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: DIMINISHING RETURNS AS A FUNCTION OF DISTURBANCE
Review round: 2
Reviewer: 1

Output:
Basic reporting: As I mentioned in my previous review, the literature review is not complete. In the revised version, the authors didn't act upon this comment. As a reply they mention that they refer to the articles I mentioned. Indeed, these articles are referred to but the paper lacks a thorough discussion of these papers (and others). The single page introduction section does not do a sufficient job in explaining the background and highlighting what problem the authors are solving (and why this problem needs to be solved)
Experimental design: The methods are fundamentally flawed. The authors use an ad-hoc approach to simulate their data (they draw g scores from a normal distribution, and next, they draw test scores that are correlated to those g scores and add disturbances).
To study a topic like this, an explicit statistic model is needed from which data is simulated. This can be a factor model or an item response theory model, but not an intuitive, ad-hoc approach like this.

In my review I also pointed out that the authors should use more contemporary approaches to study differentiation. The authors argue that they *do* apply these approaches. This is not true. Although they indeed use the conceptual idea of heteroscedastic residuals and non-linear g loadings, they test for these ideas using -again- ad hoc approaches (e.g. correlating the g scores to the log-residual variances).  This is nowadays really not admissible. If you want to test for heteroscedastic residuals you really have to apply a statistical model (e.g., that of Hessen & Dolan), consult model fit, consult standard errors, etc.

Finally, the authors argue in their reply that PCA should be studied as it has been used previously. I dont agree. I dont think we should study what is wrong with the methods we used in the past. We should study what is wrong with the methods we use *currently* and PCA is not one of them.
Validity of the findings: Because of the above, the results of the present paper are not interpretable. The authors should use proper methods and not the ad-hoc procedures that they are currently using.
Additional comments: This paper really lacks rigor both in the theoretical underpinning and in the statistical treatment of the research problem.


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: DIMINISHING RETURNS AS A FUNCTION OF DISTURBANCE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The issued raised in the review have been adequately answered. If the traditional methods stay, it should be highlighted that they are suboptimal and only serve the purpose the authors described in the rebuttal: "By including the method and showing that it is very (more so than more contemporary methods) sensitive to the influence of disturbance, we might contribute to putting the traditional method to a well-deserved rest."
Experimental design: Answer is accepted.
Validity of the findings: Answer is accepted.
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: DIMINISHING RETURNS AS A FUNCTION OF DISTURBANCE
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors chose to argue away most of our concerns. To be honest, I wasn't asking for the extra information for my sake. Rather, I was expecting them to explain why they chose not to include it, if that was their decision. In any case, I appreciated the inclusion of some of our suggestions.


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

Input:
Title: DIMINISHING RETURNS AS A FUNCTION OF DISTURBANCE
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the most recent revision, reviewer suggestions were incorporated and as a result the manuscript is more balanced and clearer than previous versions.


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: IDENTIFICATION OF NOVEL MUTATIONS IN RNA-DEPENDENT RNA POLYMERASES OF SARS-COV-2 AND THEIR IMPLICATIONS ON ITS PROTEIN STRUCTURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This is a very interesting observation of RdRp mutations in SARS-CoV-2 in a group of viruses isolated from India. Three mutations were predicted to influence secondary structure of RdRp, in particular one of the most common mutations found currently in the GISAID database (P323L). The authors suggest that this P323L mutation will cause resistance to anti-virals. However, since generally viruses trend towards attenuation, could this be an attenuating mutation instead? Could the authors comment on this?


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: IDENTIFICATION OF NOVEL MUTATIONS IN RNA-DEPENDENT RNA POLYMERASES OF SARS-COV-2 AND THEIR IMPLICATIONS ON ITS PROTEIN STRUCTURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Chand G.B. et al compares the available RdRp sequences of SARS-CoV-2 from Indian isolates and ‘Wuhan wet sea food market virus’ sequences.
The authors report seven mutations observed in Indian SARS-CoV-2 isolates and three unique mutations that showed changes in the secondary structure of the RdRp protein at region of mutation. They also studied molecular dynamics using normal mode analyses and found that these mutations alter the stability of RdRp protein. Based on their analysis, the authors then propose  that RdRp mutations in Indian SARS-CoV-2 isolates might have functional consequences that can interfere with RdRp targeting pharmacological agents.
Experimental design: The experimental design is sound. The research question is well defined. The investigation standard are good. Methods are straightforward.
Validity of the findings: The findings are novel, their impact important. Conclusions are well stated.
Additional comments: The authors compares the available RdRp sequences of SARS-CoV-2 from Indian isolates and ‘Wuhan wet sea food market virus’ sequences.
They report seven mutations observed in Indian SARS-CoV-2 isolates and three unique mutations that showed changes in the secondary structure of the RdRp protein at region of mutation. The authors  also studied molecular dynamics using normal mode analyses and found that these mutations alter the stability of RdRp protein. Based on their analysis, they then propose  that RdRp mutations in Indian SARS-CoV-2 isolates might have functional consequences that can interfere with RdRp targeting pharmacological agents. The manuscript is clearly written, the analysis is well performed and the data are clearly presented. A few typos need to be corrected.


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: IDENTIFICATION OF NOVEL MUTATIONS IN RNA-DEPENDENT RNA POLYMERASES OF SARS-COV-2 AND THEIR IMPLICATIONS ON ITS PROTEIN STRUCTURE
Review round: 1
Reviewer: 3

Output:
Basic reporting: This paper reports mutations in RdRp from SARS2-Cov-2 from Indian isolates.
Experimental design: Based on sequence alignment and structural analysis, the authors identified these mutations in the RdRP and determined their possible effects on the structure integrity of RdRP through the use of molecular dynamic (MD) modeling.
Validity of the findings: The RdRP mutations include A97V, A185V, I201L, P323L, L329I, A466V and V880I.  The modeling studies suggest some of mutations can stabilize or destabilize RdRP. In addition, the authors should use the recent structure of RNA-RdRP complex structure to analyze these mutations on the replication ability of RdRP (PDB code 7BV1 and 7BV2 in https://science.sciencemag.org/content/early/2020/04/30/science.abc1560.abstract).
Additional comments: Overall, this is an interesting paper and should be considered for publication ASAP.


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: CONTEXTUAL VIOLENCE AND ITS LINK TO SOCIAL AGGRESSION: A STUDY OF COMMUNITY VIOLENCE IN JUÁREZ
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	In the introduction in the section on Demographis section it is important to describe the current state of community violence.
2.	Although the document as a whole observes the good use of the concept of community violence and one of its main effects is contextual victimization, an error is observed in line 79 between parentheses must say contextual victimization. The same happens in lines 84 and 87 where contextual victimization of community violence should be indicated (see Echeburúa 2004).
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This is a really interesting topic, this paper is highly relevant in terms of the topic discussed, and correct in terms of the method used. However, there are some important issues that need to be corrected and improved.  Specifically, the following modifications are considered necessary:
1.	In the introduction in the section on Demographics section it is important to describe the current state of community violence.
2.	Although the document as a whole observes the good use of the concept of community violence and one of its main effects is contextual victimization, an error is observed in line 79 between parentheses must say contextual victimization. The same happens in lines 84 and 87 where contextual victimization of community violence should be indicated (see Echeburúa 2004).
3.	Since a robust data analysis is used, it would be important to deepen the discussion of the results, especially since only two predictors (neighborhood and school) remain in the model. Differences by sex would also be interesting to discuss in greater depth.


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: CONTEXTUAL VIOLENCE AND ITS LINK TO SOCIAL AGGRESSION: A STUDY OF COMMUNITY VIOLENCE IN JUÁREZ
Review round: 1
Reviewer: 2

Output:
Basic reporting: I would suggest adding some details in the tables and figures which could be useful for the readers, as follows:

•	Table 1, it is better to include sample size in the title of the table, e.g. (N=298).
•	In Figure 1 to Figure 4, the titles and the notes are repeated.
•	In Figure 1 to Figure 4, the author provided whether the effect of contextual violence reaches statistical significance and the R-squared for some paths but not all paths. For example, in Figure 1, the author stated “Post-traumatic stress also has positive predictive eﬀects on disposition to aggression”, but whether the coefficients reach statistical significance at P<0.05 level. Similar problems are observed in Figure 2 to Figure 4.

In terms of the results:

•	In the initial model (Figure 1), some factor loadings are too low, e.g. CV school and CV witness. Aware of this, the authors applied a new model (Figure 2) to estimate the effect of contextual violence. However, in the new model, the author adopted “factors of contextual violence at school and neighborhood” (line 267-269). Please explain why use the two factors and why the latent variable of the contextual violence is no longer used in the new model?
•	RMSEA of both the initial and new models (Figure 1 and Figure 2) are above 0.05. Further justification is needed.
•	The authors calculate the indirect effect of contextual violence on AQ (Line 280-281, Line 307-308). Do the indirect effects reach statistical significance at P<0.05 level?
Experimental design: My comment on the research design are as follows:

•	The authors didn't provide enough information to the reader on how this manuscript fills an identified knowledge gap. The authors need to further expand upon the knowledge gap being ﬁlled by this manuscript.
•	In the line P77-80 the author defined “contextual violence” as “hearing and witnessing violent act in one’s community” and use the term throughout the entire manuscript. However, in line 58-76, the paper reviewed is mostly concentrated on “violence exposure”. If the two concepts are different, the author needs to explain what is different between “contextual violence” and “violence exposure”. If the two concepts are the same. Why the author use “contextual violence” given the “violence exposure” applied more frequently in their review part?

