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: LIVING ON THE EDGE: GENETIC STRUCTURE AND GEOGRAPHIC DISTRIBUTION IN THE THREATENED MARKHAM’S STORM-PETREL (HYDROBATES MARKHAMI)
Review round: 1
Reviewer: 1

Output:
Basic reporting: As I describe in the General Comments to the authors, there are a number of issues with regard to the writing. Some of these issues may stem from English being a second language for the authors, but some are also matters of clarity and depth of description. The abstract and the discussion sections have the most issues, though there are a number throughout the manuscript. I think the writing needs a good polishing before it can become suitable for publication.

Also, some citations are missing from the reference list. I suggest the authors check this.

Otherwise, the background provided is sufficient and relevant, the data collected and the analyses conducted are relevant to address the question specified and provide insights that will be useful to conservation of a threatened species. The structure of the manuscript is professional, as are the tables and figures, though the latter require some revisions to allow them to stand on their own and to make them more easy to see/interpret.
Experimental design: The research question is well defined and relevant. It fills a knowledge gap and will be important for conservation of a threatened species.

However, I believe the methods are not currently described in sufficient detail for them to be independently replicated. Please see the General comments to the authors for more information, but in general there are a number of missing details (e.g. PCR recipe, how literature records were filtered, etc).

I also have some concerns about the data analyses carried out (again pleae see the general comments to the authors for more information). None of these are fatal issues, but I suggest some more thoughtful description and potential reanalysis could show that they are robust and valid.
Validity of the findings: Overall, the results found are novel and provide useful information. I had some concerns about the robustness of the specific data analyses implemented (described above) but the general approach is sound.

Some assumptions are made (e.g. that storm petrels have strong fidelity to their nesting habitat), which are not supported with the literature. Also, some conclusions are made to strongly and the data/results presented in the manuscript are not sufficient to support them. They need to be more clearly identified as speculation, or removed/rephrased.
Additional comments: In this paper, the authors describe genetic and geographic distribution model analysis for a threatened seabird, Markham’s storm-petrel.

Overall, I think the data/results presented in this paper are important for conservation on this seabird and would be worth publishing.  However, in its current form the manuscript lacks clarity on many aspects, has some issues regarding data analysis, and makes some (strong) conclusions without sufficient evidence/data. I think these issues would need to be addressed before the manuscript could be suitable for publication, and I have detailed them below, along with some more minor comments. I hope the authors find these comments helpful in revising their paper.

Writing: I can appreciate that English may not be the first language of the authors, however there are many places throughout the manuscript where the writing needs to be revisited, either for grammar or for clarity. The abstract and the discussion in general have the greatest number of these issues. However, there are other instances scattered throughout the manuscript. Some examples (but NOT an exhaustive list):
•	Line 44 – What do you mean by inefficient conservation policies? Can you be more specific?
•	Line 60 – Artificial light can’t cause predation, so something seems missing here.
•	Line 65 – It would be best at first use to describe what you mean by saline areas in a desert
•	Line 143 – “Bootstrap support values were passed to the tree with the highest likelihood among the 1,000 independent tree inferences.” Please clarify as this currently doesn’t make sense. Perhaps you mean something like ‘Support values from 1000 bootstrap replicates were annotated on the tree with the highest likelihood’?
•	Line 262-266 – This description of the results is too vague. Please be more precise and descriptive, rather than referring just to the figures.
•	Line 331-332 - ???

There were several issues with data analysis that I think need to be addressed prior to publication. I have listed them below by section.
•	Phylogenetic analyses: Coalescent constant size was used as the tree prior for the BEAST analysis, and the justification provided is that these are population level analyses. That’s true, but if you include the outgroups (e.g. Albatrosses) there is quite a deep set of sequences used in the analyses, and presumably these are only a single sequence per taxon. I worry that this might have been an invalid prior (perhaps this is why you needed to run for 100 million generations with such a small data set?). I suggest repeating the analysis using a different tree prior (Birth-death or Yule) to see how it might influence your results.
•	Occurrence: The methods here are not clear enough to allow the work to be reproduced. For example, a total of 972 georeferenced records were initially identified and then these were reduced to 75 (line 160) but the criteria for removing all these records was not described. Further, if all the records correspond to breeding sites that were already known, what was the value of doing the literature search in the first place? Where did the anthropogenic disturbance data come from, specifically? Can it be found in the papers listed (e.g. supplemental data?) or from some databases? If the latter, then give the name of the database and how to access it.
•	Occurrence: The variance inflation factor was used to remove variables that were related to each other, and the threshold set was 10. These thresholds have been found to have some issues (e.g. see A caution regarding rules of thumb for variance inflation factors RM O'brien - Quality & quantity, 2007). I suggest defending your choice of threshold more thoroughly, and also acknowledging the limitation of this kind of analysis in the text. Further the Quinn and Keough 2002 citation is not in the reference list – I presume this is the textbook?
•	Geographic projection for future scenarios: Please explain/defend why you chose those three climate models (CCCMA, CSIRO, MIROC). How do they differ?
•	Migration constraints: Line 219 – please provide more information about the three thresholds selected (300, 500, and 700). Are these km? That will help put the dispersal analysis into a better context for the reader.

There are also a number of instances where the interpretation of the results is too broad or too strong for the data presented in the paper. Here are three examples from the genetics section:
•	Line 282 – I think you need to add the word potentially here. The observed pattern with the haplotypes could be do to gene flow, but it could also be due to incomplete lineage sorting of ancestral variation.
•	Line 297 -299 – I think there is not enough data presented in this paper to support this statement. Figure 1 shows that the north lineage is sister to a clade of more southern lineages. There is no dating or ancestral state reconstruction evidence to support this statement.
•	Line 307 – 312 – There is not enough evidence in this paper to support this statement. Wallace et al. has the proper sampling to address this, whereas this paper does not. Further there is no evidence to date the splits among the Hydrobates storm petrels to say whether divergence occurred before/after/at the same time as the origin of the Sechura and Atacama deserts.

The authors seem to assume that these storm petrels can only breed in these desert areas. I think some discussion of the evidence for this assumption is warranted, as it drives the interpretation of the future predictions of suitable habitat. I know some burrow nesting petrel species (e.g. Pterodroma petrels and Shearwaters) will burrow in rocky outcrops AND in soft soil, plus they have bred in man-made burrows for conservation purposes. This suggests some plasticity. Also, the birds must have some level of dispersal, otherwise there would not be multiple colonies of them. Finally, double check the use of the word phenology (e.g. see line 317). I think it is usually used with regard to timing rather than habitat.

Data accessibility: I noted that there were Genbank accession numbers provided for sequences obtained from the database, but there were no accession numbers provided for the new sequences described here (though I see a BankIt submission file to Genbank). The accession numbers should be provided before publication. Also, there is reference to material presented in an Appendix (e.g. lines 170, 174), but I could not find any file/document that I interpreted to be the Appendix. Please either clarify or add it during the submission process.

Figures: The figures for the habitat suitability and future projections (Figures 3 -5 in the main text, and other supplemental figures) are very difficult to see because the range of the storm petrel is rather narrow and long. Is there anyway to make this easier? For example, if there is a large portion of unsuitable habitat in the middle or at the bottom, maybe you could just say that’s unsuitable in the text/figure legend and then ‘zoom in’ onto the areas where there are changes or differences between the models. Perhaps the Zoomed in figures could be presented in the main text and the full figures (as they are now) could be provided in the supplement.

Supplemental Figures S2, S4 – Why are some combinations of parameters not present here? E.g. RCP2.6 with threshold 300 in Figure S4 and RCP 8.5 with threshold 700 in Figure S2.

Minor comments

Title – This title suggests that the main emphasis of the paper will be describing a complex conservation scenario. I took this to mean, e.g. translocations, etc. The paper doesn’t really discuss the conservation scenario necessary, so I would revise. Perhaps something like “Living on the edge: genetic structure and geographic distribution in the threatened seabird Markham’s Storm-Petrel”

Line 72 – Refer to figure 1 to help the reader get a sense of distance between breeding areas

Line 75 – Are you referring to chick hatching here, or chick rearing/fledging? I’m surprised that the dates are so wide. Do they differ by colony or does each colony really exhibit the full range of dates?

Line 115 – Please provide details of the PCR reaction (e.g. the amount of buffer, type of taq used, etc). If it’s described in the same paper as the primer sequences, then please state that.

Line 123 – Is this previously published Arica sequence from the same breeding colony as the others?

Line 128 – Please paraphrase this description of the saturation test better. It seems to be very similar to text from another publication.

Line 132 – I think it would be better if you mentioned the outgroup sequences that you got from Genbank here. I found them later on, but think it makes more sense to describe them at the end of this paragraph. Also, please list all the species used as outgroups (‘representatives of Oceantitidae, Diomedidae and Procellariidae’ is too vague). Double check spelling of Procellariidae – it should have two i’s.

Line 137 – What criterion did you use to select the substitution model? AIC? BIC? AICc?

Line 153 – You list some parameters here that were not described later in the results (e.g. Fst, which the sample size is too small for anyway). Please double check what you list here vs. the results you present.

Line 203 – I find it a bit confusing that you give the future climate variables different names (bio 6, bio 8, etc) because then I have to look up what they are. I suggest just using a brief description of the variable (e.g. min temp for bio 6).

