ objects during stress.
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: RESPONSIVENESS OF DOMESTICATED GOATS TOWARDS VARIOUS STRESSORS FOLLOWING LONG-TERM COGNITIVE TEST EXPOSURE
Review round: 2
Reviewer: 2

Output:
Basic reporting: Although the language used is largely very good, there are a few suggestions I would like to make:

L32: ‘in ‘the’ form of visual …
L38: You should make clear animals were isolated from conspecifics as they were not completely isolated given the presence of experimenters
L71: First = Initial? And also has = have
L82: those= these
L83, L541-542, L565, L565, L669: the term explorative although technically correct, is little used, and I would consider using exploratory instead
L157: 'they are usually kept in ‘petting zoos’ or by hobby breeders in Europe'
L234: ‘segment changes:’ this terminology is made clear after reading the methods section, but given it is mentioned in the introduction, readers at this stage will not understand what this means
L280: ‘except of eight animals’ = with the exception of eight animals
L345-L366: It needs to be made a bit more clear that the order of which the tests are explained is the order in which they took place
L459: would be best to add the test you are using, i.e. Spearman’s rank rather than just relying on the shorthand rs
L675: not unambiguous = ambiguous
Experimental design: no comment
Validity of the findings: I would just additionally like to ask, why for the novel human and object test did you use space utilisation of the inner versus outer segments instead for example time spent in or adjacent to the square where the object/ human is positioned?
Additional comments: I would like to make clear these suggestions are only for very minor improvements, and if largely unheeded, it will not make a substantial impact to the quality of the manuscript which I feel is very high. The author has largely acted upon or replied to my previous comments and would like to thank them for doing so. I am very satisfied with their responses and revisions and feel the manuscript has improved since I last read it.


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: SYNERGISTIC EFFECTS OF CLIMATE AND LANDSCAPE CHANGE ON THE CONSERVATION OF AMAZONIAN LIZARDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article fails to meet standards for one (the first) of the four criteria listed for Basic Reporting. Mainly, but not exclusively, the introduction and methods (especially), were challenging to understand and needed much rewording, more specific language (general or abstract language was common throughout the manuscript), and addition and reorganization of sentences/paragraphs to improve clarity and logical flow of information. Generally, the text information appears well supported with citations except where noted (very infrequent). Generally, article structure was good; appropriate figures and tables, including raw (or at least supporting Supplemental data sets derived from raw data) supporting objectives were provided although data in Tables S3 and S4 could be summarized and provided in other main text tables to aid interpretation of the results by the reader.  Modifications to Figures 1 and 2 are suggested. I particularly like Figure 3. Please see specific comments for each section of the manuscript provided below and inserted within the pdf manuscript document itself. By appropriately addressing all of the attached outstanding comments, I think the article should then be considered for a "pass" by PeerJ.

INTRODUCTION
The introduction would benefit from some moderate reorganizing and added details (about climate change effects on Amazon lizards). Some sentences required further clarification or rewording to be clearly understood.

I suggest rearranging text about SDMs of lines 74-79 by moving it nearer to the bottom of the Introduction, where you begin to discuss your research objectives on line 112. If you can combine the SDM paragraph with the one beginning on line 106 into 1 paragraph that should improve the logical flow of your Introduction.

Either at line 80 or line 99- Insert a new paragraph ahead of the one that is there. The new paragraph should describe the literature about climate change in Amazonia region and the resulting effects of those changes on range shifts or other projected impacts to lizards in that region. A minor suggestion- wherever you decide to insert the paragraph, I suggest you re-arrange the order of listing “climate change” before or after habitat loss/fragmentation wording when referencing them together, to match the order they are presented in paragraphs (for example, in line 63).

MATERIALS & METHODS SECTION

Sentences should be reorganized within and among paragraphs in this section and including the addition of a new subheading for a climate projections section and/or a scenarios section that fully describes the climate projections and levels of deforestation in each scenario.

Paragraph starting on line 130- the paragraph is missing a strong topic sentence summarizing the paragraph as a whole; please add one. You may need to add other following sentences to lead into the current one on line 130. Additionally, some sentences should be reorganized to reflect sequence methods were conducted (see specific comments in pdf).

Lines 137-139- important- What is meant by "related to"? Can you reword so that the relationship isn't about the centroid but with the muni itself? Reword to something like “occurrences were located within municipalities”. What exactly does it mean to be related to a muni ecologically? Because I thought a muni reference in the sentence above was only used to determine lizard locations and didn't have any real ecological significance but this sentence seems to make a distinction b/w being in a muni or not but it's not clear why it matters. It appears you forgot to add some sentences with details about the significance of being in a muni vs. not.

RESULTS SECTION

Line 253-254- It’s unclear what “V” is. Is it year? If so, 2016 isn’t the future (future identified as 2050 in methods) and 1953 isn’t current. P-values should be accompanied by test statistics values. Most importantly what is the effect size (with units)? Table S3 (cited line 252) only provides unsummarized results for these reported values.

Lines 264-5- It would help the reader for you to label these cited locations on Fig. 2 maps.
Line 269- citation Table S4- as with table S3, a table would be helpful that summarizes the data (distills it down), perhaps by grouping species in a way that highlights main points for conservation that you present in abstract and main text and by calculating the these percents for each group.