The method is described with sufficient detail and information to replace. This section is also well presented with many details. My comments are as follows:

•	The authors investigate contextual violence->PTSD->aggregation using SEM. However, the direct effect of contextual violence on aggregation is not estimated. Why?
•	Since all the measurements are based on well-established psychological inventory, why the authors applied exploratory factor analysis (Line179-180) instead of confirmative factor analysis?
Validity of the findings: no comment.
Additional comments: This paper "Contextual violence and its link to social aggression: a study of community violence in Juáre" aims to address an interesting question on how contextual violence affects social aggression through changing the level of PTSD. Generally speaking, the paper is well-formatted and articulated. I commend the authors for their extensive data set, compiled over many years of detailed ﬁeldwork. Besides, the manuscript is written in a professional, unambiguous language. My comments are listed above, which should be considered before acceptance.


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: CONTEXTUAL VIOLENCE AND ITS LINK TO SOCIAL AGGRESSION: A STUDY OF COMMUNITY VIOLENCE IN JUÁREZ
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1. It is necessary to present conceptual definitions of direct, indirect and contextual violence, indicating the authors on which they are based.
2. It is important to rely on a psychological model that indicates the relationships between the variables considered and it can justify statistical model tested in the study.
3. The introduction of the acceptance of violence variable should be justified in the background.
Experimental design: 1. You must indicate how the study sample was selected
2. A data analysis section should be created explaining the analyzes performed, as well as to justify why it was considered to measure the neuroticism factor.
3. An important limitation is that the fifth instrument in the Mexican population has not been validated. It is indicated that it was validated in a Spanish context but cultural differences between Mexico and Spain are important and that validation is required.
Validity of the findings: 1. It was not measured if the participants suffered direct or indirect violence in their childhood. This may be an important limitation of the study since the results could be explained by the experience of direct or indirect violence, and not so much by contextual violence.
Additional comments: 1. Post-traumatic stress and violence acceptance variables must be correctly codified.
2. The Mexican validation reference of the Checklist for PTSD Traits Scale must be updated, as it has already been published in 2020.


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: CONTEXTUAL VIOLENCE AND ITS LINK TO SOCIAL AGGRESSION: A STUDY OF COMMUNITY VIOLENCE IN JUÁREZ
Review round: 2
Reviewer: 1

Output:
Basic reporting: Added the description of the current prevalence of community violence in Ciudad Juárez.
The use of the term contextual victimization was corrected
Experimental design: No comment
Validity of the findings: Differences by sex are included
Additional comments: The article in its second version includes the suggested corrections. As it is a relevant topic and of great interest, I recommend its publication


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: CONTEXTUAL VIOLENCE AND ITS LINK TO SOCIAL AGGRESSION: A STUDY OF COMMUNITY VIOLENCE IN JUÁREZ
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 addressed all of my 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: CONTEXTUAL VIOLENCE AND ITS LINK TO SOCIAL AGGRESSION: A STUDY OF COMMUNITY VIOLENCE IN JUÁREZ
Review round: 2
Reviewer: 3

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
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: SSRENRICHER: A COMPUTATIONAL APPROACH FOR LARGE-SCALE IDENTIFICATION OF POLYMORPHIC MICROSATELLITES BASED ON COMPARATIVE TRANSCRIPTOME ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors illustrate the development of a new software called SSREnricher to enrich polymorphic SSRs through the comparative transcriptome analysis. The issue is quite interesting and certainly of considerable applicability, reducing the lobariosity of traditional metodologies for microsatellite identification within species. Anyway, it is not clear whether this software can also work using genomic data or it is limited to EST-SSR.
The manuscript is written in an extremely simple and clear English language, although sentences without grammatical construction or too short often recur. Bibliography as well as the scientific discussions should be enriched.
The article structure should be modified with “Results and discussion” and “Conclusions” instead of “Results” and “Discussion and conclusions”. Figures and supplementary files should be better labeled and accompanied by more detailed captions.
Experimental design: The research is in accordance with the aims of the journal. However, in my opinion, it has been described in a too reductive way, because the potential of this software with genomic data is missing, for example, as well as the description of the performed wet experiments.
Validity of the findings: The research is very interesting and of considerable application impact, thus meeting the needs of scientific community. Anyway, not always well described or in a detailed form. The manuscript needs major revisions and some aspects to be clarified before pubblication.
Additional comments: Introduction:
raws 44-47 and 60-61: please, add some important references.
Mat&Met:
The verbs in this section are usually in a past tense. Furthermore, since the research include bioinformatic analysis and validation by wet experiments, this section should be enriched with protocols and details of the lab work done (sample preparation, DNA extraction, PCR reaction, agarose gel and capillary electrophoresis). If they refer to already known protocols, they should be mentioned as references. Moreover, punctuation must be corrected and short sentences without grammatical construct must be avoided (ex. raws 75-77).
Raw 86: please, specify the meaning of “c1” and “p”.
Raw 87: this sentence is not clear. Perhaps authors mean that SSRs less than 50 bp long have been eliminated? Please clarify.
Raw 88: replace “cluster” with “clusters”.
Results:
95: I would suggest “Results and discussion”.
96: please, provide the scientific names of both species here and remove them from raws 101-102.
98: “ecotypes”? Or genotypes? Please clarify. Moreover, replace "with" with “obtained from”.
103-106: “Primers were… .. software”. These sentences should be moved to Mat&Met. Moreover, since real experiments have been carried out (DNA extraction, PCR, agarose gel, capillary electrophoresis), all information about that must be added in M&M section in detail.
107: It would be important to indicate the number of rice and carp samples used to validate the identified SSRs and how many polymorphic alleles were found per each SSR. I guess the supplementary "primer" table could be actually included in the main text, with a detailed caption. A figure illustrating polymorphic peaks of some selected SSRs would also be useful.
I suggest to include the discussion in this section, rather than “discussion and conclusions”. Anyway, the scientific discussion is actually poor and of course needs to be extended.
Discussion and conclusions:
115: please replace with "Conclusions".
Figures:
Fig.1 needs a more descriptive caption.
Fig.3: Please, clarify the meaning of “traditional” in caption and remove all the spaces between brackets and commas.
Supplementary material:
The main text of the manuscript is missing about any reference to the additional files, as well as the corresponding captions, which anyway must be detailed and must also clarify the presence of symbols in the table itself (e.g. what do they mean by N in primer tab? The number of alleles?).
Moreover, locus name must be corrected with Os and Ci, respectively, instead of Orsa and Ctid  (e.g. Os-SSR1 and Ci-SSR2 and the same for all loci). As already mentioned, the primer tab could be included in the main text.
Furthermore, header is completely missing in the polymorphic_SSR tab; what does each column of the table indicate?


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: SSRENRICHER: A COMPUTATIONAL APPROACH FOR LARGE-SCALE IDENTIFICATION OF POLYMORPHIC MICROSATELLITES BASED ON COMPARATIVE TRANSCRIPTOME ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is not clear, the language is not professional and there are several mistakes in the text.
The references are too old and some important referenceses are totally missing.

The article is too short and several steps and descriptions are not present.

The raw data are correctly shared.

The results are supported by the experimental work.
Experimental design: The aim of the article is appropriate to the scope of the journal.

The reserach question is well defined but not efficiently supported.

The investigation is not so rigorous.

Methods are not well described.
Validity of the findings: All  raw data and primer sequences are provided, but is not well explained the real impact of the research. The discussion is not a real discussion, because is totally missing a comparison with other similar tools.
Additional comments: Dear authors, in my opinion the manuscript needs major revisons.
The introduction contains references too olds, and it is incomplete.
The materials and methods are not well explained, the validation of SSR si totally missing.
The results section is confused and reports some steps of methods.
The discussion is not a discussion: there are no comparisons with others similar bioinformatic tools.
In my opinion there is a large part of work that is missing, and the article cannot be published in this form.