Line 239 – Were the four haplotypes described here what you found with your new sequences? It would be helpful to clarify. Also, you should describe the whole data set, including the genbank sequences. How many haplotypes there were in total, etc.

Line 244 – Is the strange Arica sequence from Genbank or from your own field work? Could there be an misidentification error/typo for that collection locality of that individual?

References: Please check that all the citations are in the reference list. I found a couple that were missing.

Table 1 and Table 2 – Say what analysis this table of results is for. The table should be able to stand on their own and it’s not clear from the caption what these data are about.

Figure 1 – The branch supports are too hard to read, please make the text larger. The phylogeny is too hard to see, I suggest making the lines darker. What is the orange colour in this figure?


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: LIVING ON THE EDGE: GENETIC STRUCTURE AND GEOGRAPHIC DISTRIBUTION IN THE THREATENED MARKHAM’S STORM-PETREL (HYDROBATES MARKHAMI)
Review round: 1
Reviewer: 2

Output:
Basic reporting: This article is aimed at describing the phylogeographic structure and the role of environmental variables affecting the geographic distribution of an endangered (Markham’s storm-petrel), making inferences about future distribution expected under different climate change scenarios/models. My general assessment is that the manuscript contains interesting and novel results for an endangered and little studied species, but that in an attempt of being succinct felt short in more detailed explanations for many of the methods used.

Importantly, figures are blurred, and the legend for Fig 5 (which seem to be key for further interpretations) is barely readable. In addition, it would be of great benefit providing insets showing a zoom to current nesting areas. The scale of the maps (Fig 2-5) is also too exaggerated; there is need of showing half of South America, specially if the study is focused on the three major nesting areas, and a more restricted map such as the one presented in Fig. 1 would do a better job.
Experimental design: One of my main concerns is the little connection made between the phylogeographic analyses and the SDM. These analyses (along the migration analysis) should be closely tied among each other. For instance, given the existence of these three large spatial clusters with clear differences in phenological breeding, would not be wiser to carry out a SDM for each subpopulation by separate? I guess you sorted that out using the MigClim approach, but that should be explicit in the text.

The migration analyses was quite novel (I did not even that existed!), but I would like to see a more in-depth explanation of MigClim simulation (I do not know what columns in Table 2 mean). Please note that the migration is not even one of the declared aims of the study, and appears out of the sudden in methods. Also, the results of the simulation are not even discussed. The MigClim simulation should be part of aims of the study, along with one or several hypotheses. Please also note that given the existence of these three large spatial clusters, it would be possible to estimate migration among subpopulations (with software such as MIGRATE) and complement the MigClim simulation. I am no expert of phylogeographic analyses, authors may correct me if this migration analysis is not suitable with the used markers.

Regarding the SDM, I applaud the use of good practices such as a curatorial procedure to discard bad spatial occurrences, but please indicate if that was carried out manually of using some automated procedures (e.g., Zizka et al 2019, Methods Ecol Evol. 2019;10:744–751). I agree with other decisions made by the authors, such as controlling by predictor collinearity, and spatial autocorrelation using of the iCAR model. However, these methods/decision are introduced without any context. There are legions of papers addressing these topics, and it would be good to re-direct readers to these original sources motivating your methodological decisions. Keep in mind that many authors use SDM as a black box, e.g., using Maxent by default with little regard of all caveats and assumptions needed.

On a minor note, a recent article discuss the importance of adopting standardized practices reporting the output of SDMs (Zurell, D., J. Franklin, C. König, P. J. Bouchet, C. F. Dormann, J. Elith, G. Fandos, X. Feng, G. Guillera‐Arroita, and A. Guisan. 2020. A standard protocol for reporting species distribution models. Ecography 43(9):1261-1277). It would be good for the article to adopt such practice.
Validity of the findings: I am puzzled by author's interpretation that "...conservative and more severe climate change scenarios suggest reduction and strong reduction of the distribution of H. markhami..." (lines 315-316). I simply fail to see a reduction in the geographic areas of distribution (comparing the blurred and out scaled Figures 2-4, please see my recommendation for improving figures above). Judging by the figures, I see that all three main nesting areas will remain habitable under all models and scenarios. Then how a strong reduction in distribution is concluded? I guess that the MigClim simulation is leading that interpretation, but since it is barely explained in the results (and not in the discussion) my question remains. If this is the case, and the whole case for the 'complex conservation scenario' is rooted on the MigClim simulation, then this approach should be properly highlighted in the introduction.

The second paragraph of Discussion (lines 300-312) is a bit disconnected from the actual results of the study. However, these ideas may be better connected by further phylogeographic analyses. For instance, a Bayesian skyline plot may inform about the temporal trends in the effective population size, which may be connected with Pleistocene/Holocene paleoclimatic reconstructions of the Atacama Desert.
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: LIVING ON THE EDGE: GENETIC STRUCTURE AND GEOGRAPHIC DISTRIBUTION IN THE THREATENED MARKHAM’S STORM-PETREL (HYDROBATES MARKHAMI)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised version of this manuscript addresses most of my previous concerns. Authors have done a great job filling the gaps of the previous version, without a cost for the manuscript total length. Figure quality and resolution have also been improved.

A minor note
In the legend Figure 5the ‘pixel that have never been occupied (…)’ should be in gray symbol, not white (which is the sea in your map).
Experimental design: The new version of the manuscript addresses three major concerns I had before: 1) better connection between phylogeographic and SDMs analyses, 2) better explanation of curatorial procedures used to ensure data quality for SDMs, 3) a better contextualization and explanation of the MigClim simulation, and 4) an expanded explanation and justification for the use of the iCAR model.
My only remaining concern is that the raw database of geo-occurrences was not made available. Or is it already available in Barros et al. 2019? As far as I remember, having all the raw databases available is mandatory before final acceptance in the journal.
Validity of the findings: Now it is much clearer that the conclusions of the study are driven by the MigClim model. In fact, the MigClim is now more integrated to the rest of text.
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: SPATIOTEMPORAL PATTERNS OF THE COVID-19 EPIDEMIC IN MEXICO AT THE MUNICIPALITY LEVEL
Review round: 1
Reviewer: 1

Output:
Basic reporting: The papers applied a few spatial statistics to the COVID-19 data in the country.
Experimental design: fine
Validity of the findings: too simple
Additional comments: Suggestions:
1.	Figure 4. Calculate and add the spatial stratified heterogeneity (www.geodetector.cn)
2.	Compare and interpret the statistical findings in epidemiology in deep.


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: SPATIOTEMPORAL PATTERNS OF THE COVID-19 EPIDEMIC IN MEXICO AT THE MUNICIPALITY LEVEL
Review round: 1
Reviewer: 2

Output:
Basic reporting: The work is interesting, but it lacks many points, detailed below, for being able to be accepted. Several references need to be added, more suitable technical words be used and clarity is needed in several parts.
Experimental design: The question is well-defined and the methods are explained in detail.
Validity of the findings: I am missing a longer discussion, taking into account that the COVID-19 literature is likely to be used for policy-making. Then, I suggest the author to contextualize his results and discuss more broadly their consequences.
Additional comments: Fig. 1: this figure does not show the distances, it only displays some straight lines between the centroids of the geographical units. I suggest adding distances using a color code. I think that all the distances used in the work need to be included in this figure, illustrating the differences.
The word contaminated, which appears several times throughout the manuscript, is not the technical work used in the context of epidemics.
L19: 'at this date' is ambiguous, as the article may be read at any time, maybe 2050. I suggest saying 'By December 1, 2020'.
L21: 'and,'->'and'
L41: can you include some references?
L60-64: can you provide the digital sources of these datasets?
L71: references needed, I suggest to cite Pullano et al. Nature 590, 134-139 (2020), and Eguíluz et al., Frontiers in Medicine 7, 563455 (2020).
L78-79: not clear. 'Low levels of testing' means low positivity rates or low testing effort. Please clarify and, if possible, quantify this sentence. I do not agree with the statement saying that the number of cases will not change if testing effort is modified, taking into account the high number of asymptomatic cases.
L101-104: please reference Fig. 3 here.
Eq. (1): if I understood correctly, I depends on time, as it is computed weekly, but there is not time-dependence in this equation. Please specify it.
Question: what defines a cluster? Are the authors trying with random combinations and random sizes and keeping only those statistically relevant? These is an effort to introduce this in the manuscript, but more clarity is needed.
Fig. 4: I think that, for understanding better the spreading, this figure needs several panels, additionally to that already included: 1) temporal evolution of the number of clusters, 2) temporal evolution of the cluster geographical coverage (individual, average, or median).
L243: I think that 'amount' should be replaced by 'strength'
As I mentioned above, the final section needs more background and discussion.


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: SPATIOTEMPORAL PATTERNS OF THE COVID-19 EPIDEMIC IN MEXICO AT THE MUNICIPALITY LEVEL
Review round: 1
Reviewer: 3

Output:
Basic reporting: I believe this manuscript has potential, but requires additional work:

Authors should note more explicitly the assumptions in their analysis, e.g. lines 65-68 they are essentially assuming age and co-morbidity are distributed uniformly across municipalities (since it is excluded from the analysis) and lines 82-85 assuming no imported cases. They could then revisit this in comparison with the comment from line 137. For this reason, I also don't think the authors can conclude lines 250-252 since those effects were aggregated out of the model.