Line 297-299- I suggest providing the effect size (change in area between present and scenarios) and test statistic info that accompany the P-values in Table S5. I also suggest reporting in the text, the mean effect sizes for averages across all the 4% (mean across all these) and 19% (mean across all these) of species respectively.

Fig. 1- For panel (C) optimistic and pessimistic scenarios have very similar curves. If this panel only serves as an example and not actual results of the modeling, I suggest you adjust the curve for one or the other scenario so that optimistic is more “optimistic” and pessimistic is more “pessimistic”, or use a lizard species with more divergence in scenarios as an example, to better distinguish the 2 scenarios.

DISCUSSION SECTION

All comments are inserted in manuscript pdf. Some sentences in text require more detail/specifics and suggested rewording for sufficient clarity about authors’ intended meaning.

CONCLUSIONS SECTION

See comments inserted in manuscript pdf- minor text revisions and suggested rewording.
Experimental design: The article failed to meet standards for the 4th criteria because key information about the SDM, climate projection, and deforestation modeling processes were lacking sufficient detail as described more below. It's uncertain whether the article fails the 3rd of the 4 criteria because methods were inadequately described in enough detail to determine whether scientific rigor was sufficiently achieved. By appropriately addressing all of the attached outstanding comments, I think the article should then be considered for a "pass" by PeerJ.

MATERIALS & METHODS SECTION

Lines 134-137- Can the authors explain how well the habitat attributes of distributions of the lizards that were used in SDMs (training and validation data sets) represent the habitats of those lizards when they were collected? The concern is that if habitats have changed greatly since collections, that habitat determined using recent mapping technology (it’s still unclear to me how baseline/current attributes of habitat were determined- PRODES satellite imagery on line 177?) may not be the habitat  that was suitable for those lizards during collections, especially if lizards are in areas/municipalities where lots of deforestation and development has been occurring for decades. I think this is a very important issue the authors need to address because SDM results could misrepresent areas of relatively high deforestation as being suitable areas for lizards, when at the time of collection there was a lot more forest.

Lines 141-144- I'm not sure I understand. Please reword. Did you interpolate your predictor variables from  bioclimatic variables in WorldClim? You should define variables from WorldClim that you used to create your predictor variables, define the variables that were created by them (i.e., your predictor variables), and describe in enough detail how those predictors were created and used in your analysis. All those things don't necessarily need to be included at this point in your paragraph if some of it would logically flow better somewhere else, say in the analysis for example.

Lines 144-150- you describe a little, only one climate scenario here, yet on lines 230-231 you indicate 3 scenarios. These lines are confusing because you refer to using “all” pessimistic projections of RCP8.5 from apparently multiple climate models (which ones specifically?) and it’s unclear how you combine these projections for your scenarios (are these ensemble averages?)  and what variables you use (temp, precip)?

I think you need to add a new section and subheading to describe the climate projections used for your modeling scenarios in your analysis and justify why you used these and include which climate scenarios, variables, how they were extracted for each species occurrences or average latitude/longitude locations for each species, and any other details the reader would need to understand how you created your modeling scenarios.

Lines 154-158- More detail here is needed. Why were all 3 models needed? And how are “RBF” and “C = 1” important in the modeling and why these values were used and not others? Also, what are the predictor variables and response variables used in these models- these need to be fully described here and using table(s).
Line 159-160- Why would you remove all highly correlated variables? I think you’re not describing a step- selection among those correlated variables to include them in your models (because you don’t need to remove all of them only some of them that are correlated with the others). This comment applies to all Supplemental tables and figures of Table S1 is the first: there are no table headings or figure captions to describe what, how, when, where of each, making it hard to understand what they apply to (not stand-alone either); I’m not sure what PeerJ criteria are but this seems like a deficiency.

Lines 166-168- Reword and clarify. How did you average ranges across scenarios? Which scenarios? If you introduced the scenarios in a new section above then you won’t need to define them unless it was a subset of scenarios you averaged. Define “consensual map”; how was there consensus? by whom?

Paragraph for lines 175-191- This paragraph needs more clarification in multiple places. Has this deforestation model been published? If so, provide citations in the first sentence. If not, has it undergone stakeholder review or other review for accuracy, realism, performance, etc.? Would the Soares-Filho et al. 2006 citation apply toward this model so that you could cite it in the first sentence? If there hasn’t been any technical review other than from those that developed the model, this is concerning for your analysis. What is the PRODES imagery, define it? To make your BAU and GOV scenario deforestation rates comparable, it would help if you could add a little extra text translating the 55% reduction in deforestation to the “expected forest reduction” as you cited for the BAU scenario. Simply, would that be calculated as 0.37 * 0.45, or BAU rate * rate relative to the BAU rate? I’m having a very hard time understanding the last 2 sentences (lines 186-191) to be able to provide helpful comments for these lines. If you are extrapolating a models results to an area outside of its domain area, it’s important for the reader to know how you did that and understand the limitations with the new resulting area it’s extended to.

Paragraphs for lines 207-218- If the criteria of Rodrigues et al. 2004 was indeed “adapted” and not “adopted” then you should describe what changes were made for the criteria. As written (lines 209-210), it’s unclear what the criteria is measuring. Why species with <1000 km2 of stable habitat area (this could range down to 0 km2 by the way; I think you need a lower limit to put a cap on it) would have 100% of their distribution in PAs, >250 with 10% in PAs (again this could range to >1000 so not sure how these levels are mutually exclusive). In other words, I’m not sure the logic for these percentages by areas of stable habitat area. There should be some explanation of the criteria indicated I think. Lines 214-18: “all categories of PAs”; I don’t recall these being defined above and you probably need to do it here if not above.

