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: GOING ROGUE: WHAT SCIENTISTS CAN LEARN ABOUT TWITTER COMMUNICATION FROM “ALT” GOVERNMENT ACCOUNTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is generally readable and well written. There's a bit of awkward language here and there (266-269 runs on and uses "from" in an odd manner for example), but nothing which confuses.

1. The term sentiment is used in place of emotion to reference the DepecheMood attributes.  In their original manuscript (Staiano and Guerini 2014) the authors of the lexicon deliberately distinguish their emotional lexicon from sentiment based lexicons.

2. I do not agree with the statement that Staiano and Guerini 2014  "confirm automated sentiment coding as an effective and time saving proxy for manual coding".  They concluded "showing significant improvements over state-of-the-art unsupervised approaches" not equivalence to manual coding.  This is, admittedly, a fairly minor nitpick but the ANGER affective dimension was firmly in the middle of the pack for matching human annotation.
Experimental design: 1. This manuscript does not seem to fit into the scope of the journal.  The focus of the manuscript is the application of statistical approaches to the analysis of data regarding microblogging about science topics.  The manuscript explicitly states one of the goals is to analyze "communication strategies" (117-118) and that another is to test the applicability of "non-parametric tests, multivariate ordinations ... "  (118-119) I would recommend targeting a journal with a focus on communication, science communication, or statistical analysis.

2. The DepecheMood lexicon annotates each term with eight "affective dimensions" not seven.  The eighth "don't care" dimension is not mentioned in the manuscript.

3.  No github link or version number is provided for the DepecheMood lexicon.  There are currently two versions available (1.0 and 2.0 aka DM++).  In order to replicate the work please specify which version was used.

4. It's not clear how "all official U.S. federal Twitter accounts with an "alt" or "rogue" corollary ..." (132 - 134). were identified.  What strategy was used to identify official accounts and, more problematically, "alt" versions?  Please describe how the search was accomplished.

5. How the data was collected is unclear (132-144).  This would be less problematic if the collected data was provided in the supplementary material, but as it isn't more clarity would be appreciated.  The tool used to gather the data could also be more clearly specified as "Get Old Tweets" rather than "a Java project" as the latter is more ambiguous.
Validity of the findings: 1. A number of the conclusions seem to be overstated based on the relatively small sample size and lack of context.  For example, the conclusion that "researchers should feel comfortable using common science-based terms ..." (299-300) is based on "peer reviewed", "knowledge", and "justify" being the most retweeted terms in the evidence category.  It's not clear from the manuscript, however, whether these are the most tweeted tweets.  There is no comparison to tweets on the same topic which did not contain "common science-based terms" so it's difficult to come to the conclusion that science-based terms don't decrease attention.

2. The tweet IDs and/or their associated tweets do not appear to be provided in the supplementary material.
Additional comments: The manuscript is fairly solid and the application of statistical approaches to this kind of data is well worth examining.  With the addition of supporting data, patching up of materials and methods, and a some pulling back of some of the conclusions it makes a decent manuscript.  Unfortunately, it does not appear to fall within the scope of the journal which is the only reason for rejection rather than revisions.  Good luck.


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: GOING ROGUE: WHAT SCIENTISTS CAN LEARN ABOUT TWITTER COMMUNICATION FROM “ALT” GOVERNMENT ACCOUNTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: My comments have taken into consideration.
Experimental design: As the authors have refocused the paper, now the manuscript is clearer.
Validity of the findings: My comments have been taken into consideration.
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: CHARACTERIZING THE SPATIAL DISTRIBUTION OF CORAL REEFS IN THE SOUTH-CENTRAL COAST REGION OF VIET NAM USING PLANETSCOPE IMAGERY
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The article is well written. I have a few minor comments

Line 33: I believe PlanetScope is a commercial satellite. Therefore, it is not clear what you mean by “public domain”
Line 34: The phrase “high spatial resolution” is a relative term. High in reference to what? For example, the 3 m resolution of PlanetScope may not be as high a resolution as for a satellite that has a spatial resolution of 0.5 m.
Line 56: Please explain what high resolution means in this context.
Line 57: Define SCUBA
Line 68: Please check if there is a 5-m resolution PlanetScope. I think it should be 3-m only. The 5-m resolution should be RapidEye.
Line 87: Please check if PlanetScope is free. As far as I know, it is a commercial satellite. Nonetheless, it has free imagery of 10,000 km2 per month. Depending on the size of the area you are looking at, this could be enough to monitor coral reef in your study area.
Line 255: I suggest that you use a consistent unit (use km2 instead of ha).