It is unclear to me what the purpose of the comparison mentioned in lines 101-104 adds since the analysis is done for Mexico only and the hypotheses presented by the authors do not mention comparison with other nations. For this reason I would omit Figure 3 and lines 206-213. The comparison in instead with Kang et al. 2020 and might be good to mention that this is work analysing cases in China.
Experimental design: Authors should specify timing to allow for increased options for reproducibility. Some of the current "dates" are not informative/defined
line 24 (end of March)
line 109 (end of March)
line 201 (beginning of the epidemic)
lines 203-204 (end of November)
line 226 (beginning of the epidemic)
Additionally "as the epidemic began in Mexico one month after Europe" is very vague.

Authors should add the time mentioned in their expression for Moran's I index. They mention is it time-dependent and so should have something like I_t, x_{it} and \bar{x_t} where $t$ denotes week and \bar{x_t} = \frac{1}{n}\sum\limits_{k = 1}^n x_{kt}.

Authors should note what the constant k represents (equation 3)

I would also add expressions for/calculations of D_c and D_{nc} by d_{ij} from (3)

What is the inset in figures 5, 6, and 7?

Authors should note what a good value for per cent relative difference is (Figure 8)? Is this a large value in absolute terms (indicating the algorithm's ability to detect clusters? Authors should explain this in more detail, including why some are negative.
Validity of the findings: The first peak mentioned on line 218 does not correspond with what is shown in Figure 4 where the first peak seems to occur around week 10

Line 254: My understanding is that scan statistics do not seek to prevent epidemic spread but rather detect anomalies (hence outbreaks/clusters) and so I think the wording is a bit too strong here

The authors might want to mention (line 221) that deaths might be a more "true" measure since all COVID-19-related deaths can be expected to be reported (since severe cases will lead to deaths) and so the authors could revisit their brief mention of the effect of underreporting mentioned earlier in the manuscript

The authors might want to note that the use of weekly case counts allows them to disregard day of the week effects (or they are subsumed), but that this is at the expense of having less information/a shorter time series.

Should extend final sentence (line 184) further.

The phrasing of the sentence in line 238 is a bit odd.
Additional comments: The colours mentioned in the text (lines 186-194) do not correspond with those used in Figure 1

I currently do not have access to a printer so I have not been able to check if all of the figures print well in black and white and would encourage the authors to do so.

Line 19 - I would suggest the authors add the year as there is a chance COVID-19 might still be around in Dec 2021

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Additional literature the authors may want to consider:

Akhmetzhanov et al. https://doi.org/10.1101/2020.04.24.20077800 includes some similar expressions to equation 3 and I believe use the same package in their calculations

Xia et al. https://doi.org/10.1086/422341 for use of a gravity model in an infectious disease application may provide insights into expanding the sentence on lines 183-184

Considerations regarding popultion density: https://www.scienceinparliament.org.uk/wp-content/uploads/2020/08/johnson_PSC.pdf

-------

Miscellaneous minor corrections:
* line 134 should be "tend" as "algorithms" is plural
* line 218"COVOID-19" should be "COVID-19"
*When defining calculations, authors should not note . Remove "RR is the relative risk" (158) and move "RD is the relative difference between the two sets to distances" (line 195) to before the equation.
* Inconsistencies in terminology in  paragraph starting on line 214 ("Morans's I statistic", "Moran index", and "Moran") and again in line 236 ("Moran statistic")
* Suggest authors add "in 2020" to caption for Figure 2
* Remove apostrophe in caption for Figure 6
* Add space after apostrophe in caption for Figure 7
* I am not sure the long/lat axes are needed for figures 6 and 7
* Inconsistencies in figure captions wrt whether a full stop is included at the end or not
* Inconsistencies in in-line mathematical symbols, would suggest authors go through the text and ensure everything is in italics to match equations (e.g. lines 158-159, 196-198, caption Figure 1, 188, but I have missed some).
* The scale and compass marker could be removed from figures 5, 6, and 7 as they do not add much
* Suggest adding "from a spatial perspective" after "contagion" on line 243


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: SPATIOTEMPORAL PATTERNS OF THE COVID-19 EPIDEMIC IN MEXICO AT THE MUNICIPALITY LEVEL
Review round: 2
Reviewer: 1

Output:
Basic reporting: please see my last report
Experimental design: please see my last report
Validity of the findings: please see my last report
Additional comments: 1.	The paper applied Moran’s I and Satscan to illustrate hotspots of COVID19 in Mexico. It is still too simple to be an academic paper and little interesting.
2.	Considering a highly diverse population, measure and attribute the spatial stratified heterogeneity (SSH) of the diseases and their change in time. Interpret the statistical findings in epidemiology.
3.	My concerns are not appropriately addressed.


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: SPATIOTEMPORAL PATTERNS OF THE COVID-19 EPIDEMIC IN MEXICO AT THE MUNICIPALITY LEVEL
Review round: 2
Reviewer: 2

Output:
Basic reporting: I did not like this revised version, basically because the authors try to avoid addressing some of the points raised by the reviewers, and even in one case they say they did something which in fact does not appear in the submitted manuscript.
First of all, the authors should revise the basic geometry concepts of path and distance for addressing my comments about Figure 2. A path is a line, while a distance can be calculated using that line, but it is a number. Also, in that figure, the color code for the lines ('distances') is not explained.
Authors mention that they have cited the suggested works but they did not, or at least these references do not appear in the manuscript that I received.
When authors mention that they applied spatial scan statistic, they do not mention to what they applied this algorithm (to cases, deaths, distances?).
Additionally, the new figure 7 looks interesting and may align with my previous comment, but it would be nice to see how a specific cluster evolves in consecutive weeks, to illustrate, for example, the spatial spread phenomena.
Experimental design: No comments
Validity of the findings: No comments
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: SPATIOTEMPORAL PATTERNS OF THE COVID-19 EPIDEMIC IN MEXICO AT THE MUNICIPALITY LEVEL
Review round: 2
Reviewer: 3

Output:
Basic reporting: As an additional author is now listed, authors should note their Contributor Roles Taxonomy (CRediT)

Authors should add results to the abstract (suggest placing before "This study helps us understand...").

"head city" is not an English term, suggest "largest" or "capital" city used instead.

Authors should note on line 195 that area under ROC curve abbreviates to AUC

In line 197 the authors should use the number 1 to be consistent with the rest of the text (explanation of I index etc.)

In line 200 the authors should note what n is (number of neighbours considered) to aid the reader
Experimental design: Authors dates are consistent in terms of both formatting (full versus abbreviated month names) as well as content (23rd February 2020 and 7th March 2021 vs 6th March 2021). They should ensure the correct and consistent study dates are used.

Authors may wish to use a negative binomial rather than a Poisson model, allowing for more variance.

Lines 319-320 Why haven't the authors done this? Such information should be available

Please explain in more detail how the false positive and true positive rates are calculated and enter ROC/AUC
Validity of the findings: Lines 202-211: For the reasons outlined by the authors it makes little sense to compare them; would suggest the authors retain focus on Mexico

Please split Figure 5 into two separate plots with separate y-axes and add a line where RR = 1 to show where no difference in relative risk occurs

It is a bit of a stretch to conclude the gravitational model is best with the amount of overlap seen in Figure 11
Additional comments: Please format the y-axis labels as numbers (Figure 4). Could use par(las = 1) to have them horizontally.

What determines the bin width in the histograms in Figures 8 and 9?

If noting which references are pre-prints, it seems De Anda-Jauregui should also have such a label


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: SPATIOTEMPORAL PATTERNS OF THE COVID-19 EPIDEMIC IN MEXICO AT THE MUNICIPALITY LEVEL
Review round: 2
Reviewer: 4

Output:
Basic reporting: This study was well conducted and reported. However, there were several limitations in the manuscript like failure to report asymptomatic cases, focusing on only two municipalities without stating any justification for this action. There is no discussion of any prophylactic treatments and vaccinations in Mexico which gives the impression that only the Jornada Nacional de Sana Distancia campaign was being used to curb COVID-19. It would be interesting to see the author incorporate these issues and information on recovered individuals in the municipalities.
Experimental design: The research questions and hypothesis are properly addressed. The methods are also well explained.
Validity of the findings: Further discussion of the results and their impact on policymaking on COVID-19 prevention measures like lockdown and vaccination is needed. These measures in addition to prophylactic treatments have been reported to reduce on the magnitude of the COVID-19 pandemic. A study in the USA reported that with limited protection against infection, vaccination could have a substantial impact on mitigating COVID-19 outbreaks (Moghadas et al., 2020). This could help better inform the audiences.
Additional comments: Abstract
Line 12-17: the abstract is quite informative, but more details should be incorporated. It seems to be focusing mainly on the methods used in the study. I suggest stating a brief COVID-19 background, Euclidean, gravitational, least cost, resistance distances. Gravitational distance was the best model to predict new infected municipalities though the AUC was relatively low and varied over time.

Introduction
Line 20: I would suggest adding some references.
Line 26: I think a brief inclusion of the mode of transmission of COVID-19 would be helpful to future readers.
Line 27: did this include complete restriction of movement and enforcement of continued use of PPEs by the public. Were there any pharmacological treatments during this period. If not please state it.
Line 54: It is not clear why it is a bold header, but I suggest creating a “data sources” section in the methods instead.
Line 67: are there records of the municipality of the health unit where patients consulted?
Line 88: the records discarded were based on the difference in the state of residence and state where the patient did the consultation. I would imagine that since the records of municipality address are available, using this and the municipality where the patient did consultation would be better since the stay is at the municipality level.
Line 89: has this been reported before? Or it is a hypothesis. If yes, is it statistically tested?