Lines 194-218 or 221-239- somewhere in one of these sections you should briefly describe the process for identifying patterns 1-5 relating to Figure 3. I know you go into some general detail in describing the patterns in the results section but you didn’t provide background info or explain criteria/observations on how you determined these patterns that should probably reside in the methods. Similarly, you didn’t describe the process in using future scenarios to map these patterns in panels C and D. For example, it is unclear whether C and D are the product of the pessimistic and optimistic scenario results being combined in some manner to produce a single pattern 3 and single pattern 5, or did you map 3 and 5 only for the pessimistic scenario because this is the only future scenario presented in figure 3 (panel B). In other words, I would expect 2 sets of pattern results- 1 each for pessimistic and optimistic, but there is only 1 set and it’s unclear which scenario the patterns belong to or if it’s some joint combination (average)? How you created the pattern results in figures 2 and 3 need to be clearly described in the methods.

Lines 230-31- “Thus, we produced three curves for each species: one for the present day (2019) and two for the 231 future (2050), considering pessimistic and optimistic scenarios.”
This reminds me that you need to create a paragraph that describes each scenario thoroughly and perhaps a table that lists each current period, future optimistic, and future pessimistic scenario and their attributes, including the type of climate projection and deforestation scenario that characterizes each one (a full description of each).
Validity of the findings: Assuming that the study was conducted with suitable and well-designed methods (high rigor, 3rd criteria of 2. Experimental Design above), the article would appear to meet all 4 validity of findings criteria. Below are specific comments as well as comments provided in the  attached pdf document. By appropriately addressing all of the attached outstanding comments, I think the article should then be considered for a "pass" by PeerJ.

RESULTS SECTION

Lines 260-261- You report results for pessimistic scenario but not other 2; report them as well.

Fig. 2- Why are there no results for the optimistic scenario? Please add optimistic scenario results, in which case you will have a new panel (in total panels A-E). Please see comments above for the methods section, lines 194-218 or 221-239. Some brief text from the methods that you use to address those comments will need to be inserted in these figure captions to describe which scenario(s) are presented in these figures.

CONCLUSIONS SECTION

Lines 420- Circling back to an earlier comment, habitats modeled in SDMs may underrepresent the natural forest land cover needed to support lizards because of the temporal lag between when lizards were collected for scientific collections and when the landscape was characterized for this study. Unless I’m mistaken (which is possible but methods were not detailed enough for me to determine the timing of mapping), you should describe this problem with the analysis and how that could also affect the results to then affect conservation of lizards as you did in the previous lines.
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: SYNERGISTIC EFFECTS OF CLIMATE AND LANDSCAPE CHANGE ON THE CONSERVATION OF AMAZONIAN LIZARDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Suitable.
Experimental design: Suitable, except for some issues on Methods, as described on the fourth section.
Validity of the findings: Suitable, except for some issues on statistical procedures, as described on the fourth section.
Additional comments: The manuscript “Synergistic effects of climate and landscape change on the conservation of Amazonian lizards” provides a pertinent and important approach to how climate change and landscape changes could affect 63 species of Amazonian lizards. The introduction and hypotheses of the work are very clear and well grounded! Material and Methods is well-written, but there are points I would like to see revised and better clarified: (i) I think that updates on climate variables and future climate scenarios from the WorldClim database (CMIP6 rather than CMIP5) should be incorporated into this study; (ii) considering that there is a widespread recommendation that the construction of potential species distribution models be carried out based on at least 25 occurrence records (or, alternatively, that models with less than 25 records be evaluated in a peculiar way, such as jackknife “leave-one-out”), how do the authors justify the use of at least 10 records?; (iii) the authors used 3 Machine Learning algorithms, but they do not justify not using other classes of algorithms. Why did they only use this class of algorithms?; (iv) lines 144-150: despite saying that they used only the pessimistic scenario (RCP8.5), results are presented for both the optimistic and the pessimistic scenarios - please revise; (v) considering that the present study has a conservationist implication, what is the advantage of using the Receiver—Operator Curve” (ROC) as a threshold?; (vi) the AUC metric is out of use as a way of evaluating models in SDM, due to its lack of robustness. I suggest using TSS or another (in the case of models with less than 25 records, jackknife could be an alternative). In conclusion, line 419, please state which other environmental factors were not considered and which would also be important for the models.


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: SYNERGISTIC EFFECTS OF CLIMATE AND LANDSCAPE CHANGE ON THE CONSERVATION OF AMAZONIAN LIZARDS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Dear editor,

I am very thankful to review the article entitled “Synergistic effects of climate and landscape change on the conservation of Amazonian lizards”. The article talks about the synergistic effects of landscape modification, climate change on lizards species in the Amazon. The article is well written and highlight the urge needs to amazonian conservation. But I have some doubts and question that I will be listing below.
Experimental design: The processes modeled by author occurs in the different temporal and spatial scales. Climate change will be occur on a broad-scale perspectives (spatial) and around 50 years (time). The landscape changes act on biodiversity on a narrow-scale (spatial) and in few year (time). These difference in processes acting on biodiversity cause difficult to understanding the synergistic effects. In my opinion, the authors do not use spatial and time in  the correct form: Normally, climate and landscape are used as ecological filtering, here I don't see it.