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: SSRENRICHER: A COMPUTATIONAL APPROACH FOR LARGE-SCALE IDENTIFICATION OF POLYMORPHIC MICROSATELLITES BASED ON COMPARATIVE TRANSCRIPTOME ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors provided an improved version of their manuscript compared to the first one, answering almost all my requests (someone is still open). The addressed issue is certainly useful, English language is definitely improved, bibliography and the scientific discussions have been enriched. Anyway, the manuscript still needs some features to be assess and some minor corrections, before publication. For instance:
1)	How many carp and rice samples did authors test for SSR validation? The sample size is relevant for SSR polymorphisms and discrimination power.
2)	In accordance to the sample size, the number of SSRs selected for wet validation takes on importance.  Now it represents the 4% of the computationally identified SSR in both species.
3)	Authors ignored the hypothesis to add a picture of the identified polymorphic profiles (alleles by capillary electrophoresis).
4)	The references to supplementary materials are missing in the main text.
5)	Please correct le abbreviation of specie Latin names.
My detailed comments are on the pdf file.
Experimental design: The research is in accordance with the aims of the journal. In this new version the experimental design has been deeply described and became reproducible.
Validity of the findings: The research has high potential with regard to applicability and needs of the scientific community.
Additional comments: Abstract:
raw 31: replace semicolon with a dot.

Introduction:
raws 44-46: single o multiple references must be added following exactely the refer research, i.e: “SSRs have emerged as one of the most popular genetic markers in a wide range of applications in population genetics (specific ref), conservation biology (specific ref), and marker-assisted selection (MAS) (new ref).” Please correct and add proper references.

Mat&Met:
raw 31: replace “the developed” with “our”.
raw 104: replace "with" with “obtained from”.
raws 106-107: I would remove this sentence since it’s a repetition.
raws 106-109: I still believe that the number of used rice and carp samples for SSR validation could be of considerable importance.

Results:
Table 1: please check apics.
Fig.3: please provide ANOVA results in both captation and graph.
Moreover, a figure illustrating polymorphic peaks of some selected SSRs should be useful.

Supplementary material:
The main text of the manuscript is still missing about any reference to the additional files. Moreover, locus name have not been corrected jet according to the conventional scientific language (Oriza sativa is shorten as Os!).


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: SSRENRICHER: A COMPUTATIONAL APPROACH FOR LARGE-SCALE IDENTIFICATION OF POLYMORPHIC MICROSATELLITES BASED ON COMPARATIVE TRANSCRIPTOME ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article is improved, however the large part of the citations is too old. The introduction and discussion are not sufficient. The added table 1 is not suitable for a scientific publication (i.e. number instead of N or No)
Experimental design: Also if material and methods were improved the experimental design is not clear and does not fit with the discussion.
Validity of the findings: The discussion session is poor and does not fit with the experimental work:
In material and methods you report:
"To validate the developed software, we used it to develop SSR markers in rice (Oryza sativa L.) and in grass carp (Ctenopharyngodon idella), the transcriptomes of which are publically available. Analysis in these two species allowed for the validation of our pipeline in both a plant and animal species."

In the discussion you write:

"The frequency of polymorphic SSR marker development by traditional methods and HTS approaches is relatively low (usually, the frequency ranges between 10% and 50%), especially in species with low polymorphism, such as Pigeonpea (Cajanus cajan) and ovate pompano (Trachinotus ovatus)(Dutta et al., 2011; Xie et al., 2014)."

What does it mean? I need to have a comparison and discussion with other systems validate in rice and grass carp.
Additional comments: Dear authors I tried to give you an opportunity to publish this article, but unfortunately this revised version is too far from a corrected version suitable for publishing.


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: ELEPHANTS IN THE NEIGHBORHOOD: PATTERNS OF CROP-RAIDING BY ASIAN ELEPHANTS WITHIN A FRAGMENTED LANDSCAPE OF EASTERN INDIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a straightforward account of human elephant conflict.  It’s a serious problem — lots of people die — and it makes sense to do what the authors have done.  They want to know when in the day, when in the year, where exactly, and from which elephants the conflict arises.  This they do entirely sensibly.  They provide compelling explanations for what drives the conflict.

My one surprise was the introduction of alcohol into the discussion.  It wasn’t clear initially whether this was humans drinking or (as happens in Africa) elephants eating fermented fruit.

Alcoholism  is a major driver of human-elephant conflicts in this region with tea estate workers and farmers  being the primary victims of fatal elephant attacks. As a consequence, an annual sum of USD …

Ok, so it’s human, and I’d change this to “human drunkenness…”

Then, as I read the paper, it became clear that this is a key result — it accounts for a lot of the mortalities.  The authors claim that these are clustered around the places that make the beer.  It’s also a result that could lead to practical actions — and indeed, the authors suggest them. However, the authors don’t map these sources and don’t do a good job of analysing the data in terms of them.

They write

Our results also suggest that elephants raided villages where alcohol production (haaria- rice beer) was prevalent. Alcoholism is a major driver of fatal elephant attacks in this region and drunk people have been reported to harass and chase elephants from villages, crop fields (Naha et al., 2019).

I wished for more!  Can’t they show these villages that produce beer and where the incidents are?  This struck me as being one of the more useful results of the paper.
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: ELEPHANTS IN THE NEIGHBORHOOD: PATTERNS OF CROP-RAIDING BY ASIAN ELEPHANTS WITHIN A FRAGMENTED LANDSCAPE OF EASTERN INDIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: •	Generally, you have provided relevant references for this field of study. However, you need to make it clearer how your work fits into this literature.

•	Please carry out a thorough grammatical check on your manuscript, as there are many small errors throughout.

•	I would like to see more flow in the paper.

•	The introduction and discussion could be shortened in length and made more concise.

•	Please check all your references carefully, as there are a number of times where you have misused references. See my comments below relating to specific sections.

•	1.1. Abstract
-	Lines 12–16: Please rewrite this section. Biodiversity decline is as a result of habitat loss and fragmentation, which is attributed to increasing human populations, and human activities such as agriculture...to name but a few.
-	Line 30: You need to clearly state the result here, as you just say ‘showed a similar spatial pattern’. It is unclear as to what this pattern is.
-	Line 32-33: Please really think about the conservation takeaway from this paper. ‘Improving the condition of forest patches’ is very vague and unspecific. This is also not captured in the Discussion section. I would like to know what measures could be taken to improve the forest.

•	1.2. Introduction
-	Lines 40–50: Please rewrite the first paragraph, as you have made broad and vague statements, e.g major consequences for ecosystem processes. Please provide detail about why human-wildlife conflict is arising and why it is such a challenge in conservation
-	Line 47-50: This statement is not accurate as there is a lot of research into HEC in Asia. Please rephrase or delete. For example, here are some references from studies in Asia:

-	Chen, Y., Marino, J., Chen, Y., Tao, Q., Sullivan, C.D., Shi, K., Macdonald, D.W., 2016. Predicting Hotspots of Human-Elephant Conflict to Inform Mitigation Strategies in Xishuangbanna, Southwest China. PLoS One 11, e0162035

-	Choudhury, A., 2004. Human–Elephant Conflicts in Northeast India. Hum. Dimens. Wildl. 9, 261–270.

-	Goswami, V.R., Medhi, K., Nichols, J.D., Oli, M.K., 2015. Mechanistic understanding of human-wildlife conflict through a novel application of dynamic occupancy models. Conserv. Biol. 29, 1100–1110

-	Gubbi, S., 2012. Patterns and correlates of human-elephant conflict around a south Indian reserve. Biol. Conserv. 148, 88–95

-	Linkie, M., Dinata, Y., Nofrianto, A., Leader-Williams, N., 2007. Patterns and perceptions of wildlife crop raiding in and around Kerinci Seblat National Park, Sumatra. Anim. Conserv. 10, 127–135.

-	Webber, C.E., Sereivathana, T., Maltby, M.P., Lee, P.C., 2011. Elephant crop-raiding and human–elephant conflict in Cambodia: crop selection and seasonal timings of raids. Oryx 45, 243–251.

-	Wilson, S., Davies, T., Hazarika, N., Zimmermann, A., 2013. Understanding spatial and temporal patterns of human–elephant conflict in Assam, India. Oryx 49, 140–149.

-	Lines 53-54: This is an inaccurate statement and also does not have a reference.
-	Lines 59-61: Please remove this as the figures are inaccurate. The Kenyan Wildlife Authority have not published the number of problem elephants which have been killed since 2010 – 2017 so I am unsure about how you got these figures. Also, the reference you have used here (Mariki et al 2015) is incorrect and is a study from Tanzania.
-	Lines 62-68: You cannot make this bold statement without backing it up with references. Poaching could be argued as an indirect impact of HEC not a direct impact, although there have not been any studies to date looking at this correlation. Discussing poaching is also unrelated to this paper. Please remove these lines completely.
-	Lines 93-95: Please provide references here.
-	I suggest that you make improvements from lines 116-129 in the introduction to provide more justification for your study. You need to make it clear here (and throughout the study) how your study is contributing to knowledge in this field.
-	Line 105: the term used here ‘Cathemera’ is not suitable to describe elephants. You could say that elephants show risk avoidance strategies by avoiding areas of high human settlement and crop raid at night etc.
-	Lines 93-107: Please add a few sentences about the fact that females crop raid too. There are a number of studies that show this in parts of Asia and Africa
-	Lines 111-112:  This is a bold statement. Alcoholism cannot be a major driver. You need to rewrite this section. Do you mean that there is a problem with alcoholism in the area, which is leading to people walking in the middle of the night when elephants are around, thus coming into more contact with elephants?