Methods
Line 114: I assume Mar 7, 2021, is a typo?
Line 115-117: please define x̅ t in equation (1), (average of the number of cases in the entire region)
Line 125: please include the reference for this cluster definition.
Line 164: what is the rationale of using Kilometer as the metric units, is this the metric unit used in the coordinate system you chose? I think the metric units should be the same.
Line 192: please include more references on ROC analysis.

Results
Line 204: Figure 3 does not show the drastic reduction of the number of deaths but rather the deaths are constant between 50 and 200 days. Please review the figure
Line 206-211: I would suggest including this in the discussion.
Line 224: In Figures 4,5 and 12; are “abr” and “dic” typos for the months of April and December respectively?
Line 240: figure 7; why was only Mexico City include in the inset map? Could it be that Mexico City reported the highest number of cases and clusters? I would suggest including a table showing the number of cumulative COVID-19 cases population and population density of the municipalities. This will help inform an international audience on what was referred to as a densely populated municipality in this study.  Please label the most densely populated municipalities, they could also reveal a unique trend.
Line 244: “It is also worth noting that 26% of the municipalities were included in a cluster at some moment during the period of study.” I suggest stating the specific weeks of the mentioned period.
Line 247: in figure 10: why did not the author stick to reporting Mexico City and reported Guanajuato’s municipality instead.
Line 260: I would suggest reporting the actual AUC values in Figure 11 description.
Line 270: please choose a better line pattern for the gravitational model. The current one makes the figure challenging to comprehend.

Discussion and Conclusion
Line 273: please specify the territory being referred to here.
Line 274: do you anticipate that most of the new COVID-19 cases were due to infected individuals returning from Europe?
Figure 8 & 9: I would suggest a further discussion of why the clusters remain for longer periods of time in the most populated cities.
Line 285: I agree with the authors, however, it would be helpful to state how robust the testing, prophylactic treatment, and vaccination are in Mexico.
Line 309: could head city be meaning capital city?
Line 320: did this affect your spatial analysis?
Additional discussion inclusions: I think testing being biased towards symptomatic cases could be a limitation in the study since asymptomatic cases could also transmit the disease. I think with the emergency and adoption of COVID-19 vaccines, commenting about it as a preventive strategy and how it could affect these spatial-temporal patterns is ideal. Analyzing spatial tendencies of the recovered cases could also help in modeling control and prevention measures of COVID-19.
Additional comments
Line 16-17: typo help not helps. Please double-check the “and” used numerous times.
Line 46: include “such”/ rephrase the sentence for better understanding.
Line 52: typo inter-municipality not intermunicipality
Line 270: figure 11 typo newly not new
Line 293: typo - distributed not distribuited

Reference
Moghadas, S. M., Vilches, T. N., Zhang, K., Wells, C. R., Shoukat, A., Singer, B. H., Meyers, L. A., Neuzil, K. M., Langley, J. M., Fitzpatrick, M. C., & Galvani, A. P. (2020). The impact of vaccination on COVID-19 outbreaks in the United States. MedRxiv : The Preprint Server for Health Sciences, 1–16. https://doi.org/10.1101/2020.11.27.20240051


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: SPATIOTEMPORAL PATTERNS OF THE COVID-19 EPIDEMIC IN MEXICO AT THE MUNICIPALITY LEVEL
Review round: 3
Reviewer: 1

Output:
Basic reporting: 1. My concerns are not appropriately addressed so the problems remained unsolved;
2. Compare gravity model and SEIR model in prediction of infection transmission;
3. Figures 3-6. Try to interpret the time series using behind spatial process.
Experimental design: see the above
Validity of the findings: see the above
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: SPATIOTEMPORAL PATTERNS OF THE COVID-19 EPIDEMIC IN MEXICO AT THE MUNICIPALITY LEVEL
Review round: 3
Reviewer: 2

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: I look forward to reading the point-by-point responses to the previous comments from reviewers 1 and 2 in the next revision. For this reason, I only provide a few comments at the current stage:

The references in the current version seem to be broken; they all render as "?".

Ad. my comment about using a negative binomial: I believe there may be some misunderstanding. Using a Poisson model is not a more "general purpose" approach, as it necessitates that the variance is the same as the mean. A negative binomial distribution would allow for more heterogeneity to be captured. It would seem that negative binomial options have been explored by the authors of the methodology used, cf. https://doi.org/10.1111/j.1541-0420.2010.01412.x so I hope the authors are able to try such an approach to see what effect that has on the results and provide their analysis code as open source.

Having multiple axes in figures remains confusing and I would suggest that less is more. Figures 4-6 and 14 have different time scales at different magnitudes. Readability would be improved if the x-axis above the chart were the main axis (i.e. place below the chart) and remove weeks. Similarly, in Figure 5 it would be easier to read the values of the two time series if they had separate y-axes as they are not on very different scales, one even seems to be a log scale. Having a Figure 5 with two side-by-side panels would be easier to decipher. While the descriptive plot in Figure 3 (it does not arise from the methods used and so should not really be in a Results section) is more readable, I would like to reiterate to the authors that unless case and death definitions and surveillance systems (testing, reporting, etc.) are the same across countries, it makes little sense to compare countries. I would once again suggest they retain focus on comparing the different options for distance metrics.

RE: Lines 37-41 There has not been much fomite transmission from contaminated surfaces for SARS-CoV-2 virus (https://doi.org/10.1016/S1473-3099(20)30561-2), please update to reflect that COVID-19 is a mostly airborne disease, see e.g. recent WHO review https://doi.org/10.12688/f1000research.52091.1


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

Input:
Title: SPATIOTEMPORAL PATTERNS OF THE COVID-19 EPIDEMIC IN MEXICO AT THE MUNICIPALITY LEVEL
Review round: 3
Reviewer: 3

Output:
Basic reporting: The authors properly report their methodology and findings.
Experimental design: Please see my last report
Validity of the findings: The author states that they mentioned the “huge effect of vaccination on mitigating the COVID-19 outbreaks”. However, this effect is not described in the manuscript.
Additional comments: Comments for the author
Abstract
The current incorporation of the methods and results by the author is very informative to an international audience.

Introduction
Line 28-30: It is not clear why you have the (March 23, 2021) date in brackets. Please rephrase and improve the grammatical flow.
Line 54: Are these global delays and shortages of COVID19 vaccines?
Line 57: Please rephrase “A large body of research”, I recommend “enormous research”.

Materials and methods
Line 88: Please maintain the “tense” throughout your reporting to remain chronological.
Line 89-91: I think you should state (but not the municipality) on its own and rephrase the sentence to give better meaning.
Line 93: What do you mean by “robust sampling” in this case?
Line 96-107: I would suggest stating the limitations after reporting your research findings. Perhaps in the discussion.
Line 115-118: I would suggest reporting this at the end of the methods section after describing how you obtained your results.
Line 123: Is “Mas (2021)” an abbreviation or a typo?
Line 129-132: Is there a justification for choosing France as a representative country for the whole of Europe?
Line 130-132: Please state the figure being referred to.
Line 171 “likelihood ratio” not “likelihood”
Line 172: I think p should be stated as p-value because it’s not defined as an abbreviation here.

Results
Line 269: Please rephrase the sentence for more clarification. “similar pattern than the index-based in new cases”.
Discussion and conclusion
Line 315: what does the author mean by “founded”?
Line 355: I suggest explicitly stating this as “High population density”
Line 359: Is stating “As point by Jo et al, (2021)” necessary in this case? Please clarify.

Additional comments
Line 19: The “and” between cost and resistance distances isn’t necessary.
Line 28: Is this comma “2,70 million” required?
Line 67: The sentence is not grammatically correct, I think it’s “carrying out”.
Line 78: I think typo “number” not “numbers”. The author needn’t make this plural.
Line 148: I think you should include “which is widely used to detect clusters” to improve the grammatic flow
Line 205: Typo determine not determinate
Line 205-211: Please maintain the grammatic tense in the manuscript
Line 232: typo municipalities not minucipalities.
Line 250: I think strength is a more precise word than strongness.
Line 265: figure 5 typo: right y-axis not rigth y-axis.
Line 269: Please include the hyphen “index-based”


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: BACTERIAL DIVERSITY AND POTENTIAL RISK FACTORS ASSOCIATED WITH SALMONELLA CONTAMINATION OF SEAFOOD PRODUCTS SOLD IN RETAIL MARKETS IN BANGKOK, THAILAND
Review round: 2
Reviewer: 1

Output:
Basic reporting: The structure of the article is adequate, as well as the tables and figures presented. The language is clear, and literature references support the findings and discussion.
Experimental design: The research presents a regional focus on the contamination of some seafood. However, the high contamination rates and testing the association of E. coli and Salmonella results bring insights that can be verified in other products in locations beyond Bangkok. Therefore, although it is a verification of the contamination of a regional product, the authors overcome this limitation of the study through data analysis and discussion of the paper. Furthermore, the methodology used was adequate and well described, the research question was clear and there was a rigorous investigation.
Validity of the findings: Data were well explored and properly analyzed. My only question is whether it would not be possible to use a T test to compare bacteria and different types of samples (Figure 4). I understand that due to the difference in sample N of each type of seafood, this comparison may be compromised. However, I suggest that in the next papers with this approach you carry out the sampling standardization between foods, or if this was the previous objective of this study, describe in the text why some products have a different N than others.