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: CHARACTERIZING THE SPATIAL DISTRIBUTION OF CORAL REEFS IN THE SOUTH-CENTRAL COAST REGION OF VIET NAM USING PLANETSCOPE IMAGERY
Review round: 1
Reviewer: 2

Output:
Basic reporting: - The article does not provide clear progress on how the PlanetScope image was use to mapping coral reefs.
- Several sentences and information have not been cited (line 72).
- The satellite remote sensing images were acquired on 28 June 2019 (ST) and 08 August 2019 (CLC) with 0% of cloud cover and coordinate system projection. However, August is the beginning month of raining season in central Vietnam, so the impacts of rain, typhoon, river run-off would be the significant effects on water quality and water transparency. The authors should include these negative impacts on the manuscript.
- The structure of the article should conform to an acceptable format of ‘standard sections’ (see our Instructions for Authors for our suggested format). Significant departures in structure should be made only if they significantly improve clarity or conform to a discipline-specific custom.
- Figures should be relevant to the content of the article, of sufficient resolution, and appropriately described and labeled.
- All appropriate raw data have been made available in accordance with our Data Sharing policy.
- The mapping results need to consider because did not provide any spectral difference on each classes (1LC, 2LC, 3LC, 4LC, HB, S and D).
- The validation results need to be rechecked, especially the Kappa Coefficients
Experimental design: The submission should clearly define the research question, which must be relevant and meaningful. The knowledge gap being investigated should be identified, and statements should be made as to how the study contributes to filling that gap.

The author should add the number of sample site observations used in the survey (sample bottoms). For example: 47 1LC, 20 2LC, 30 HB, 40 S, 50D,…
I wonder that nothing has been said about atmospheric correction during processing. I think dust, gas, aerosols, and air molecules in the atmosphere could affect surface.
The author can add more information about the Ki/Kj ratio of the PlanetScope band pair and reflectance spectrum correlation coefficients for each band pair (R2)
This study use OA, PA, UA, Kappa to assess accuracy. Therefore, total different accuracy measures are four, not seven (line 214).
All Figures: please provide each map a letter a-c and clarify these in the caption. It would be good to add a scale bar and scale text (ratio) and a north arrow for each of the maps. It is also required the map frame to present the coordination of the study areas.
Table 1: Class description is not clear. LC > 0-25% or Cover of LC from 0-25%??? I suggest rewrite this part.
Table 1: The author should add the depth of live coral, Sand, Hard bottom. I suggest rewrite this part.

Methods were not described with sufficient information.
The author can add more information about wavelengths and radiometric resolution characteristics of PlanetScope.
Line 130-131: It is not sure the authors used 25’s method in a grid of 5 x 5 m using underwater camera.
Line 134: Provide more details of accuracy and GPS model of Garmin GPS unit (USA).
Line 136-137: A total of 1,543 photographs in ST and 1,560 photographs in CLC were collected. Please provide more details about the photo locations and captured days. The depth of captured photos.
Need to describe more details about accuracy assessment. What is the cited references?

In the methodology section, it is presented 1,543 photographs in ST and 1,560 photographs in CLC were collected and used for accuracy assessment. However, the Table 3 – Confusion matrix of bottom types in Son Tra Peninsula and Table 4 - Confusion matrix of bottom types in Cu Lao Cham Island were presented very few number of points. This is a huge flaw that requires further work to address.

Moreover, the Abstract section presents “The overall accuracies of the classifier (the Kappa coeficient) were 78.08% (0.78) and 76.78% (0.76%) for ST and CLC, respectively”. This conclusion is also make a remarkable flaw as in the Table 3 and Table 4 present the results in the reverse figures.
Validity of the findings: The novelty of the work is unclear. Methods use for determining coral reef types at a higher spatial resolution are not new, and understand spatial distribution of coral reef categories have been quantified in many studies. If the knowledge gap is site specific, it would suffice for local reporting while for an international journal, a more global knowledge gap needs to be addressed.

The paper seems interesting. However, the novelty of your research is relatively low in the journal study to be considered as a research article. As the research methods were not descripbed clearly and used very traditional/ old methods, lower-performing parametric classifier, none robust contribution.

The findings of this study can provide a comprehensive understanding of coral pattern and support policymakers/planners to develop appropriate coral reefs conservation policies in coastal regions.

Discussion and results should be divided into two part. I suggest rewrite this part.
The author should compare the accuracy of the classification to other areas of Vietnam.
The main potential problem of this manuscript is the local framing of the research, which preset as a case study.
The methods were not clearly and correctly presented. Further improvements to the methods are strongly required. The results were not inconsistent, sufficient robust and poteintial flaw.
I think it could be published if the authors could improve the manuscript and framing their work in international literature and comparing to others reported in the literatures.
Additional comments: - This manuscript is not well prepared.
- The manuscript should be rewritten, hence, discussion and results should be divided into two separated parts.
- The author should compare the accuracy of the classification to other areas of Vietnam.
- The main potential problem of this manuscript is the local framing of the research, which preset as a case study.
- The methods were not clearly and correctly presented. Further improvements to the methods are strongly required. The results were not inconsistent, sufficient robust and potential flaw.

I think it could be published if the authors could improve the manuscript and framing their work in international literature and comparing to others reported in the literatures.


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: CHARACTERIZING THE SPATIAL DISTRIBUTION OF CORAL REEFS IN THE SOUTH-CENTRAL COAST REGION OF VIET NAM USING PLANETSCOPE IMAGERY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have committed to addressing my comment.
Experimental design: The authors have committed to addressing my comment.
Validity of the findings: The authors have committed to addressing my comment.
Additional comments: The authors have committed to addressing my comment.