•	1.3. Study Area
-	Figure 1: A good and informative study site map. However, I would like to see labels for the Himalaya and Gangetic Plains. Also, I would like to see labels of the Protect Area names and major roads added to this Figure.
-	Lines 149-150: Please add the total human population for this area.

•	1.4. Discussion
In general, your Discussion needs some restructuring. I would suggest breaking it  up into sections, following the structure of the results. There is some misuse of references and it is also quite long (like the introduction).
-	Line 272: term ‘loner’ is not appropriate. Please see comments in the ‘Experimental Design’ section about terminology for elephant group type.
-	Lines 273-279: Please can you emphasise how these findings relate to other studies.
-	Line 286: the references from Sitati and Chen do not support your statement that one-third of HEC victims were drunk. Please find new references.
-	Line 290: Again, you need to be clearer on how you define the group composition, i.e small herds - is that cows and calves or mixed groups? See comments below in ‘Experimental design’ regarding group composition.
-	Lines 300–304: Reference needed.
-	Lines 306-309: Please include references from other studies here and see my comments in the Conclusion section (Lines 401-412).
-	Line 342: This is an incorrect use of Sitati’s reference, as his study was in Kenya not Tanzania.
-	Line 368-371: Is the ‘Aus’ variety of paddy not impacted? Why could this be?

•	1.5. Conclusion
The structure of the conclusion is a bit like a list. This whole section should be restructured, as the recommendations that you give should come in the Discussion not the Conclusion. Also, there is a lot of repetition here from the Discussion.

-	Lines 393-394: This is very vague and unspecific. How would you restore forest patches?
-	Lines 401-412: Authors need to reference other sites where these different techniques are working and could be applied here. Again, this is just a list of mitigation measures. What could work in this region given the results of this experiment and the wider literature of studies in similar study sites?
Experimental design: •	The authors’ research aims and objectives are clear. However, the authors need to emphasize how their research is meaningful and how it fills a gap in knowledge.

•	This study is very similar to Naha et al 2019, as the authors used a hot spot analysis, looking at crop depredation, not human injury/death. I don’t think there is enough reference to Naha et al, 2019 study, considering it is exactly the same study site. I would like to see how this current study has improved on knowledge since the Naha et al 2019 study, which looks at data from 200-2006.

•	I commend the authors for their data collection over such a large study area and it appears that the analysis has been conducted to a high standard. The ‘hot-spot’ analysis is a very useful tool to identify areas in need of HEC mitigation and could be an important analysis that conservation managers use. However, I would like to see in Supplementary Materials more detail as to how Maxent was used in the analysis. This would be helpful to determine how replicable this analysis is.

•	I would really like the authors to state why they conducted their analysis at a spatial scale of 5 km2 and how they accounted for spatial autocorrelation. I would like to see the results of the analysis at different scales (even if it were to be provided in the Supplementary Materials), as one could then see how changing the spatial scale changes the results.

•	The authors need to clearly define the group composition of elephants, as currently there are different terms used throughout the paper such as herd and solitary bulls. Bulls can be in herds and so it is unclear as to exactly the type of group that are crop raiding. I would recommend you to define groups as:
-	Cow/calf group (females and offspring)
-	Mixed groups (females, calves and bulls)
-	Bull groups
-	Lone bulls

•	2.1. Study Area: Please include information to address the following questions: What are the different seasons? What are the main crops grown and when are these harvested? What is the population of elephants in this area? This is key information as it gives us an understanding of the numbers that these communities are dealing with. Also, authors need to provide a description as to whether the National Parks are connected to each other.

•	2.2. Methods
Throughout the methods I would like to see more detail added in certain areas, or more justification for particular aspects. See comments below:
-	Lines 116-119: Why did you not look at distance to villages, slope, elevation?
-	Need to introduce the study by Naha et al 2019 as it is very similar. Explain how this study is contributing to research from this previous study.
-	Lines 162-164: How many teams? How many respondents in a team?
-	Lines 168-177: Unclear. Were the results on herd composition based on elephants seen in the community? If so, how did you avoid bias in the number of elephants? i.e. often people over report the number of elephants they see in their farm.
-	Line 182: Unclear how you categorized your data to carry out the Chi Square test. What significance in the patterns were you looking for?
-	Line 184: Why was 5km2 used?
-	How did you account for spatial autocorrelation? Is 5km a fine enough scale?
-	Table 1 (Line 186): I would like to see in Table 1 a referenced justification for why each variable was used.
-	Lines 189-190: Was this per grid cell? Currently this is unclear.
-	Line 191- What was the format of the census data. Population by village, district etc? How did you overlay this data?
Validity of the findings: •	Parts of this study, as I interpret, are a replication of Naha et al 2019. This is great as the authors could compare their results across the two studies. However, the authors do not provide a rationale for this replication and do not clearly state how this study adds value to the literature.

•	The conclusions in general have been appropriately stated and linked back to the original research questions. However, I would like to see the structure of the Discussion (see comments in 1.4) improved so that the interpretation of the results are clear (i.e how do they link back to original research question, and do the data support your hypothesis? Are your results consistent with what other investigators have reported? What further research would be necessary to answer the questions raised by your results?)

•	Finally, I would like to see more evidence in the paper about how your study fits into the big picture.

•	I thank the authors for providing the raw data. However, there are sections of this data that are unclear, such as ‘number of humans on the field’ - in the excel cell there are dates given not numbers. Also, ‘Species raided,’ does that mean eaten by the elephants or damaged?

•	3.1 Results

-	Seasonal and temporal patterns of crop raiding: I would like to see a graph showing the number of crop raiding incidents per month across your study period correlated with monthly rainfall.
-	Line 218: Elevation is not in the list of predictor variables that you stated you tested.
-	Line 254: To what extent? What area of agricultural fields did it then start to decline?
-	Line 257: What was the population density and area of human settlement?
-	Lines 260-262: There needs to more of a description of Figure 2 here.
-	Figure 2: The scale bar needs redoing here as it is very unclear.
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: ELEPHANTS IN THE NEIGHBORHOOD: PATTERNS OF CROP-RAIDING BY ASIAN ELEPHANTS WITHIN A FRAGMENTED LANDSCAPE OF EASTERN INDIA
Review round: 1
Reviewer: 3

Output:
Basic reporting: In the introduction, the authors have comprehensively provided the background of the research contextually, in a clear and unambiguous manner. However, the flow of the first three paragraphs in the introduction i.e. lines 40 – 78 can be greatly enhanced, where for instance, the first paragraph can be used to describe attributes on elephant biology, ecology and behavior that predispose the species to Human Elephant Conflict (HEC). This can then be followed by a paragraph on the global context of HEC (comparing Africa and Asia), then zero in to the Asia perspective, culminating with the documented drivers of conflict.
Contrary to the indication by the authors, in lines 46, 47 & 48, HEC has been studied extensively (De Boer et al., 2013, Evans and Adams, 2018, Vezina et al., 2020, Tiller et al., 2017, Pant et al., 2016). However, both the ecological and anthropological drivers to HEC are highly dynamic. A better statement, therefore, would be on the needs to improve the understanding of HEC to match the high dynamics in the field.
Some statements in the introduction such as in lines 64 – 66, 86 - 87 & 111 - 113 are subject to misinterpretation hence might be necessary to either rephrase or have supporting reference.
Experimental design: The genesis of this paper is to identify the drivers of HEC and propose mitigation measures. This is adequately captured in the hypotheses, study design and results. The methods are detailed and can be replicated.
The authors have, however, only provided the questionnaire set of data. It would be valuable to see data on the other predictors, especially after extraction to the 5 km2 grids.
The figures presented in the manuscript are of good quality. If possible, in figure 1, the authors may consider displaying the tea plantations as polygon rather than point data as presented.
Validity of the findings: If the ultimate goal is to suggest mitigation measures to HEC, some of which include community response to conflict incidents, it would be valuable for the authors to investigate and comment on the severity of HEC in relation to human activity as provided in Column N of the csv data file. This would help to further describe the statement by the authors in line 275 – 276, to for instance, include the contribution of active guarding to abate HEC.
Additional comments: I commend the authors for the great effort and their contribution to the understanding of HEC. The two year period (2018 -2019) when data were collected might not have been sufficient enough to conclude on seasonality but the authors have referenced similar studies to support the findings. While the findings substantive, it is encouraged to establish a long term monitoring program within the study area to enable document seasonal changes in future.