Moreover, a clear gap in the present paper, as mentioned in the conclusion, was the failure to verify the causes of this high contamination. However, although this matter has not been determined I believe that this work brings important information and will contribute to a better understanding of the contamination of these seafood.

Furthermore, I strongly advise you to carry out a more in-depth study trying to determine the causes of this high contamination, in order to obtain a microbiologically safe product.
Additional comments: I suggest the authors review the use of bacterial names in italics in the text. In conclusion section all bacterial names are not italicized.


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: BACTERIAL DIVERSITY AND POTENTIAL RISK FACTORS ASSOCIATED WITH SALMONELLA CONTAMINATION OF SEAFOOD PRODUCTS SOLD IN RETAIL MARKETS IN BANGKOK, THAILAND
Review round: 2
Reviewer: 2

Output:
Basic reporting: No main comment, limitations of statistical analysis could be discuss in the section Discussion and the results were not analyzed according to the period of sampling on several months.
Experimental design: Table 1 needs to be revised (Regional source of seafood)
Validity of the findings: Some confounding variables or interpretation bias could occur during the statistical analysis
Additional comments: See the additional review file


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

Input:
Title: BACTERIAL DIVERSITY AND POTENTIAL RISK FACTORS ASSOCIATED WITH SALMONELLA CONTAMINATION OF SEAFOOD PRODUCTS SOLD IN RETAIL MARKETS IN BANGKOK, THAILAND
Review round: 2
Reviewer: 3

Output:
Basic reporting: The English language should be improved some examples include lines 79, 96.

Authors should improve the information of the epidemiology of these pathogens in Thailand. They provide an extensive information about the seafood consumption and production.

Figure 1 should be improved, standard errors should be showed. VC, VP should be indicated. No figure captions were provided.
Experimental design: Authors should indicate if the analysis were performed in duplicate or triplicate.

L95- 99 Authors mention that individual seafood sample was purchased in the early morning (5 to 7 a.m.) and samples were kept on ice during transportation and submitted to the laboratory within 3 h. You should specify what temperature each sample was at the point of collection and how the samples were transported to the laboratory.

L149 Confirmation of Vibrios spp. should be determined by PCR to specify serogroups and pathogenicity factors. Furtheremore, Viable but nonculturable (VBNC) state strains in fish and seafood samples should be determined because this state is a survival strategy when bacteria suffer from stressed conditions such as low temperature. Bacteria in this state cannot be detected by culture-based methods, but they still maintain metabolic activity, and are even able to produce virulence proteins (Alleron et al., 2013; Morishige et al., 2015). It is a significant concern for food safety and public health.

L146 Specify the commercially antiserums (S&A Reagents Lab Ltd., Lat Phrao, Bangkok, Thailand) used to determine the serotype of Salmonella.
Validity of the findings: Authors should indicate that the average concentration of indicator bacteria occurrence is the general or total average concentration, as they are the averages of the population sampled.

The risk factors associated with Salmonella contamination, data shown in table 5, are not adequately explained. Authors should indicate, according to the OR and P values, if the variable represents a risk factor. Moreover, temperature and %RH were not considered as risk factors. However, authors mentioned that most of the ice used to store seafood was at risk of rapidly melting due to high ambient temperatures in open air conditions.

From my point of view, Conclusions are limited to the results, which were obtained by microbiological methodology without confirmation by molecular biology. Today, a large number of designed assay formats using these technologies are available commercially for the detection in foods of practically all major established pathogens and toxins, as well as of many emerging pathogens. Molecular biological assays are more specific, sensitive, and faster than conventional microbiological methods.
Additional comments: Authors should validate their findings and improve the interpretation of their results and discussions.


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: BACTERIAL DIVERSITY AND POTENTIAL RISK FACTORS ASSOCIATED WITH SALMONELLA CONTAMINATION OF SEAFOOD PRODUCTS SOLD IN RETAIL MARKETS IN BANGKOK, THAILAND
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors corrected the paper as requested.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors satisfactorily answered the questions addressed.


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: QUANTIFICATION OF ANTIBIOTIC RESISTANCE GENES AND MOBILE GENETIC IN DAIRY MANURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general, the manuscript is well-structured, and the literature is well referenced.

There are a few sections where the language should be improved, including:
•	Lines 90-91.
•	Lines 97-99.
•	Lines 117-126
•	Line 159

Table 3 and Figure 2 show the same results; therefore, they are redundant. It would be better to keep only the figure in the manuscript and provide the table as supplementary material.

Besides, table 43 is too crowded and hard to read. It has some values that do not make sense such as 4.18E+00, or 0.00E+00. Again, I consider it could be removed from the manuscript and provided as supplementary material.
Experimental design: In general, I consider this study had a solid and well-planned experimental design.

However, my main concern is that the analysis is based on the average of the samples from different farms, which, in my opinion, could mask the real fate of the ARG on the different stages of the manure management system.
Validity of the findings: This study found not very significant differences in ARG levels in different stages of a manure management system. The results are interesting since most of the studies have found different ARG levels under different conditions, which is generally attributed to the different microbial populations present in each stage. It would be useful to include some data about the general microbial composition of the different sampling points to strengthen these results.

My major concern is that these small differences in ARG levels on the different stages could be caused by averaging out the results of the different farms sampled in this study. If possible, it would be useful to group and present the data by farm and stage and then evaluate if there is a difference within each farm’s samples (along the process). That is the only way to conclude that the different stages/conditions of the manure in operating farms affect or not, the fate of ARGs since the condition on each farm could be very different.

It would be useful to include some more discussion on the putative microorganisms hosting ARGs. There are plenty of studies that have found interesting correlations that fit with the results reported in this study. This could strengthen the manuscript.
Additional comments: There are some other minor corrections suggested to the manuscript:

It would be helpful to include a short description of the reason for the high concentration of antibiotics in manures (low absorption in the body) to make a better transition between the paragraph ending on line 55 and the new one starting in line 56
.
Line 58:  Dairy cattle are considered potential mediators, reservoirs, and disseminators of resistant bacteria and/or ARGs. Shouldn’t it be dairy/cattle manures?

Line 82: …the understanding of regulating ARG in the process is still unknown. It is not clear what the authors are trying to express.

Line 87: lower instead of lowest.

Line 92-93: Most manure conditions were simulated at the bench scale, and only few analyzed commonly used practices in dairy farms. This sentence is not aligned with what is described in the paragraph, which states the finding on ARGs present in manures of different operating farms.

Lines 106-116. I am not sure if this paragraph provides valuable information for the manuscript since it mostly describes the limitation of a culture-based approach for the identification of antibiotic-resistant bacteria. It would be better to describe the advantage of using qPCR or a justification of why using qPCR to do this and not the use of culture-based or metagenomic approaches.

Lines 117-118: The goal of this study was to investigate the fate of ARGs and MGEs under dairy farm manure management. Include the fact that the manure management is a flushed system.
Lines 118-120: I consider that the author should refer to the “different manure management conditions” as different stages of the manure in the flushed system or something similar so the readers could quickly and easily associate them with the system they studied.

Line 130-131: Sample information is described elsewhere and 16S rRNA gene sequencing results were published. This sentence does not make sense in this place.

Line 132: have or account for instead of has.

Line 144: L instead of liter.

Lines 153-155: Liquid samples with clear-to-low turbidity were processed by the MO BIO PowerWater® kit, and 10 - 200 mL of each was filtered through a Millipore filter (0.45-μm pore size). Just wondering, could this filtration have retained some microbial biomass and affect the DNA isolation?

Line 226-227: Four manure management groups, FP, FM, PL, and SL, had similar positive percentages of gene types. You should refer to the manure samples (conditions or stages of the flushed system) consistently through the whole document to avoid confusion.

Line 241-242: DNA templates for qPCR were the same batch of extractions as PCR. Include reference to Table 1.

Line 325-326: This study showed that tnpA and sulII abundance were significantly correlated, which indicates that the sulII was possibly related to the HGT by transposons. This sentence is wrong or there is not data supporting it. It is true that sulII was correlated to tnpA but there is not evidence in this study of gene transfer of any sort. Please support this sentence or modify it.

Lines 340-343: Although current animal waste treatment systems in dairy farms are not specifically designed to remove ARGs, it is important to understand the potential impacts of existing manure management practices on removal of ARGs. ARGs are considered as an environmental contaminant, which may adversely impact human health. This part should not be part of the conclusions, it does not fit here and it is not necessary.


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: QUANTIFICATION OF ANTIBIOTIC RESISTANCE GENES AND MOBILE GENETIC IN DAIRY MANURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: I found the introduction a bit too long, with several examples for each idea the authors want to highlight. I think this could be shortened. If all the references are too be maintained, more general sentences could be written.

Figures S1-S6 are not provided by the authors.