I) I) the temporal climate models and landscape variables in not the same. You inferred the effects of climate change on species richness and after use landscape variables, but in the future this landscape variables is not real. It is a simplistic scenario that can be changed depending of a policy decision, causing noise on models results and conservation discussion. Why did you used models only under current landscape and climate scenario using a hierarchical approach (climate broad and landscape narrow), is sound good and the results is important to discuss amazonian conservation.  I also believe that  more landscape metrics is important your models building.

II) the species richness is related not only habitat amount (habitat area). The species (the basis of species richness)  suffer with landscape  configuration as connectivity, heterogeneity, matrix quality and others. So, use only a single landscape variable (habitat area) is not a real scenario to effect of landscape modification on species richness. I suggest the use o other landscape variables to simulate the landscape changes effecting on species richness. Only habitat area is unreal and can be causing extintic debt noise - more predict species than the real (see species x area relation and species debt).

Iii) Why did you use of Pearson correlation? Why 0.8 as threshold? I suggest the use of PCA eigenvalues as variables. See De Marco & Nobrega 2011 and the ENMGadgets R package.

iv) why did you use only Maxent and RF? You used only machine learning methods? Why? The machine learning sounds good to model species with broad distribution. But species with restricted distribution others algorithms are better.
Validity of the findings: Based on my comments, I would like to see a novel analysis approach and novel findings.
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: SYNERGISTIC EFFECTS OF CLIMATE AND LANDSCAPE CHANGE ON THE CONSERVATION OF AMAZONIAN LIZARDS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I suggest that a "Pass" for basic reporting be assigned to this manuscript. The authors addressed all of my most substantive comments of my initial review. There are only a few relatively minor comments I suggest that the authors address. I provided one of the main comments below that follows on the authors' rebuttal response (In order you will see my comment from my first review, authors' response, then my follow-up response to theirs. Other minor comments and suggested edits can be found in the attached manuscript PDF with my comments included.

Reviewer 1, first review: "Lines 137-139- important- What is meant by "related to"? Can you reword so that the relationship isn't about the centroid but with the muni itself? Reword to something like “occurrences were located within municipalities”. What exactly does it mean to be related to a muni ecologically? Because I thought a muni reference in the sentence above was only used to determine lizard locations and didn't have any real ecological significance but this sentence seems to make a distinction b/w being in a muni or not but it's not clear why it matters. It appears you forgot to add some sentences with details about the significance of being in a muni vs. not."

Authors' response: "We agree with the reviewer that the wording in these lines was confusing. We meant to talk about the resolution of some geographic coordinates used as occurrence records. Therefore, we re-wrote these sentences and re-allocated them in the previously mentioned new subheading, “Species occurrence data”. "

Reviewer 1 follow-up review: "Previous review comments about municipalities and centroids (Why species locations are referenced based on coarse municipality areas?, what it means for occurrences to correspond?) were not fully addressed here either. Please address.

I found some partially clarifying information, only by reviewing one of your cited references. Below is what I found in Ribeiro-Júnior, M. A., & Amaral, S. (2016a). You may need to summarize this information for yours? Or otherwise add pertinent details about specimen locations.

"Geographical coordinates associated with the examined specimens were used when available, otherwise specimen locations were georeferenced based on collector’s field notes, personal information, or any other source that could identify the locality with confidence. In such cases, coordinates were identified with gazetteers (e.g. [22]) and Google Earth [34]. After searching every available gazetteer to identify the precise geographical position of a record, when specific localities could not be retrieved, the record was not considered for analysis. Records with incomplete information (e.g. Pará, Brazil) were still cited in Appendix 1 and used for distributional purposes."
"Ribeiro-Júnior, M. A., & Amaral, S. (2016a). Diversity, distribution, and conservation of lizards
699 (Reptilia: Squamata) in the Brazilian Amazonia. Neotrop. Biodivers. 2, 195–421.

"
Experimental design: I suggest that a "Pass" for experimental design be assigned to this manuscript. The authors addressed all of my most substantive comments of my initial review. There are only a few relatively minor comments I suggest that the authors address. I provided one of the main comments below that follows on the authors' rebuttal response (In order you will see my comment from my first review, authors' response, then my follow-up response to theirs. Other minor comments and suggested edits can be found in the attached manuscript PDF with my comments included.

Reviewer 1, first review: "Paragraphs for lines 207-218- If the criteria of Rodrigues et al. 2004 was indeed “adapted” and not “adopted” then you should describe what changes were made for the criteria. As written (lines 209-210), it’s unclear what the criteria is measuring. Why species with <1000 km2 of stable habitat area (this could range down to 0 km2 by the way; I think you need a lower limit to put a cap on it) would have 100% of their distribution in PAs, >250 with 10% in PAs (again this could range to >1000 so not sure how these levels are mutually exclusive). In other words, I’m not sure the logic for these percentages by areas of stable habitat area. There should be some explanation of the criteria indicated I think. Lines 214-18: “all categories of PAs”; I don’t recall these being defined above and you probably need to do it here if not above."

Authors' response: "We changed the wording to address the suggested points. The exception is that the criteria are cited exactly as Rodrigues et al. (2004) proposed, so we maintained it. "

Reviewer 1 follow-up review: "There was an error in “250” (revise). See comment on PDF document line 264- comment: “Revise, this should be "250,000" (replacing decimal with comma), which I discovered after looking up Rodriguez et al. (2004). After revising, this set of criteria will now makes sense. Initially, I thought 250.000 was just an overstatement in the value's precision.”