References
DE BOER, W. F., VAN LANGEVELDE, F., PRINS, H. H., DE RUITER, P. C., BLANC, J., VIS, M. J., GASTON, K. J. & HAMILTON, I. D. 2013. Understanding spatial differences in African elephant densities and occurrence, a continent-wide analysis. Biological Conservation, 159, 468-476.
EVANS, L. A. & ADAMS, W. M. 2018. Elephants as actors in the political ecology of human–elephant conflict. Transactions of the Institute of British Geographers, 43, 630-645.
PANT, G., DHAKAL, M., PRADHAN, N. M. B., LEVERINGTON, F. & HOCKINGS, M. 2016. Nature and extent of human–elephant Elephas maximus conflict in central Nepal. Oryx, 50, 724-731.
TILLER, L., HUMLE, T., AMIN, R., DEERE, N., LAGO, B., LEADER-WILLIAMS, N., SINONI, F., SITATI, N., WALPOLE, M. & SMITH, R. 2017. 3. An uncertain future: changing seasonal, temporal and spatial crop raiding trends in a human-elephant conflict hotspot. Tragically, Peter passed away in February 2017 and never got to read this thesis. I hope I would have made him proud., 61.
VEZINA, B. I., SMITH, R. J. & TILLER, L. N. 2020. A neglected aspect of human–elephant conflict: fence damage by elephants in the Trans Mara, Kenya. Pachyderm, 78-87.


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: ELEPHANTS IN THE NEIGHBORHOOD: PATTERNS OF CROP-RAIDING BY ASIAN ELEPHANTS WITHIN A FRAGMENTED LANDSCAPE OF EASTERN INDIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I am satisfied that the authors have addressed all my comments, and I think that the manuscript has been greatly improved. With a few more minor revisions (see below) and another round of grammatical editing, I think that the paper is worthy of publication, and is a valuable contribution to the literature.

-	Figure S1 – I think this would be more appropriate in the main paper. Also, it would be clearer if the ‘number of crop raids’ were presented as bars, and the ‘rainfall’ as a line.

-	Table 1 – I think this would be more appropriate in the supplementary materials section.

-	There is a missing reference in the ‘References’ section: Pokharel et al., 2018.

-	Please add a few more details about ‘human drunkenness’. In your response to my comments you gave details about this, which would be appropriate to include in the introduction. I.e Intoxicated people chase/harass elephants near settlements, crop fields and are attacked (findings from Naha et al. 2019). Rice beer (alcohol production) is also frequent within some of these villages and elephants are reported to visit such areas and damage crop, property.

-	Line 209 – 210: Crop raids lead to retaliation and elephants are also killed by local communities (Sukumar & Gadgil, 1988; Gubbi et al., 2014). This is unclear.

The sentence should be: Crop raids can lead to retaliatory killings of elephants by local communities.

-	Line 223 – 224: Reference needed here.

-	Line 316: more suitable references would be Sitati et al 2003 and Chen et al 2016.

-	Line 676: please reword this sentence as bull elephants do not go into estrus. They enter a period of musth every year, but this is not estrus.

-	Line 691: This line is repetitive and also is inaccurate as bull elephants do not go into estrus.

-	Discussion: please add details about what measures could be put in place to try and tackle the problem of alcoholism in the area.


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: EFFECTS OF VEGETATION RESTORATION ON SOIL QUALITY IN FRAGILE KARST ECOSYSTEMS OF SOUTHWEST CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Understanding the effect of restoration activities on soil quality is a relevant issue, especially in karst ecosystems with erosion prone soils.  However, the analysis presented cannot support the inferences made in the study.
Experimental design: My major concern is the experimental design. Need a better description of the study sites, including the size of each restoration site and the separation within the each plot of the restoration treatment to ensure independence of the each sample.  The study tries to compare different restoration techniques, but to make this comparison; restoration treatments should have replication.
Validity of the findings: One possible analysis that could be made is the effect of a pre-restored (control sites) and restored sites.  Even this approach will need the standardization of the variability of the restoration techniques. Another possible analysis with the current data will be to compare the treatment of economic forests with natural restoration techniques and the control.  The analysis of the Soil Quality Index still could be use the PCA, but using the treatments previously suggested.

I am aware that having replication in restoration projects is always a limitation.  Therefore, it is a good practice to avoid the use of parametric test, One-way analysis of variance, if the initial project did not consider the treatments during the restoration activity.
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: EFFECTS OF VEGETATION RESTORATION ON SOIL QUALITY IN FRAGILE KARST ECOSYSTEMS OF SOUTHWEST CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: 1.	The workload of the article is relatively large, but thinking and logicality of the article have some deficiencies. The article needs major revision.
2.	Why do you choose the cropland as control and how to consider the effect of vegetation.
3.	The research of this article is of great significance, but the conclusion was just the collection of results. A high quality of conclusion should be summarized and abstracted form these results. The authors need to refine the research results to enhance the significance of the research.
4.	The latest research results should be selected for literature citation as far as possible.
5.	What's the purpose of the first paragraph of the introduction? It seems not closely related to the content of this article.
6.	Line 70, the hypothesis is not consistent with the objectives of this research.
7.	Line 79-80, the reasons of choosing this index were not explained in the introduction of this paper. Suggest to cite relevant references to explain.
8.	The spatial distribution of sites is far away, and how to exclude the influences of uneven elements such as climate.
9.	Why did this research studied three soil layers and it need to be explained and discussed deeper.
10.	The discussion of this article should be discussed deeper.


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: EFFECTS OF VEGETATION RESTORATION ON SOIL QUALITY IN FRAGILE KARST ECOSYSTEMS OF SOUTHWEST CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I think the author has adopted the opinions of the reviewers and carefully revised the manuscript. The manuscript has been significantly improved, and the quality is now suitable for publication in terms of ecology and environment.


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: EFFECTS OF MICROBIAL EVOLUTION DOMINATE THOSE OF EXPERIMENTAL HOST-MEDIATED INDIRECT SELECTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: -	Literature used is relevant. However, the authors fail to cite or discuss the following paper, which directly addresses similar questions in the same model organism:
o	Bacterial Adaptation to the Host’s Diet Is a Key Evolutionary Force Shaping Drosophila-Lactobacillus Symbiosis Martino et al., Cell Host & Microbe 2018 https://doi.org/10.1016/j.chom.2018.06.001
This is a major oversight and should be addressed.
-	The title of the article sounds catchy but implies a conclusion that is not supported by the experimental results. The authors showed that the microbiota composition changed over the course of the experiment, but they did not determine whether it evolved in response to their experimental manipulation or simply changed due to random chance. Even if the microbiota changed in response to selection (be it the “random” control or other), the study did not assess how they changed (e.g. faster growth rates in the diet) or what aspect of the experimental regime they responded to. Simply put, the manuscript does not answer the question “what does the microbiota want?” Neither does it demonstrate that microbial evolution ‘overtook’ the effects of indirect selection. The data show that indirect selection and microbial evolution were not correlated, but without more info about what exactly changed in the microbiota it is too speculative to say those changes ‘overtook’ or overpowered the impacts of indirect selection.
Experimental design: -	Description of the methods: A few important details are missing from the methods.
o	Was the humidity controlled? If so, at what value? Humidity can have significant impacts on developmental rates in Drosophila.
o	The recipe for the fly diet used is not complete. At present it states “..reared on standard media with 45g of cornmeal/100g of sugar.” Then says “For no-sugar diet, sugar and cornmeal were removed.” This would suggest the no-sugar diet contained nothing! A full accounting of the ingredients in the diet is needed for the reader to evaluate the manuscript. In particular – did either the stock vials or experimental ones contain preservatives? Also, did they contain yeast? Many labs use live yeast in their fly cultures, which is a dimension of the microbiome that you did not assess.
o	About how many flies developed per vial? This is an important figure to include because of the effects that density can have on development time.
o	Were samples pooled from the three sub-replicate vials for 16S sequencing? This seems to be the case, but it is not specifically stated.
-	16S sequences do a very poor job distinguishing between Acetobacter species (and even between related genera in the Acetobacteraceae) See references below. So, it is quite possible that the microbiome changed a lot on the no-sugar diet but you did not see it based on the resolution of the tools used.
o	Matsutani, M., Hirakawa, H., Yakushi, T., & Matsushita, K. (2011). Gen- ome-wide phylogenetic analysis of Gluconobacter, Acetobacter, and Gluconacetobacter. FEMS Microbiology Letters, 315, 122–128.
o	Chouaia, B., Gaiarsa, S., Crotti, E., Comandatore, F., Degli Esposti, M., Ricci, I., ... Daffonchio, D. (2014). Acetic acid bacteria genomes reveal functional traits for adaptation to life in insect guts. Genome Biology and Evolution, 6, 912–920.
Validity of the findings: -	Rigor of the investigation.
o	The random selection control is key and the main conclusion of the study hinges on the fact that it changed in the same direction and magnitude as the experimental population. However, it is hard to assess the significance of the change observed without additional replication (see below) or an assessment of the variance in the development time of stock flies or of axenic animals over the same timeframe (what the authors refer to in the discussion as a ‘host-stability’ control). Having some independent development data would clarify the extent to which the changes observed were the result of the experimental manipulations vs. chance environmental factors.
o	The lack of replication is a serious concern for me. If there were parallel technical replicates whose microbiota were passaged and sequenced independently, it would be possible to see if the changes that occurred in the microbiota were non-random (i.e. reproducible and resulting from selection – via diet or host). Also, if there had been independent replication (same experiment at a different time), this would enable us to see if the selection regime (random or not) had the same biological effects on distinct groups of flies and microbes. One example of where significant variance can arise in experiments tracking development time of gnotobiotic Drosophila is the circadian rhythmicity of oviposition. The time of day during which egg laying is initiated and the time when collection occurs prior to surface sterilization influences the age of embryos at the start of the experiment. Given that a 24 hour window was used there could be enough variation in this window to produce the differences you observed in eclosion time across selection rounds. Was the same time of day used for these steps during each round?
Additional comments: Line 62 – you seem to define microbiome engineering using the word engineering. Would ‘modifying and transferring…” or “evolving and transferring…” work here?