In Table 2, it is not easy too the eye to understand what the “average column” refers to.
Experimental design: Animal manure is often used as organic fertilizer as an alternative to chemical fertilizers for arable soils of low fertility, and antibiotic residues, bacteria and antibiotic resistant genes are frequently detected in manure and manure amended soils. As the health of people is closely connected to the health of animals and our shared environment (One Health), it is important to explore antimicrobial resistance genes content of manure and the optimal management practices to reduce that content, once resistance may compromise the effectiveness of antibiotic treatment of bacterial infections. The authors show that different management practices don’t significantly impact the amount of ARGs in manure, highlighting the need for improved practices.

Some information is missing in the methods. For instance, detection of intI and tnpA genes is not mentioned anywhere in the text. Additionally, the primers listed in Table 1 for intI do not present the original primers reference, which is Barlow et al. 2004 AAC. Nothing is also mentioned about the fact that the tnpA primers only target the transposase of the IS6 group, not all tnpA in general; this changes the way results must be interpreted.
Validity of the findings: No comment
Additional comments: The authors screen for the presence of two genes that are usually part of a MGE, but are not a complete MGE by themselves. For instance, the intI gene is not a MGE; it is usually part of class 1 integrons. And detecting the intI1 gene does not ensure that the complete integron is present. This should be clarified; intI1 may be used as a marker or an indicator of a MGE presence, but it is not a MGE. An evidence supporting this observation is the fact that the authors did not find a correlation between intI and sulI, which are both part of the conserved segments of class 1 integrons.

Another thing that should be clarified is that HGT occurs by conjugation, transduction or natural transformation. HGT is not mediated by transposons (unless they are conjugative) nor integrons (lines 67-68, 317-318, 326, 350). Acquisition of naked DNA by natural transformation may also be relevant in the context of ARGs dissemination, especially in this type of environment.

The sentence in lines 321-2 needs to be re-written. Plasmid RSF1010 is not integrated into transposons; rather, the transposons mentioned in the reference (Cain et al 2010) carry genes that are derived from that plasmid, which is different.


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: QUANTIFICATION OF ANTIBIOTIC RESISTANCE GENES AND MOBILE GENETIC IN DAIRY MANURE
Review round: 2
Reviewer: 1

Output:
Basic reporting: In general, the manuscript is well-structured, and the literature is well referenced.
There are a few sections where the language should be improved and/or contains typos, for example:
Line 50: causND ing
Line 58: manures instead of manure.
Line 93: haveL

Thorough proofreading is recommended.

Figures and tables are well presented now.
Experimental design: In general, I consider this study had a solid and well-planned experimental design. The authors have addressed the recommendations provided in the first review.
Validity of the findings: The study provides interesting insight into the ARG evolution in manure treatments. Most of the previous concerns were addressed by the authors.

It would be useful to include some more discussion on the putative microorganisms hosting ARGs. For instance, Line-297-299: “Network analysis indicates intI1 and Psychrobacter, ermF and Pseudomonas, were significantly correlated (p < 0.05). This suggests Psychrobacter was potential hosts of intI1, and abundance of ermF could be attributable to the presence of Pseudomonas.” This paragraph is short and should be expanded to include some of the potential microbial species hosting ARG reported in previous studies and briefly discuss if the finding of this study agrees or not with them. A few studies provide interesting results on this, for example:
Duan, M., Gu, J., Wang, X., Li, Y., Zhang, R., Hu, T., & Zhou, B. (2019). Factors that affect the occurrence and distribution of antibiotic resistance genes in soils from livestock and poultry farms. Ecotoxicology and Environmental Safety, 180, 114–122. https://doi.org/10.1016/j.ecoenv.2019.05.005
Flores-Orozco, D., Patidar, R., Levin, D. B., Sparling, R., Kumar, A., & Çiçek, N. (2020). Effect of mesophilic anaerobic digestion on the resistome profile of dairy manure. Bioresource Technology, 315, 123889. https://doi.org/10.1016/j.biortech.2020.123889
Zhang, Q., Xu, J., Wang, X., Zhu, W., Pang, X., & Zhao, J. (2021). Changes and distributions of antibiotic resistance genes in liquid and solid fractions in mesophilic and thermophilic anaerobic digestion of dairy manure. Bioresource Technology, 320, 124372. https://doi.org/10.1016/j.biortech.2020.124372
Additional comments: There are some other minor corrections suggested to the manuscript:
Line 53-54: “The high concentrations of antibiotics in excreted manure is potentially due to low absorption in the cattle body”. A reference for this is missing. You can use (Jjemba, 2002),
•	Jjemba, P. K. (2002). The potential impact of veterinary and human therapeutic agents in manure and biosolids on plants grown on arable land: A review. Agriculture, Ecosystems and Environment, 93, 267–278. https://doi.org/10.1016/S0167-8809(01)00350-4

Line 277-279: “Previous studies showed various responses of ARGs to biological conditions such as anaerobic lagoons and composting”. Include a citation here.

Line 300 – 301: “In general, ARGs are persistent in the environment, and it is well established that many putative microorganisms hosts ARGs”. I feel this sentence does not fit in this section, please remove.


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: QUANTIFICATION OF ANTIBIOTIC RESISTANCE GENES AND MOBILE GENETIC IN DAIRY MANURE
Review round: 2
Reviewer: 2

Output:
Basic reporting: Some references are lacking, especially in newly added sentences.
Experimental design: Authors included the original reference of intI primers, but kept the previous reference. Why?
Although the authors said they included a sentence regarding the limitation of tnpA and IS6, they did not do it.
Validity of the findings: Some limitations of the study were not stated.
Additional comments: First of all, it would have been helpful if the authors have included the lines where changes were done in the responses to the reviewers. It was difficult to find where were the changes they said they did it.

In my opinion, the authors did not put effort in improving the manuscript, but rather copied comments from both reviewers, without including supporting references. I think they need to re-read the comments from the first review and really improve the manuscript.

Additionally, the authors said they deleted some information, but they did not. For instance, it is still written that “The plasmid was also found to be integrated into transposons” (line 306), which is wrong.


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: QUANTIFICATION OF ANTIBIOTIC RESISTANCE GENES AND MOBILE GENETIC IN DAIRY MANURE
Review round: 3
Reviewer: 1

Output:
Basic reporting: Thorough proofreading is still required, especially in the content added in this new revision.

Line 323-331: “Elevated levels of ARGs and IntI1 were found in many farm samples such as chicken farms, pig farms, and beef cattle farms”.   This sentence requires a citation.
Experimental design: Line 151-155. “While this study evaluated the presence of two genes that are usually part of a MGE, it is important to note that they are not complete MGE by themselves. For instance, the intI gene is not a MGE; it is usually part of class 1 integrons, and detecting the intI1 gene does not ensure that the complete integron is present. The intI1 may be used as a marker or an indicator of a MGE presence, however, it is not a MGE”. I feel this part does not really fit in this section. It sounds more like a discussion of the limitations rather than methodology.
Validity of the findings: The quality of the manuscript has certainly improved. However, some minors corrections are required, especially in the new content.

Line 285-290. “While IS6, family members of bacterial and archaeal insertion sequences are known to play crucial role in spreading antibiotic-resistance genes, overproduction and inhibition phenomenon presents challenges (Munoz-Lopez and Garcia-Perez, 2010; Harmer and Hall, 2019). The use of transposons, tnpA, has disadvantages due to the generally nonspecific nature of target site selection (Fickman and Dyda, 2016).”
These new sentences should be developed a bit more and framed better. For example, the part “…overproduction and inhibition phenomenon presents challenges” should include some details in the challenges. I imagine that the idea was to say that the overproduction and inhibition of IS6 do not necessarily mean an increase or decrease in HGT.
The part “The use of transposons, tnpA, has disadvantages due to the generally nonspecific nature of target site selection” is only focused on the limitation and does not provide an actual discussion on the meaning of the finding. The author could mention that even though the presence of tnpA does not necessarily mean an increase ARG spread, it could suggest a greater risk of HGT, or some similar ideas.

Line 323-340 : “Elevated levels of ARGs and IntI1 were found in many farm samples such as chicken farms, pig farms, and beef cattle farms. Further many ARGs such as tetX, sul1, sul2, and tetG were dominant in farm soils (Duan et al., 2019). Studies (Duan et al.,2019; Zhang et al., 2021) focused on evaluating the impacts of manure on soil’s ARGs, and …”. I consider this part does not belong to the paragraph. It could stand as a separate paragraph.

Line 338-340. “In addition, this will help in developing improved manure management practices, which are capable of unwanted microbial population and ARGs in manure.” This sentence needs to be corrected. I assumed the intention was to say that manure management practices are capable of reducing microbial populations potentially harboring ARGs.
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: QUANTIFICATION OF ANTIBIOTIC RESISTANCE GENES AND MOBILE GENETIC IN DAIRY MANURE
Review round: 3
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: Misunderstanding of some concepts limit the validity of the discussion. More details are added below.
Additional comments: The authors overlook my comments about fully copied paragraphs from reviewers suggestions, including lack of references (for example, lines 93-99, 151-155 and 301-2). The idea present in lines 93-99 is already partially included in lines 53-62; the ideas should be included only in one place. Lines 151-155 were included in material and methods, when they are more a limitation of the manuscript as it was discussed. Regarding transposons, lines 300-1 have the opposed meaning of lines 301-3. The same idea is here: “Network analysis showed that sulII was significantly correlated with HGT by transposase gene” (lines 349-350) cannot be said, as tnpA does not promote intercellular movement by HGT, it is only able to promote intracellular movement.