Otherwise, author revisions have addressed these comments.
"
Validity of the findings: I suggest that a "Pass" for validity of the findings be assigned to this manuscript. The authors addressed all comments of my initial review.
Additional comments: Dear Authors,
I enjoyed reading your revised manuscript. Thank you for your effort in addressing my initial comments and I provided only a few minor comments for additional clarification of your methods plus a few minor edits, which you can find in the attached manuscript PDF with my comments. Please make the appropriate text edits in response to my comments, though I see NO reason to review your next manuscript draft.
Best regards,
Reviewer 1


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: SYNERGISTIC EFFECTS OF CLIMATE AND LANDSCAPE CHANGE ON THE CONSERVATION OF AMAZONIAN LIZARDS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: I thank the authors of the manuscript “Synergistic effects of climate and landscape change on the conservation of Amazonian lizards” for the corrections and justifications for my comments. I would like to ask you, please, to check the possibility of inserting in the text the justification given in the reply letter for the use of the Receiver Operator Curve (ROC) as a threshold. Added to that, considering the current advantages of remote access to workstations, I insist on the possibility of redoing climate models according to CMIP6, possibly even including them as a comparison with CMIP5.
Validity of the findings: No comment.
Additional comments: No comment.


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

Input:
Title: RAPID PARAMETER ESTIMATION FOR SELECTIVE INVERSION RECOVERY MYELIN IMAGING USING AN OPEN-SOURCE JULIA TOOLKIT
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper is well written and clear.

1 - The paper does not set itself properly in the context of existing literature. Importantly, it does not cite the original qMRLab paper (Karakuzu, A., Boudreau, M., Duval, T., Boshkovski, T., Leppert, I. R., Cabana, J. F., … & Stikov, N. (2020). qMRLab: Quantitative MRI analysis, under one umbrella. Journal of Open Source Software, 5(53), 2343. doi: 10.21105/joss.02343). qMRLab provides an SIR FSE fitting routine and I think a comparison of the two would be interesting to readers (https://qmrlab.readthedocs.io/en/master/qmt_sirfse_batch.html).

1b - Other toolboxes which do not include SIR FSE, such as QUIT (Wood, (2018). QUIT: QUantitative Imaging Tools. Journal of Open Source Software, 3(26), 656, https://doi.org/10.21105/joss.00656), are of relevance to the discussion of toolboxes and languages.

2 - With regards to the SIR model itself, the recent work of van Gelderen (https://linkinghub.elsevier.com/retrieve/pii/S1053811920301877) is not discussed. van Gelderen calls into question the assumption that R1m=R1f, and instead indicates that R1m is approximately 4 per s at 3T. The authors should discuss how this contrasts with their fitting assumptions and would affect their results. Furthermore, papers such as https://linkinghub.elsevier.com/retrieve/pii/S0006322314007501 suggest that the exchange rate can change due to pathology (specifically inflammation), which suggests that the assumption of fixed kmf may not be safe.

3 - Much extraneous detail about the Julia language are included in the paper, which could be replaced by a reference or link to the Julia educational materials. For instance, I would remove the mention of multi-dispatch in the discussion, as I doubt any readers who have not previously used Julia will know what this is (and neither is this paper the correct place to explain the concept). It is unnecessary to give the specific names of code files or Julia functions. It is sufficient to only cite the papers that these are from. Similarly, the sentence on choice of editor should be removed. While I also use Visual Studio Code, it is of little relevance to the reader.

4 - Throughout the paper citations often appear after the full stop of the sentence they refer to, i.e. . (citation). This should be corrected.
Experimental design: The methods are described with sufficient detail and the analysis code is open source.

5 - The research question is not particularly well defined, and this is the central weakness of the paper as currently written. The core message of the paper is that the authors took an existing method and implemented the analysis in a new programming language which was faster. While the speedups are impressive, there is not significant new scientific content in the paper.

There are then two other stated goals - one is implementing the method directly on the scanner, and the other is the extension of their code into a toolbox for multiple techniques. However, these goals are merely aspirational and are not actually addressed in this paper. I think the passages about these goals should be removed or de-emphasised, and more emphasis given to why the work presented in the paper is interesting in its own right.
Validity of the findings: The main findings are valid.

6 - With reference to point 4, the authors do not discuss the numerous practical difficulties for implementation on the scanner such as motion correction and integration with the scanner image database. Motion correction in particular is not currently discussed in the paper at all. As in point 5, I  suggest the passages about implementing the method on the scanner are removed or significantly rewritten as the authors to address such issues.

7 - It is not clear to me why the authors report the run time with differing numbers of Julia threads. It appears that performance decreases with an increasing number of threads, which is counter-intuitive. Furthermore it is not clear what is meant by "only improved fits by a small amount" on line 257. Both points should be clarified.

8 - The noise analysis in figures 1 & 2 is an interesting way of visualising the performance across a range of parameter values simultaneously. However, a diverging color map should be used for figure 1 C/D, and I suggest that the differences are scaled into  fractions or percent, i.e. the presented variable should be (Fitted - Truth) / Truth. Currently due to the grayscale colorbar and use of absolute units, it is difficult to asses how the noise varies with parameter values.