Line 93 – Whether Drosophila melanogaster has a core gut microbiome is a matter of debate.
Wong et al. essentially show that the microbiome is inconstant and there is not a core microbiome in lab flies. (Wong AC, Chaston JM, Douglas AE. ISME J. 2013 Oct;7(10):1922-32. doi: 10.1038/ismej.2013.86) This view has critical implications for your experimental design because the starting microbiome likely represented a random subset of the strains in the laboratory population.

Line 261 – This seem an apt place to discuss Martino et al. (mentioned above). Your manuscript does not mention an estimated number of bacterial generations that passed (though you speculate “many” at line 328). This is relevant if significant adaptation to the culture medium was occurring that the level of individual species vs. changes in the relative abundance of species in the microbiome, or both.

Lin 266 – Host-mediated (hyphen)

Line 284 – “interacting” is an odd word choice here

Line 281 (and elsewhere) Your language causally links the changes in the microbiome to changes in fly phenotypes. However, your data do not show this causality. They show a correlation. Without replication (see comments above) or the ‘host stability’ controls referenced in the discussion, there is still doubt that the microbiome change caused the change in fly phenotype.

Figure 1: There are two “step 2” and no “step 3” in the diagram

Figure 2: The labels in the key are confusing. Either get rid of the first “selection” in the diagram, or keep it and change the other labels to “random” (instead of control) and “phenotypic” or something along those lines.


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: EFFECTS OF MICROBIAL EVOLUTION DOMINATE THOSE OF EXPERIMENTAL HOST-MEDIATED INDIRECT SELECTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Arora et al. reports findings from a microbial engineering experiment wherein experimental evolution is used to select for microbes that reduce Drosophila melanogaster eclosion time. The results show that it was possible to select for faster eclosing Drosophila lines; however, the microbiome was not a contributing factor to host developmental acceleration despite a change in microbiome composition over the selection cycles.

A major strength of the manuscript is that a random-selection control rather than a null inoculation control was used. Overall, the manuscript is well-written and the results support the conclusions. The raw data and code to produce the figures and statistical findings have been shared.

Please add the following references to Line 97:
Wong, C. N. A., Ng, P. & Douglas, A. E. Low-diversity bacterial community in the gut of the fruitfly Drosophila melanogaster. Environ. Microbiol. 13, 1889–1900 (2011)
Ridley, E.V. et al., 2012. Impact of the resident microbiota on the nutritional phenotype of Drosophila melanogaster. PloS One, 7(5), p.e36765.

Please clarify the following issues:
Figure 2: I am guessing that each of the circles appearing below or above the plots are the individual replicates. If this is the case, why do only some of the box plots have circles? The circles should be described in the legend.
Figure 3:  According to the methods, 3 sub-replicates are made from each selection cycle. Does each circle represent a sub-replicate? If so, why is it the case that only 2 circles appear for some rounds?

Minor corrections:
223-230: Many of these details have already been described in the Methods section. Please edit to remove redundancy.
Figure 1: Step 2 appears twice; there is no Step 3. Also, the table for “Step 2” is very faint and may not reproduce well.
Experimental design: The research question is well-defined and of interest to the field. The experimental design is sound.
The following should be addressed in the methods:
118: It would be helpful if the authors provided a brief rationale for why high-sugar and no-sugar diets were used.
137: Details about the media and numbers of flies placed in each vial should be provided since these parameters can affect host life history traits and microbiome composition. In particular, the authors should address the following:
How many flies were placed in each vial and was this number kept consistent across all conditions and selection cycles?
Was the ratio of males: females constant across all conditions and selection cycles?
Please provide recipe for fly media in terms of % (w/v) rather than “g”.
What was the volume of food placed in each vial and what were the dimensions of the vial?
What was the temperature and humidity of the incubator?
Validity of the findings: Shortcomings and alternate explanations are provided in a thoughtful discussion section. Many in the field will  appreciate the conclusions section, especially with respect to recommendations for experimental design.
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: EFFECTS OF MICROBIAL EVOLUTION DOMINATE THOSE OF EXPERIMENTAL HOST-MEDIATED INDIRECT SELECTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: Arora and colleagues – PeerJ Review –

“The microbiome wants what it wants: microbial evolution overtakes experimental host-mediated indirect selection. “

The manuscript, entitled ‘The microbiome wants what it wants: microbial evolution overtakes experimental host-mediated indirect selection’, by Arora, Brisbin, and Mikkeyev, examined the effects of ‘Microbiome Engineering’ on the microbial flora of fruitflies in an experimental setting.

The paper describes a method in which host phenotypes are experimentally selected upon in fruitflies (developmental time) using clonal population of hosts. Hosts are genetically identical, so the only phenotypic variation that can be selected upon is driven by community differences in the hosts’ microbial flora. The experiment selected on host developmental time in two diet backgrounds, used replication, and employed control lines in which no selection occurred. Microbes were transferred from the feeding dishes / media where the flies lived so included a mix of fecal-generated communities and other flora that were growing in the media. In my opinion this transfer method was the fundamental flaw in the experiment such that the experiment did not produce any of the expected results (or really any results that made sense given the protocol). Because of this flaw little / none of the empirical data are of publishable quality. I found this really disappointing, as the experiments were otherwise fascinating.

The authors take this challenge somewhat in stride and then use the paper to motivate how to better design microbiome-engineering experiments. I found this discussion thoughtful and quite coherent, but it unfortunately gave short shrift to the central flaw in the experiment: that the community that was passaged was incorrectly designed to passage the gut flora community of the flies. Rather than passing the community of microbes that was in the gastrointestinal (GI) tract of the flies, they passaged the community that was on the feces / food /media which likely included a different mix of genotypes than would be optimal in the GI tract. This was clarified within the paper but not at all emphasized in the key points (it came near the end – see below). I really struggle with whether -- and in what venue -- data like this are useful for a journal like PeerJ.

Detailed comments.
1.	The abstract and throughout the paper the authors describe the term ‘independent microbial evolution’. This needs to be better defined. It might be better described as community drift except for the fact that the phenotype of the hosts did move in one consistent direction. See esp. Abstract / lines 85-86 / line 257.
2.	Lines 36-37. I do not really agree with this statement. It seems like a matter of opinion though, especially since no reference or specific data are offered.
3.	Lines 103-108. Given that the authors are explaining the results at this juncture, some explanation should be given of how this shift occurred even though there was no treatment effect.
4.	Line 115 The authors should explain what they mean by minimizing host evolution, perhaps my saying something about segregating variation.
5.	Lines 135-136. What motivates the diet treatments?
6.	Lines 154-155. I cannot understand why this method was used. Given the extreme amount of effort that shaped the rest of the experiment, this seemed like such an unexpected choice rather than grinding up the guts of a few flies to passage. Is there something that I am missing?
7.	Lines 182- 183. The benefit of using CSVs is to gain extra resolution over binning genotypes in OTUs. This method is circular in generating CSVs but then binning them at the genus level, which seems very strange. What motivates this?
8.	Lines 195-197. Data exploration sounds worrisome in a big -data setting like this. I trust these authors fully, but It would be better to explain exactly what was done and the motivation for each analysis. The statistical analysis should not be shifted to a website that is not part of the publication.
9.	Large parts of the results should be moved to the materials and methods section including Lines 212-215, 223-233
10.	Lines 304-316 seem unnecessary
11.	Lines 325-331 is when the authors finally spell out the fundamental flaw in the experiment. This should be moved to the top and then the rest of the paper will make a lot more sense.
Experimental design: As explained above, the core passaging protocol was flawed.
Validity of the findings: Not sure if this is applicable, since there no findings that linked to the protocol used.
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: EFFECTS OF MICROBIAL EVOLUTION DOMINATE THOSE OF EXPERIMENTAL HOST-MEDIATED INDIRECT SELECTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors have done a thorough job of addressing my concerns.


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: EFFECTS OF MICROBIAL EVOLUTION DOMINATE THOSE OF EXPERIMENTAL HOST-MEDIATED INDIRECT SELECTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: Arora PeerJ43924_UGMReview20Apr2020

I evaluate here the comments of the 3 previous reviewers, the authors' rebuttal letter, and the revised manuscript.  I have only minor comments on the main manuscript, all of which are easy to address, and my comments below address mainly the reviewer comments.

The authors' rebuttal letter addresses all reviewer comments in great detail.  I disagree with some reviewer comments, but I also agree with a few reviewer comments.  The reviewer comments are very constructive and overall positive, and I agree that this manuscript is appropriate for publication in PeerJ.