Additionally, the limitation of the use of primers for tnpA of IS6 group is not included. The authors mention IS6 once in the discussion (line 285), without a previous explanation before. The readers won’t understand that IS6 is mentioned because the primers the authors use for tnpA detection are specific to this family. Furthermore, the sentence used (from Hickman and Dyda) does not apply here; the nonspecificity is related with the target site sequence. In this study, the authors amplify the transposase gene by PCR, which is different and specific for this IS family.


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: QUANTIFICATION OF ANTIBIOTIC RESISTANCE GENES AND MOBILE GENETIC IN DAIRY MANURE
Review round: 4
Reviewer: 1

Output:
Basic reporting: Some typos:
Line 35: “…which are considered to be responsible for the dissemination of ARGs in environment”. Should be “… in the environment”.
Line: 46: “concernt” instead of concern.
Line 104: there is a dot at the beginning of the sentence.
Line 111: “under” instead of under.
Line 201: “useing” instead of using.
Line 348: “… limited dairy farms,. Further…”
Line 350: “an” instead of and
Experimental design: NA
Validity of the findings: I still feel that the discussion on the cooccurrence of ARGs, MGEs, and microbial communities is weak and should be structured better because it is a little hard to follow. For instance, it should start stating the main correlations found in the study and then proceed with the discussion.
Also, the following sentences need to be frame better in the context of this study since they seem disconnected:
•	Line 318-319: “The sulII gene was reported on a broad-host plasmid RSF1010 (Rådström and Swedberg 1988; Yau et al. 2010)”. This sentence needs to be aligned with the previous sentence on the importance of determining ARG transfer mechanisms.
•	Line 319-320: “Huang et al. (2019) reported abundance of ARGs and transposase genes, which were decreased during anaerobic digestion of swine manure”. It would be better to say that Huang et al. (2019) found a reduction in ARG and transposase genes levels during AD of pig manure, which suggested that transposase genes could be correlated with ARG or something of the sort linked to your findings.
•	Line 324: the discussion on the microbial species could stand as a separate paragraph. It could start highlighting that Psychrobacter and Pseudomonas were correlated with ARG and MGE. Then describe why this finding is important, mainly what is already there but framed a little better.
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: A JOURNEY THROUGH THE CORYNEBACTERIUM PSEUDOTUBERCULOSIS PROTEOME PROMOTES INSIGHTS INTO ITS FUNCTIONAL GENOME
Review round: 1
Reviewer: 1

Output:
Basic reporting: This review paper is comprehensive with the stated topic with current information. This review have included all data compare and contrast with previous and current research related to the topic and the group of author is the expert subject matter. Well Done.
Experimental design: Well design manuscript and written and can be publish.
Validity of the findings: Yes all the data are compared and contrast with current and previous findings with subject matter of this review paper.
Additional comments: Well written and good studies have been conducted with the stated subject matter.


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: A JOURNEY THROUGH THE CORYNEBACTERIUM PSEUDOTUBERCULOSIS PROTEOME PROMOTES INSIGHTS INTO ITS FUNCTIONAL GENOME
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors are an established group studying the pathogenesis of C. paratuberculosis and have compiled a thoughtful review on the molecular basis of pathogenesis of this microbe. The review is very informative and exhaustive and the topic is not reviewed  very often. The English language needs to be improved. Also there are some redundant information in the manuscript which needs to be trimmed to make the review more crisp and meaningful. The abstract needs to be  rewritten highlighting the key thoughts from the review.
Experimental design: Specific comments
1.Lines 77-91 is not useful and should be removed.
Line 95 -what are these tools??
Line 97- rephrase. Does not make any sense; too long sentence.
Line 152- check grammar
Line 238-grammar
Line 240- bovine fetal? Please proof read extensively!
Line 260-isolated field???
Line 261- What is label based on demethylation? explain
Line 293-oxide nitric???
Lines 408-417 can be removed.
Validity of the findings: I would like a table showing the most relevant proteins which are  identified across several studies and could be immunogenic and also could be vaccine  candidates.  Also, tabulate them according to function? Which ones do the authors think are best candidates? speculate.
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: A JOURNEY THROUGH THE CORYNEBACTERIUM PSEUDOTUBERCULOSIS PROTEOME PROMOTES INSIGHTS INTO ITS FUNCTIONAL GENOME
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript submitted by Wanderson Marques da Silva and co-author is summarizing the importance and progress of methodology in the field of functional –omics and how high-throughput techniques improved our understanding of C. pseudotuberculosis.
Experimental design: A check of the methology showed that several articles are neglected by the authors. An inclusion of manuscripts would imrove the manuscript.
Validity of the findings: The manuscript would benefit from a schematic overview, which approaches were used in the past and which strains were examined. Moreover, a comparison of the results from all different studies would help the reader. A short analysis of the common findings could specify targets and the present review would give the reader an outlook for future work.
Additional comments: General remarks
(i)  Substantial English editing is recommended and several sentences need substantial rewording (see e.g.:  line 100-104; 133-124; 123-124; 276-277; 298-301; 339-341; 405-406; 415-417; 419-420; 428-431; 434-435).
(ii) There are too many self-citations (48) in the manuscript, even when considering that the senior author contributed a lot in the field. Especially the introduction part should be revised, and more proper articles should be included. For example, Dorella et al., 2006a is not a good reference to describe the complex cell wall architecture of corynebacteria (line 46-47). A check of the methology showed that several articles are neglected by the authors. An inclusion of manuscripts would reduce the amount of self-citations.
(iii) Furthermore, the manuscript would benefit from a schematic overview, which approaches were used in the past and which strains were examined. Moreover, a comparison of the results from all different studies would help the reader. A short analysis of the common findings could specify targets and the present review would give the reader an outlook for future work.
(iv) In general, description of cited research is often rather superficial without giving any details of results obtained. See e.g.:
-	Paragraph 77-91:
o	What is “a high number”?
-	Paragraph 130-147:
o	how did growth in CDM improve the identiy? (line 140)
o	how did the TPP method improve the results? (146-147)
o	Is this “bias” confirmed?
-	Paragraph 148-163:
o	You started to compare gel-based and gel free proteomic approaches
o	Could gel free approaches and bioinformatics techniques improve the quality of the results?
-	Paragraph 164-184:
o	93 different proteins were detected in this study. Please specify, if this result was strain specific or in combination. Compare the findings and list them.
o	Specify: the majority (line 174-175)
o	You listed virulence factors (PLD, FagD and Cp40) identified in C231_ovis, are they present in 1002_ovis as well? You mention that this strain is described as less virulent. Please compare the virulence factors found in both strains. A Venn-diagram can visualize the differences and commonalities.
o	Are both strains clinical isolates?
-	Paragraph 210-226:
o	Please list the 13 proteins from C. pseudotuberculosis and the 49 host proteins. This is a very interesting finding and should be explained in detail. Host-pathogen interaction networks can give a better understanding the infection process and gives hints for future research.
-	Paragraph 227-237:
o	For membrane proteomics it is recommended to use different proteases. Moreover, a filter aided sample preparation (FASP) an improve the identification. Please specify the protease
o	Please list the 13 proteins from the bacteria, isolated from lymph nodes
o	Please specify the identified 49 proteins from the host. This result shows the Host-pathogen interaction.
-	Paragraph 257-264:
o	Incorrect reference: this paragraph is about Rees et al., 2015b.
o	Are there differences in the quality of the results between label-free and label-based methods and genomics?
-	Paragraph 265-275:
o	Please specify the differences between the strains, analyzed.
o	A Venn-diagram or table would visualize the differences
-	Paragraph 276-283:
o	Rewrite the paragraph and focus on biofilm formation or show the differences between the different strains.
-	Paragraph 321-329:
o	Please explain the transposon based screening, this would also clarify the mutant strains
o	How are the mice immunized? Are they immunized with the ciuA siderophore null strain and then challenged with a virulent strain?
o	Strain differences?
o	Is this paragraph necessary?
-	Paragraph 330-345:
o	What means “best immune response”? Please specify
o	Leal et al., 2018 do not include experiments with rCP01850 (line: 341)
-	Paragraph 346-362:
o	Please list these 49 proteins with antigenic properties
o	In line 361 you mentioned the resulting proteins. For consistency, please do this as well for the other proteins mentioned in your manuscript.
-	Paragraph 363-373
o	Again the proteins are not mentioned (line 367 and 372)
o	Especially the potential targets would underline the importance of the use of proteomic strategies to evaluate the virulence potential.
-	Paragraph 391-406:
o	Please mention that the bacteria extracted from lymph nodes were compared to bacteria grown in liquid culture.
-	Paragraph 407-417:
o	Specify: pathogenic process specific (line 417)