9 - While the authors discuss the overhead of various aspects of Matlab, they do not discuss the overhead from Julia's Just-In-Time compilation. What was the start-up time of their code if running as a command-line script rather than in an interactive Julia session?
Additional comments: Overall this paper is well written, contains no substantive errors, and I think will be suitable for publication after addressing the points above. As currently written there is too much emphasis on the specifics of the Julia language itself, for which the interested reader can be directed to the educational materials, and also on potential future uses of the toolbox for which this paper does not actually present evidence of. The paper will be improved if more focus is given to the actual results and better comparisons are made to existing work.


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: RAPID PARAMETER ESTIMATION FOR SELECTIVE INVERSION RECOVERY MYELIN IMAGING USING AN OPEN-SOURCE JULIA TOOLKIT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors reported an open-source and julia-based software package that can fit the biexponential MRI signals obtained from selective inversion recovery MRI scans, producing quantitative maps reflecting bound water fraction (with very highly significant implication to research and clinical studies of neuro-degenerative disorders). Credits should be given to authors for their great efforts of implementing Julia based MRI processing codes, building jupyter-notebooks (that greatly benefit students and trainees), and providing software interface for Matlab and Python users.
Experimental design: The study includes numerical simulation (including Rician noise that is appropriate for simulating MRI signals), phantom and human MRI. Overall it is well designed and rigorous.
Validity of the findings: The experimental results from simulation, phantom, and human studies demonstrate that the implemented Julia software package is valid, with a significantly reduced computation time as compared with their previous matlab implementation. Although human data were obtained from only 1 subject, this is sufficient for validating the implemented julia software.
Additional comments: As an MRI researcher with experience in julia programming, I am convinced that the reported study and the implemented open-source software will greatly benefit neuroimaging community. It would be great if the authors could further discuss the following in their manuscript.

First, when using the implemented software procedure in human studies, it is expected that the motion artifact should be addressed before fitting the biexponential signals. Could the authors discuss if the motion artifact should be removed with Julia (in future implementation) or other tools (e.g., FSL)?

Second, the readers of this paper might be interested to know if the program could be further accelerated with GPU computation. It is my understanding that GPU computation is also available in Julia, and I would be interested to know authors' opinion on this topic.


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: RAPID PARAMETER ESTIMATION FOR SELECTIVE INVERSION RECOVERY MYELIN IMAGING USING AN OPEN-SOURCE JULIA TOOLKIT
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed my concerns on reporting and I have no further comments.
Experimental design: The authors have addressed my concerns on design and I have no further comments.
Validity of the findings: I thank the authors for clarifying the results section of the paper in response to my previous comments. First, two minor points:

1 - The sentence at lines 250/251 of the paper, as written, suggests that the MATLAB code was faster when the opposite is true. I suggest this sentence is reworded.
2 - The authors do not appear to state the number of cores/threads available on the CPUs used for processing. I believe that 8 cores/16 hyper-threads are available for the serial numbers listed, which makes some sense given the ~6 times speed-up in MATLAB when enabling threading. These numbers would be helpful to the reader.

And now a major point. I cloned and ran the author's repository to examine the multi-threading issue earlier. I am not an expert in Julia, so I followed the instructions on multi-threading at this web-page: https://docs.julialang.org/en/v1/manual/multi-threading/

I noted that the author's main script, "run_me.sh", did not have the `--threads=auto` command-line argument in the Julia call.

I first ran the script as-is, and confirmed the authors observation that adding the @threads decorator did not change the run-time. I then added a print statement to output the number of available threads, which was 1. I then added the `--threads=auto` option and re-ran. The program then reported that 6 threads were available (on my 6 core machine), I confirmed that the program used all the cores by observing the process monitor, and this led to a 2x speed-up in program execution.

I have submitted the required changes as a pull-request on Github https://github.com/nicksisco1932/The_SIR-qMT_toolbox/pull/5

I ask the authors to confirm if they can replicate this behaviour on their machines. If they can, then the results section will require re-writing. If they cannot, I think they must provide a comprehensive explanation of the threading behaviour in Julia.
Additional comments: No further 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: RAPID PARAMETER ESTIMATION FOR SELECTIVE INVERSION RECOVERY MYELIN IMAGING USING AN OPEN-SOURCE JULIA TOOLKIT
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors have addressed my concerns on reporting and I have no further comments.
Experimental design: The authors have addressed my concerns on reporting and I have no further comments.
Validity of the findings: I thank the authors for thoroughly responding to my request in the previous review. The behaviour of multi-threading in Julia is clearly more complex than is perhaps expected, but I agree with the authors that this is beyond the scope of the paper. I have no further issues with their work.
Additional comments: The authors have addressed my concerns on reporting and I have no further 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: SUBJECT SPECIFIC MUSCLE SYNERGIES AND MECHANICAL OUTPUT DURING CYCLING WITH ARMS OR LEGS
Review round: 1
Reviewer: 1

Output:
Basic reporting: English could be improved.
Experimental design: The experiment is original and the study was well conducted but the design is not well suited for the first research question (#1)
Validity of the findings: Some claims are not supported by any analysis.
Additional comments: Review of the paper entitled: Subject specific muscle synergies and mechanical
output during arms and legs cycling