Agree with reviewer comments:
I agree with Reviewer 1 that the title does not convey quite the main finding, particularly the part "The microbiome does what it wants", and I suggest to shorten the title to simply "Effects of microbial evolution dominate those of experimental host-mediated indirect selection".  The microbiome "does what it wants" if allowed to roam on a long leash (per analogy with Foster et al 2017 Nature), but microbiomes on a short leash (i.e., microbiomes under greater host control) may not be able to change uncontrolled because short-leashed microbiomes are shaped significantly by host control, and host phenotype is a more reliable indicator of microbiome properties.  Specifically for the study here, if the authors had chosen a slightly different microbiome harvesting scheme (e.g., harvesting microbiomes directly from guts by macerating flies; or by transferring flies to fresh sterile media at the end of a cycle, then allowing the flies to defecate for a very short time, then immediately harvesting the defecated microbiomes before the microbiomes can restructure/evolve outside the gut), the authors may have gotten a greater response to microbiome selection.  One key message of the entire study is that the microbiome harvesting and microbiome transfer steps are very important for microbiome selection to work, and these steps will be improved by those who will build on the present study.
Anyway, changing the title is easy to address.

Comments by all 3 reviewers are very constructive, and the authors have addressed all comments in the revision.

Disagree with reviewer comments:
I disagree with Reviewer 3 that the "transfer method was a fundamental flaw" and that therefore "none of the data are of publishable quality". I agree with Reviewer 3 that the microbiome harvesting/transfer methods can be improved, now that we know post-hoc that the particular transfer method chosen by the authors was not optimal (the methods could have been adequate, noone knew because noone tried such an experiment so far), but the study is definitely worth publishing because the study develops and test an experimental protocol that will be used widely by other researchers aiming to engineer gut-microbiomes through differential microbiome propagation in insects and other animals.
If the hologenome concept has any value, then it must be true that we should be able to select on host-microbiome associations (or on microbiomes if host genotype is held constant) and thereby obtain a response to selection in host phenotype, mediated through changes in microbiomes.  The authors develop here a protocol to do that for an animal, pinpoint an important step in the protocol (microbiome harvesting/transfer), and the findings will therefore enable others to improve the protocol.  This advances the field significantly in this fast-moving and economically important field of microbiome engineering.

My evaluation of the revised manuscript:
The manuscript is a significant and timely addition to the field; as far as I know, it is indeed the first test of host-mediated microbiome selection in an animal.
The writing is very clear and informative.
Some very important insights emerge in the discussion, for example the authors' framing of host-mediated selection within a quantitative-genetic framework (the section "Did the experiment have power to detect effects of artificial selection?").  One challenge of microbiome selection is a proper conceptualization of microbiome heritability, which is unlikely completely analogous to heritability of genomes (as in the breeder equation discussed by the authors).  To avoid irking quantitative geneticists here in the discussion, it may be worth pointing out in half a sentence that the narrow-sense heritability in the breeder equation is different from the heritability of a microbiome (or maybe define h with a subheading m for microbiome, to indicate that the heritability here is something different).  The heritability of a microbiome is less similar to the heritability of a genome, and more similar to the heritability of an infectious virus with a highly fragmented genome that gets reshuffled/recombined as multiple viruses infect the same cell, and fragments are added or lost with each replication cycle. Heritability of such viruses is difficult to conceptualize, but such viruses clearly evolve, as do microbiomes under host-mediated differential microbiome propagation.
Line 468:  A good point, microbiome evolution in the absence of host control will reduce the efficacy of differential microbiome propagation, and therefore it will be important to transfer microbiomes efficiently between hosts.  I disagree, however, that the possibility of microbiome evolution in the absence of host control "limits the utility of microbiome engineering"; instead, the main message here should be that, to engineer microbiomes through differential microbiome propagation, it will be important to devise experimental tricks or propagation schemes that minimize microbiome evolution in the absence of host control (that is, it will be important to devise propagation schemes that keep the microbiome always and consistently on a short leash). Maybe the first sentence should be rephrased to highlight less any limits if a protocol is not optimized, but highlight the importance of optimizing all steps in a selection cycle, including microbiome harvesting and transfer.

I was unable to view Figure 1, which was not included in the word.doc sent me for review.

Minor comments; all of these will be easy for the authors to fix:
Line 18: "... but microbiome engineering had no effect relative to a control treatment" OR: "... but microbiome engineering had no effect relative to a random-selection control treatment" OR the phrasing used in lines 128&129.
Line 32: Zilber-Rosenberg is misspelled several times in the manuscript (including the list of references) without the caps in Zilber-rosenberg
Lines 49, 104, 445, 476: The Mueller et al bioRxiv manuscript was updated in 2019, and the list of authors changed:  Mueller, U.G., T.E. Juenger, M.R. Kardish, A.L. Carlson, K. Burns, C.C. Smith, D.L. Des Marais. Artificial microbiome-selection to engineer microbiomes that confer salt-tolerance to plants. bioRxiv 081521.   doi: biorxiv.org/content/10.1101/081521v2
Lines 339&340: italicize bacterial genera
Line 379: delete redundant parenthesis "("
Line 477: ... does not ....
Line 483: there are three interacting processes, not only two: evolution of microbial genomes of each microbial type/species; changes in the relative abundances of microbial types/species; immigration/extinction of microbial types/species.  Immigration/extinction can be viewed as extremes of change in relative abundance (zero -> above zero; above zero -> zero), but I agree it is helpful for an experimenter to separate out immigration/extinction.
Line 484: this is an incomplete sentence. The entire paragraph needs some revision. Microbial immigration can be eliminated in closed systems/mesocosms, even with Drosophila; working with closed systems is experimentally more cumbersome (and less realistic), but not impossible.
Line 568: Random selection lines are important if there is only one selection treatment, as in the present experiment.  An alternate experimental approach is two different selection treatments run in parallel in the same experiment, and these two selection treatments effectively serve as controls for each other, then cross selected microbiomes and treatments at the end of the experiment after several rounds of selection (as in the Mueller bioRxiv experiment); for such selection scheme, random selection lines would not be necessary (though of course could also be added if resources/time permit).
Experimental design: Overall sound.  See my comments above how a future study can be improved by optimizing the microbiome harvesting and microbiome transfer steps when selecting on gut microbiomes of Drosophila.
Validity of the findings: Valid conclusions, given the data
Additional comments: Some very important insights emerge in the discussion, for example the authors' framing of host-mediated selection within a quantitative-genetic framework (the section "Did the experiment have power to detect effects of artificial selection?").  One challenge of microbiome selection is a proper conceptualization of microbiome heritability, which is unlikely completely analogous to heritability of genomes (as in the breeder equation discussed by the authors).  To avoid irking quantitative geneticists here in the discussion, it may be worth pointing out in half a sentence that the narrow-sense heritability in the breeder equation is different from the heritability of a microbiome (or maybe define h with a subheading m for microbiome, to indicate that the heritability here is something different).  The heritability of a microbiome is less similar to the heritability of a genome, and more similar to the heritability of an infectious virus with a highly fragmented genome that gets reshuffled/recombined as multiple viruses infect the same cell, and fragments are added or lost with each replication cycle. Heritability of such viruses is difficult to conceptualize, but such viruses clearly evolve, as do microbiomes under host-mediated differential microbiome propagation.
Line 468:  A good point, microbiome evolution in the absence of host control will reduce the efficacy of differential microbiome propagation, and therefore it will be important to transfer microbiomes efficiently between hosts.  I disagree, however, that the possibility of microbiome evolution in the absence of host control "limits the utility of microbiome engineering"; instead, the main message here should be that, to engineer microbiomes through differential microbiome propagation, it will be important to devise experimental tricks or propagation schemes that minimize microbiome evolution in the absence of host control (that is, it will be important to devise propagation schemes that keep the microbiome always and consistently on a short leash). Maybe the first sentence should be rephrased to highlight less any limits if a protocol is not optimized, but highlight the importance of optimizing all steps in a selection cycle, including microbiome harvesting and transfer.

I was unable to view Figure 1, which was not included in the word.doc sent me for review.