Minor points
-	Line 70: corynebacteria
-	Line 97: “there is no in the literature a work with current information” plese revise
-	Line 100 – 104: please rewrite the sentence.
-	Line 123-124: please correct the sentence
-	Line 165: What does MQD mean?
-	Line 188: that alternative sigma factors are
-	Line 191: sigmafactors
-	Line 222: “bacterial  surfaceome”
-	Line 238: For the characterization of the surface proteome
-	Line 238: VD57,
-	Line 248 249: The whole-cell proteomic strategy allows the identification and quantification of thousands of proteins.
-	Line 253 -254: to the identification of proteins
-	Line 254: virulence
-	Line 269: A DNA methylase
-	Line 276 – 277: please rewrite the sentence
-	Line 281: formation in prokaryotic organisms / prokaryotes
-	Line 296: technologies
-	Line 298 – 301: please correct the sentence
-	Line 346: post genomic era
-	Line 351-353: please rewrite the sentences
-	Line 356: what means PSE
-	Line 366: modelomic
-	Line 367: strains
-	Line 387: pathogen
-	Line 390: response_(Henderson…)
-	Line 402: binds
-	Line 405 – 406: please rewrite the sentence
-	Line 413: underscore after unveiled, please revise
-	Line 414 – 417: please rewrite the sentence
-	Line 419: proteins
-	Line 441: identification
-	Line 445 – 446: Future research, based on other
-	Please revise the reference section.
o	Do not start words with capital letters in the title
o	DOI number is missing for some references
o	Line 834: Streptococcus in italics
o	Line 936 – 937: Corynebacterium pseudotuberculosis in italics
-	I have found many spelling mistakes in the text. For this purpose, I suggest an English editing service or a native speaker.
-	The word utilization has been used excessively in the text, please revise.
-	NclassG+ is not available anymore, this should be mentioned or removed from the manuscript (line 162)
-	Please add references for the CMNR group (line 44)


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: A JOURNEY THROUGH THE CORYNEBACTERIUM PSEUDOTUBERCULOSIS PROTEOME PROMOTES INSIGHTS INTO ITS FUNCTIONAL GENOME
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have answered most of the reviewer comments. I have no further queries
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: A JOURNEY THROUGH THE CORYNEBACTERIUM PSEUDOTUBERCULOSIS PROTEOME PROMOTES INSIGHTS INTO ITS FUNCTIONAL GENOME
Review round: 2
Reviewer: 2

Output:
Basic reporting: The mauscript entitled „A journey through the Corynebacterium pseudotuberculosis proteome promotes insights into its functional genome” is interesting and enjoyable to read. The article provides information about previous and current research and future challenges. The authors improved the manuscript significantly. Nevertheless, some minor changes are recommended.
Experimental design: In some titles of references, words always start with a capital letter. Please revise (Line: 654-656; 665-669; 672-674; 718-719; 720-721; 805-806; 852-854; 858-860; 865-868; 914-915; 918-920; 999-1000; 1021-1024; 1033-1036; 1096-1097).
Validity of the findings: Abbreviations of proteins should be written consistently. Always start with a capital letter, please revise the manuscript (Line: 289; 292; 303; 323; 324; 352; 462; 464).
Additional comments: The included tables and figures improve the manuscript considerably. However, the format of Table S.2 should be improved. It is hard to assign the “x” to the respective protein and reference. Moreover, it would be helpful to include the protein identifier. Especially to distinguish between the hypothetical proteins.

English editing:
Line 66: “described as major virulence factor this bacterium” the word “of” is missing
Line 152: produced
Line 198: CDM..
Line 199: ad
Line: 212-213: please revise the sentence
Line 223: proteins
Line: 237: proteomic
Line 344: “
Line 350: “differences quantitative” please revise
Line 387 – 388: please revise the sentence
Line 504 – 508: please revise the sentence
Line 513: an
Line 515 – 517: please rewrite the sentence
Line 521: “function biological” please revise
Line 532: “expression genic” please revise


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: REDISCOVERY OF LAURA’S GLASSFROG NYMPHARGUS LAURAE (ANURA: CENTROLENIDAE) WITH NEW DATA ON ITS MORPHOLOGY, COLOURATION, PHYLOGENETIC POSITION AND CONSERVATION IN ECUADOR
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is clearly written, well-structured and uses appropriate technical language. The introduction provides sufficient background on the problem and cites all relevant previous literature. Methods, results, discussion, figures, tables, and raw data shared conforms all the standards of PeerJ. However, there are some minor issues, namely:

In the methods section, it is unclear why the authors decided to exclude hypervariable regions from the alignments. I suggest including a brief justification for this action (see comment on line 151 in the annotated pdf).

Also in methods, I consider that the part corresponding to the evaluation of the best partition schemes and replacement models using PartitionFinder should be better explained. As currently described, it is unclear how many and which partitions were selected as part of the optimal scheme. In addition, I think the claim that the same replacement model was selected for all partitions should be verified. I included detailed comments on this topic in lines 157-158 of the annotated pdf.

Several species names must be corrected in the figures 5 and S1, and in the Table S1 (see notes on Fig. 5 in the annotated pdf and in Table S1, annexed at the end of the annotated pdf).

A couple of citations are missing in the references (see lines 57 and 268 in the annotated pdf) and all the citations corresponding to the primers used in the study are also missing (see lines 126-131 in the annotated pdf). Other minor details that need to be corrected are highlighted throughout the annotated pdf.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: No additional comments


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

Input:
Title: REDISCOVERY OF LAURA’S GLASSFROG NYMPHARGUS LAURAE (ANURA: CENTROLENIDAE) WITH NEW DATA ON ITS MORPHOLOGY, COLOURATION, PHYLOGENETIC POSITION AND CONSERVATION IN ECUADOR
Review round: 1
Reviewer: 2

Output:
Basic reporting: The structure of the document is clear, concise, and includes all the elements required to comprehend the results obtained and their relevance.
The document includes the basic literature relevant to the study field. The supporting data are effectively provided by the authors. The colors of N laurae and N siren are inverted in the legend of figure 1 so it has to be corrected.
The names of the species in the phylogenetic tree (Figure 5) must be italicized
Line 179: "presented by IUCN (2012, 2019)"
Experimental design: Please provide in the Methods section the information about the museum where the specimens are deposited. Some information is available in the results section but it has to be specified before.
Validity of the findings: The phylogenetic position as well as the morphological comparisons with similar and close-related species are valuable results on this research.

The main concern about the findings is the use of only one specimen on the molecular analysis, the geographic distance between the type locality and this report, and the morphological variation on diagnosis characters (toe papillae) mentioned by the authors. Therefore, the authors must provide enough information to 1) confirm that both individuals collected are from the same species and 2) the species is not a new fourth ocellated Nymphargus species.

The authors must justify the exclusion of hypervariable regions (I suppose are those from ribosomal sequences) in the analysis.
Additional comments: I wish to congratulate the authors on their high-quality work.


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

Input:
Title: IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF THE ALTERNATIVE SPLICED VARIANTS OF BETA CARBONIC ANHYDRASE 1 (ΒCA1) FROM ARABIDOPSIS THALIANA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a very interesting study on carbonic anhydrases. The authors provide an interesting set of data covering the bioinformatics analysis and molecular structure of four βCA1 splicing variants. They provide pooled gene expression data for the four βCA1 splicing variants.
•	Data establishes that beta-CA1 has 4 alternative forms, or spliced variants.
•	The spliced variants are differentially expressed in Arabidopsis tissues.
•	Data shows specific localization of the variants to the chloroplast and cytosol.
1. The introduction addresses the important issues to make the reader understand the paper. One main concern I have throughout the paper is the language and grammatical errors, a few of which I highlight below:
a.	The abbreviation for Alternative Splicing is defined twice, in Line 68 and Line 86.
b.	Line 77 grammatical errors.
c.	Line 101, the 7 beginning sentence should be written in full.
d.	Line 126 repetitive word…’sequences’.
e.	Lines 129 and 146 correct word …’Analyze’.
f.	Line 143 plant transformation….
2. The variants were analyzed in combination as (β-CA1.1 and β-CA1.2) and for β-CA1.3 and β-CA1.4. This implies the transcript is in pools as shown in Fig 1C. However the reporting in  Lines 208-213 separates βCA1.1  and βCA1.4 as if they are individual expression of individual variants. I think the reporting needs to be consistent for the pools.
Experimental design: The methods are described clearly.
1. One issue I raise is specificity on how the plants were exposed to heat stress. It is not clear if one wanted to repeat this treatment. I refer to Line 105-107: It is not clear whether the seeds or the plants were subjected to heat.
2. On the same matter, there is inconsistency on the temperature used for heat treatment.  Line 106 in text mentions 50oC, while Fig 3 caption indicates 40oC, the Image Fig 3 shows 50oC. Please clarify.
Line 179 – 183: While the title refers to accession numbers, the Gene ID or Loci is what is provided not the accession numbers. Either remove the title, or change it to gene ID, or provide actual accession numbers.
On subcellular localization, Line 157 mentions protoplasts from Four-week old seedlings, but Figure 2 is saying one week old seedlings. Please address the inconsistency.
Validity of the findings: No comment
Additional comments: The manuscript needs language and grammatical editing. I recommend a proficient English writer or professional editing service.
a.	Additional examples of these errors:
b.	Sentence in Lines 76 – 78  not grammatically correct.
c.	Sentence Line 149- 151
d.	Line 251: The importance of …..
e.	Lines 285 -287, sentence not clear.
f.	Line 284 …’frechuntely’

RT-PCR gene expression data is great and shows expression differences. Quantitative (q-PCR) would have been more robust and could quantify expression of each individual variant.
3. There is a Table in Fabre et al., (2007) showing that Arabidopsis βCA1 has 9 cDNAs. Here you report four splicing variants. So the other cDNAs do not code functional proteins?  It might be important to bring this to attention in your discussion.