The authors investigated the intra and inter subject variabilities in the structure of muscles coordination and in their mechanical output during leg and arms cycling. They found higher inter-individual variability in the temporal structure of the upper limbs muscles than in the lower limbs despite similar variability in the torque profiles.
The study is interesting and original but some claims are not supported by any analysis.
Major
-An important conclusion of the study is that for the arms the temporal recruitment of the synergies is highly flexible without strong repercussion in the mechanical output. However, the variability in mechanical output seems greater for the arms than for the legs (it should be tested) and it is not clear whether the relation between the variability in the synergy activations and the variability in the mechanical output is different or not between the two tasks.
-Torque represents the forces applied orthogonal to the crank, not all the forces, and the movement might be much more constrained (in the sagittal plane) for the lower limbs. For the lower limbs whole body stability might be greater too. The variability in muscle activations might simply reflect these two aspects.
-The functional interpretation of the synergies is likely biased by the impact of gravity on the different segments in the two tasks. However, this aspect is probably not the most important and as the authors commented “it is impossible to attribute a precise mechanical action for these synergies” (L330).
-The author should provide evidences that the differences in the temporal structure is not due to the differences in self-selected cadences, i.e., can you ascertain that small changes in the arm cycling cadence is not associated with large modifications of the muscles activation timing.
-I recommend using statistical tools to confirm some of the claims/conclusions. For example, the spatial structures (W) appear similar across participants while it was not the case for the temporal structure (L264-265). However, VR and Pearson’s r were computed using vectors of different dimensions. It is easier to obtain great correlations when comparing small vectors, therefore the differences might not be due to different “similarities” but to different vectors size. Using the same thresholds (moderate, strong) might not be very relevant here. Intra and inter similarities could be compared more formally also. Importantly, the analysis is dependent on the number of synergies extracted for each participant. Different approaches were used in the literature that do not depend on this number (e.g., Yang et al. 2019 or Frère and Hug, 2012).
Frère, J., & Hug, F. (2012). Between-subject variability of muscle synergies during a complex motor skill. Frontiers in computational neuroscience, 6, 99.

Yang, Q., Logan, D., & Giszter, S. F. (2019). Motor primitives are determined in early development and are then robustly conserved into adulthood. Proceedings of the National Academy of Sciences, 116(24), 12025-12034.

minor
Although the reviewer is not a native English speaker, the English sounds weird at different places and could probably be improved.
Despite the exclusion criterion (L 101), it is very likely that people are more used to the leg ergometer (a bike) than the arm ergometer.
L 88. The study is not really designed to investigate the first objective. The link between muscle coordination and mechanical output was simply inferred from the results but their relation was not really evaluated.
Were the feet attached to the pedal for the lower limbs?
L 171. Was this algorithm based on the kinematic data? Is so, which one?
L 157. Was the lag corrected (zero lag filter)?
L 232. Torque and body weight do not have the same dimensions. It is not exact to speak of percentages.
L 265. What does it mean that “P03 and P06 doesn’t showed the synergy S2” if four synergies were extracted for all of them and that  “functional organization ... is shared among participants”. 2/6 of the participants represents a lot. Idem in L 282.
L 347. This interpretation is questionable: for the legs two synergies appear to push, one to make the transition (at the bottom dead center) and the last one seems to lift the leg (= pushing, lowering and pulling, respectively, for the arms).
L 364. The variability in Hug et al .2010 was related to the spatial structure.
L 373. This absence of correlation should be demonstrated.
The discussion should start with a resume of the main findings.


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: SUBJECT SPECIFIC MUSCLE SYNERGIES AND MECHANICAL OUTPUT DURING CYCLING WITH ARMS OR LEGS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: I have some concern regarding the interpretation of the results, as the analysis of the kinematics has not been shown and it could play an important role to explain the obtained results.
Additional comments: In this study, T. Cartier and co-workers analyze the muscle coordination underlying the execution of a cycling task with arms or legs. By using the muscle synergy framework, they show that the complexity of the coordination of 11 upper or lower limb muscles is the same and consists of the activation of 4 muscle synergies. By recording the crank torque, they also provide a biomechanical interpretation of the role of the identified muscle synergies. They conclude that arm cycling has a higher complexity in terms of biomechanical functions, as identified by the higher variability in synergy structure and recruitment.

The study faces an important topic related to motor control, with implications in the field of neurorehabilitation. Of particular relevance is the relationship between the neural circuits regulating the control of cyclical movements of arms and legs. The work is well introduced and properly contextualized. The method section is detailed and well described for repeatability. The results are also well presented. However, I have some doubts regarding few methodological points, as some definition should be clarified. Moreover, I have some concern regarding the interpretation of the results, as the analysis of the kinematics has not been shown and it could play an important role to explain the obtained results.

-	The authors mention that reflective markers were used to calculate 2-D kinematics of upper and lower limb, but these results are not shown. Might the variability in the motor control strategies of the upper limb be related to a higher variability in kinematics? This is an important point to be clarified. The authors could show the kinematics tracks of the upper and lower limb, or even perform the VR analysis on these tracks. I would suggest at least to insert a figure showing the ankle, knee and hip on a panel and wrist, elbow and shoulder on another panel, similarly to what done in Figure 3.

-	I have some doubts regarding the structure of the leg cycling muscle synergies. While S1, S2 and S4 have a functional significance/biomechanical interpretation in terms of co-active groups of muscles and are in line with previous literature, the structure of S3 seems quite strange. It is composed of the co-activation of GL and TF (I suppose it is Tensor Fasciae Latae, its acronym has not been defined at lines 145-148). How is it possible that GL works independently with respect to GM? And which is the biomechanical significance of such synergy?