Minor comments; all of these will be easy for the authors to fix:
Line 18: "... but microbiome engineering had no effect relative to a control treatment" OR: "... but microbiome engineering had no effect relative to a random-selection control treatment" OR the phrasing used in lines 128&129.
Line 32: Zilber-Rosenberg is misspelled several times in the manuscript (including the list of references) without the caps in Zilber-rosenberg
Lines 49, 104, 445, 476: The Mueller et al bioRxiv manuscript was updated in 2019, and the list of authors changed:  Mueller, U.G., T.E. Juenger, M.R. Kardish, A.L. Carlson, K. Burns, C.C. Smith, D.L. Des Marais. Artificial microbiome-selection to engineer microbiomes that confer salt-tolerance to plants. bioRxiv 081521.   doi: biorxiv.org/content/10.1101/081521v2
Lines 339&340: italicize bacterial genera
Line 379: delete redundant parenthesis "("
Line 477: ... does not ....
Line 483: there are three interacting processes, not only two: evolution of microbial genomes of each microbial type/species; changes in the relative abundances of microbial types/species; immigration/extinction of microbial types/species.  Immigration/extinction can be viewed as extremes of change in relative abundance (zero -> above zero; above zero -> zero), but I agree it is helpful for an experimenter to separate out immigration/extinction.
Line 484: this is an incomplete sentence. The entire paragraph needs some revision. Microbial immigration can be eliminated in closed systems/mesocosms, even with Drosophila; working with closed systems is experimentally more cumbersome (and less realistic), but not impossible.
Line 568: Random selection lines are important if there is only one selection treatment, as in the present experiment.  An alternate experimental approach is two different selection treatments run in parallel in the same experiment, and these two selection treatments effectively serve as controls for each other, then cross selected microbiomes and treatments at the end of the experiment after several rounds of selection (as in the Mueller bioRxiv experiment); for such selection scheme, random selection lines would not be necessary (though of course could also be added if resources/time permit).


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: SPAWNING OF FRENCH GRUNTS, HAEMULON FLAVOLINEATUM, IN RECIRCULATING AQUARIUM SYSTEMS
Review round: 1
Reviewer: 1

Output:
Basic reporting: MS is correctly prepared and formatted. References are properly selected. The hypothesis should be corrected for, e.g.: it is possible to spontaneously spawning of French grunt in captivity, and then verified.
Experimental design: This is the biggest weakness of MS. Four fish were kept in each pool - but in some pools there were only females (no males). This factor may interfere with the reported results because it is unknown how the presence of males and their pheromones affects egg production and their ovulation and subsequent release into water.
It is not entirely clear whether the eggs were collected collectively from pools A and B (and probably it was unfortunately so) or separately. In the latter case, it would be easier to interpret the results. One could then consider individual subgroups, e.g. 2: 2 (males: females) - this is one group with repetitions; 1: 3 - is the second group; 0: 4 - this is the third group.
There is also no data on the weight of females in MS (although the text shows that the authors have these data) - then relative fecundity could be calculated, not just total. It would be easier then to present and discuss the results.
The work does not show whether the fertilization rate was tested. If so, adding this data would greatly increase the value of MS.
Validity of the findings: See "Experimental design" notes. The value of the work would also increase the information about how many days eggs were obtained from the same pools, which will allow to conclude more accurately about the frequency of reproduction. The fact that spawning at night should also be discussed more strongly. Coral reef fishes often spawn at night or their offspring (like clownfish) hatch at night. It is a very interesting biological phenomenon, especially when compared to freshwater fish.
Additional comments: This is a very difficult decision. I have read MS many times and I think that authors should be given a chance to improve their work, because of its importance for the protection of coral reef biodiversity. The practical aspect of MS is also important.


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: SPAWNING OF FRENCH GRUNTS, HAEMULON FLAVOLINEATUM, IN RECIRCULATING AQUARIUM SYSTEMS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript submitted for review contains very important information regarding reproduction under controlled conditions of one of the coral reef fish species. This is the aquaculture topic that will be intensively developed in the near future. The manuscript is properly constructed. But my objections concern the purpose of research, hypothesis and justification for establishing research groups.
Experimental design: 1. There is no research hypothesis. In the case of these studies, it is very easy to make such a hypothesis, e.g. it is possible to breed French grunt under controlled conditions.
2. The division of fish into research groups is unclear. Indeed, the authors inform that the fish was selected in terms of their similar size and the least aggression, which in coral reef fish can lead to a drastic reduction in stocking. However, different reproductive groups were created, where the ratio of males to females was 1: 1, 1: 3 and 0: 4 (so-called negative control) and they should be analyzed this way.
Validity of the findings: The results should be presented a little differently.
1. The effects of reproduction (harvested eggs) should be presented in relation to reproduction groups 1: 1, 1: 3 and 0: 4.
2. It is necessary to get eggs from individual tanks every how many days to determine the reproduction frequency. It would be interesting to study (calculate) the relationship between temperature and reproduction frequency - here each result is interesting.
3. It would be good if the results obtained were recalculated depending on the weight of the females: in this way relative fecundity would be obtained.
4. It would be very interesting to obtain data on the total fecundity (number of eggs) from individual groups throughout the duration of the study.
5. It is also important to indicate whether the fertilization percentage was calculated or not!
Additional comments: If the authors have output from individual pools, after re-calculating, e.g. relative and total fecundity, this will be a high-quality 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: SPAWNING OF FRENCH GRUNTS, HAEMULON FLAVOLINEATUM, IN RECIRCULATING AQUARIUM SYSTEMS
Review round: 2
Reviewer: 1

Output:
Basic reporting: MS has been improved in line with my comments. I have no negative comments about the current version.
Experimental design: The MS after corrections, including the description of the experiment, are presented correctly, legibly and clearly.
Validity of the findings: No comment
Additional comments: In my opinion, the current version of MS is absolutely acceptable for printing.


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: SPAWNING OF FRENCH GRUNTS, HAEMULON FLAVOLINEATUM, IN RECIRCULATING AQUARIUM SYSTEMS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The paper has been very well improved. My comments have been taken into account. Paper is easy to read now, is understandable and clear.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: The paper, after corrections, is of high quality. It refers to very important reproduction issues of coral reef fishes.


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: HABITAT ASSOCIATION IN THE CRITICALLY ENDANGERED MANGSHAN PIT VIPER (PROTOBOTHROPS MANGSHANENSIS), A SPECIES ENDEMIC TO CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors present an interesting study about an imperiled and secretive snake. The experimental design has produced novel information about habitat selection of this species, although I note below there are some caveats that may restrict interpretation.

The English is excellent but could be improved to more precisely communicate meaning and ensure correct grammar, for example lines 43-46 and 54-56.

Lines 68-74: The objectives/hypotheses imply there is a cyclical nature to habitat selection coincident with the seasons. However, “constantly changing” implies to me there is no order to habitat selection.
Experimental design: Lines 89-101: More information is needed to explain how transects were designed, where they were placed, and whether they changed. The reader needs to be assured that there is no bias in where the transects were placed that would affect purported habitat selection results.

Line 94: A number of studies have shown reptiles select habitat at multiple scales. What does a plot of 10x10 m represent to the snake and how/why was this size plot chosen by the researchers?

The sex, age, reproductive status or even metabolic condition (i.e., hungry or digesting) of a snake may affect how it selects habitat. The authors should note this was not controlled for.

It is critical to consider how variation in detection probability has affected results (for example, see Mazerolle et al. 2009). It is possible that snakes were easier to observe in certain habitats (perhaps with less vegetation); the current analysis will indicate that snakes selected these habitats even though they could be selecting multiple other habitats where they were more difficult to observe. Further, if the authors already suspected that these vipers are associated with logs (there is reference to an independent tracking study) they may be more inclined to search in and around these logs than they are in other habitats.

I’m concerned that there were nearly as many habitat variables (14) as there were plots (20-32). Can the authors demonstrate that this is a valid statistical analysis? Even after correlated variables were removed, the number remaining seems relatively high.
I am not qualified to evaluate the fine points of the analyses, but some questions were raised as I was reviewing associated parts of the manuscript. Some more information about the analyses and specifically what each were evaluating would be helpful. For example:

Line 148: I’m unsure if three separate analyses were conducted for each season or whether season was a variable included in one analysis. Some more information related to the analytical framework would be helpful. For example, would the authors have analyzed habitat selection by season if the stepwise discriminant function analysis had not indicated that they could be distinguished?

The authors have shown that what is available to the snakes is different from what is selected by the snakes within each season, but it’s unclear whether they analyzed whether what was available differed by season, which seems necessary to tease out whether changes in habitat selection by season are due to decisions made by the snake or seasonal changes in habitat.
Validity of the findings: Line 214-215: Did habitat selection change because of habitat heterogeneity or because they were selecting different habitats based on their physiological needs and/or reproductive status, for a couple examples.

Line 251-252: This is unclear. Did the availability of water change by season? If this is the case, how can the authors know whether they are analyzing changes in habitat selection or changes in habitat availability across different seasons?

Lines 252: What tracking study were the authors referring to? Are the authors implying that snakes selected habitats near water primarily because of foraging opportunities? Do they eat less in the summer?

Line 285: this mention of poachers here in the section about seasonal differences is a non sequitur.

Lines 288-291: This sentence seems redundant.

Lines 275-301: This section seems redundant with information presented elsewhere. I suggest the authors consider condensing and combining the sections of the discussion, first explaining that habitat selection changed by season and then providing examples why it might vary, including different needs related to shelter, thermoregulation, etc.

Lines 313-322: There is little background information or context provided for these conclusions and no clear link to the research and results described in this study.
Table 2: It is unclear what ‘correctly classified’ means.

Mazerolle, M.J. et al. 2009. Making great leaps forward: accounting for detectability in herpetological field studies.
Additional comments: No additional comments