-	The definition of rinter and rintra is not clear, as it has not been explained in the text. Did you calculate all the possible pairs of r (intra or inter, escluding autocorrelation) and then use the average value? Please clarify this point.

-	Line 63: activates – activated.

-	Lines 218-220: the meaning of this sentence is not clear, I suggest rephrasing.

-	Lines 223-227: I suggest rephrasing as the whole period sounds awkward. Also, in lines 223-225 it appears that there is no quantitative analysis, but only a visual analysis performed by the authors. Why not quantifying this information?

-	Lines 232-233: what do you mean when expressing torque as a percentage of body weight?

-	Equations 2 and 3, in the VAFtot and VAFm definition, a term (1 - ) is missing before the summation.

-	I am not a native English speaker, but I think that English throughout the manuscript could be improved.


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: SUBJECT SPECIFIC MUSCLE SYNERGIES AND MECHANICAL OUTPUT DURING CYCLING WITH ARMS OR LEGS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I thank the authors for their additional analysis and for their extensive work of revision to the mansucript. All my previously raised concerns have been thoroughly addressed. I have no further comment related to the manuscript.


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

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

Output:
Basic reporting: I was very excited to read this paper as this is an important study and finding these trade-offs is really furthers our understanding of coral immunity. The use of Mote micro-frags is a great idea and has the potential to produce some very interesting data. I feel this paper could be improved by going a bit deeper into the data analysis and tightening up the main findings. The title itself is compelling but the data, as presented, don’t really tell the same story. I would love to see more hypotheses presented and tested.

Introduction
The last paragraph of the introduction could be a bit clearer on what the motivation of this study is. Is it to find the mechanisms that are allowing for faster growth in the microfragments? Or is it looking for trade-offs with growth? As I see it, this study isn’t really about the process of microfragmentation, but more looking about using this set-up as an experimental platform to study the rapid growth of microfrags and the potential trade-offs.

Results
Line 246 – seems like discussion or potentially a justification of methods that could be moved into the methods.
Why are growth rates of the coral fragments not presented? I would like to the range of growth rates and how many frags of each species had a faster vs slower growth rate.
Figure 1 – can you highlight or bold the stats that are significant or simplify the statistical output. Would an average reader understand what each of the pMCMC values mean? How do I interpret this and understand what interaction was significant?

Line 256 – “Immunity results indicate some tradeoffs between enzymatic activity and growth “ this seems like a discussion point, please give stats or this negative correlation.

Figure 2 – again can you add information to help the reader understand what is important in these graphs? Which have significant correlations between growth and immune paraments? Bold and star the significant interactions.

Also, why is log initial weight used instead of growth rate? I understand that smaller frags grow faster but its not that convincing without using the actual fragment growth rates. Also, this is the title of your paper.  Another idea is to bin the fragments into groups that all are similar growth rates and show the immune activity of those. Also perhaps grouping each fragment into fast, medium and slow growth rates and showing the immune properties/symbionts and gene expression of those would illustrate the points well too.

Figure 3 and the results of the symbiont identification could be elevated a bit. Was there different growth rates between the individuals that different symbiont populations. For example there were several M. cavernosa that didn’t have Durusdinium, what was their growth rates? Was there a significant correlation between % of any symbiont type and growth rate? Did symbiont population drive growth in any way? I think there are numerous ways to test this in your data set. Even grouping the individual stacked plots presented in figure 3 by slowest to fastest growth would make this figure more interesting.

Figure 4 and gene expression - Is DESeq the best way to get the patterns of this data if you only have a continuous variable? Why wouldn’t you bin them in groups of low, medium and high growth rates to look for DEGs or use WGCNA which is made for this type of analysis. I feel that the gene expression data could be much deeper and give more in depth results. There also isn’t an appreciation for the immune genes or GO terms that can be easily seen. Again, this is the title of the paper? Shouldn’t there have been an effort to add this trade off hypothesis to the figure? Also looking for trade-offs with the gene expression would be good, like were there any growth GO terms that were upregulated and immune processes downregulated or vise versa?

Where is Mcav gene expression data? DEGs are not the most important thing in this type of data set. I feel a lot of data was left on the cutting room floor and not analyzed completely.

I also really appreciate that you presented the uncharacterized genes, as this is a common issue with non-model organism, but are there are ways to get some basic information about these gene pFam or even eggnog annotation that will you the domains of the base ortholog? Perhaps also putting them is separate figure would help direct the focus of the gene expression figure to the known genes and allow for some deeper/different analysis of the uncharacterized genes.

The heat map could be more informational and impactful and could be a valuable contribution to the genes involved in  growth rate. Some grouping by GO term or function would be great. Show the immune genes clearly.  Also the legend is backwards from conventional heat maps.

Perhaps can you regress the gene expression to the immune parameters? These are additional phenotypes to help pull out patterns. Then you could overlay the growth data to really show nice correlation and trade-offs.

Discussion
This discussion could use some re-organizing. for example the entire paragraph on line 282 seems out of place. discussing the data as far as the trade offs in the smallest fragments and as far as growth rate of the fragments seem like two different questions to me.
Experimental design: see above
Validity of the findings: see above
Additional comments: No additional comments