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

Input:
Title: CHARACTERIZING THE SPATIAL DISTRIBUTION OF CORAL REEFS IN THE SOUTH-CENTRAL COAST REGION OF VIET NAM USING PLANETSCOPE IMAGERY
Review round: 2
Reviewer: 2

Output:
Basic reporting: It is fine in revised version
Experimental design: Also good, authors added some information about methods
Validity of the findings: Yes, it is clarified
Additional comments: The manuscript has been significantly improved. However, there are few minor things must be changed:
- the country name must be Viet Nam, it is right words as Ministry of Home Affairs of Viet Nam
- Vietnamese references must be changed, eg, from 563-568, 665-677

Correct references:

Nguyen T.T.H., H.T. Nguyen, S.P.H. Tong,  L. Nguyen-Ngoc, 2019a. Vegetation Biomass of Sargassum Meadows in An Chan Coastal Waters, Phu Yen Province, Vietnam Derived from PlanetScope Image. Journal of Environmental Science and Engineering 8:81–92.

Nguyen T.T.H., H.T. Nguyen,  V.T Nguyen, Lam Nguyen-Ngoc, 2019b. Spatial distribution of submerged aquatic vegetation in An Chan coastal waters, Phu Yen province using the PlanetScope satellite image. Vietnam Journal of Earth Sciences 41:358–373.

Nguyen V.L. 2009. Coral reef fishes in the coastal waters of south-central Vietnam. Vietnam Journal of Marine Science and Technology 9.

Nguyen V.LL, Phan HK. 2008. Distribution and factors influencing on structure of reef fish communities in Nha Trang Bay Marine Protected Area, South-Central Vietnam. Environmental biology of fishes 82:309–324.


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: PARALLEL POWER POSTERIOR ANALYSES FOR FAST COMPUTATION OF MARGINAL LIKELIHOODS IN PHYLOGENETICS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors propose a parallellisation of the PS and SS approaches to decrease computation time. For these approaches, the marginal likelihood readily splits up into independent bits that can be estimated and combined into the overall (log) marginal likelihood. The implementation and the reported performance increases are certainly interesting for these demanding estimations, but the authors need to put a bit more work into correctly mentioning other publications regarding similar work and a better estimator such as GSS.

Otherwise, the manuscript is well written and easy to read through. The figures are clear but do not offer any insights into the accuracy of the reported estimates.

Specific questions:

Lines 14-15: A more general statement would be preferred, as both these methods have slightly less impressive properties than generalized stepping-stone sampling (GSS). It’s a bit strange that GSS isn’t mentioned by name in the introduction as I think it should be, as it related to how generalizable the approach of the authors actually is. The working priors required for GSS can only be constructed from an actual posterior exploration run, and not from a power posterior pre-burnin analysis. The authors need to be clear on this issue and mention it in the manuscript. Suggested citations: Fan et al. (2011); Holder et al., 2014; Baele et al., 2016.
Experimental design: The experimental design seems correct for the task at hand, i.e. measuring performance increases through the parallel implementation. No design was set up to measure correctness again a known value in my opinion. As an alternative, the authors could compare the results against those of a single-instance analysis, although that would a comparison against an approximation / estimate.

Specific questions:

Line 168: what about a simulation study for correctness? The GSS publications have already shown that PS and SS generate biased estimates of the true (log) marginal likelihood. Performance is certainly important, but I would expect that accuracy is still the more important aspect. It would be interesting to see how much accuracy is lost (if any) when opting for maximum performance.

Line 185-189: these are rather low settings and I don’t think these should be applied to any empirical data set analyses; sampling every 10th iteration is too often in my opinion and a chain length of at least 100,000 but preferable longer will be required (I showed this in a 2014 book chapter I believe: “Bayesian model selection in phylogenetics and genealogy-based population genetics”).

Lines 223-229: This requires some more explanation, especially lines 225-226. Values from a posterior analysis are not guaranteed to offer good starting values for each power posterior analysis as the closer you get to the prior, the more these values can move away from their posterior estimates.
Validity of the findings: The findings are well represented but the overall manuscript is a bit lacking in terms of citations and acknowledging similar efforts.

This type of work has been done before, although it will be good to have this available in RevBayes. You can manually edit BEAST XML files to have BEAST estimate these separate parts of the (log) marginal likelihood as well, although it’s not fully automated to launch the separate jobs and combine the end result. Still, I think it may be good to mention that BEAST 1.10 has this ability to compare the MLE contribution of a range of powers, or just one single power at a time (although there's probably no documentation about how to do it).

The main novelty of this manuscript is to be found in the actual implementation in RevBayes. I think I may have been the first to split these types of marginal likelihoodestimations into multiple pieces although I can’t fault the authors for not knowing about this particular publication (https://bmcecolevol.biomedcentral.com/articles/10.1186/1471-2148-9-87). The authors do need to make it clear how automated such a parallel analysis is from the end user’s point of view. Is a single command required to run such a parallel analysis? How portable is the implementation across different server systems, i.e. regular servers and/or job/queue-based systems?
Additional comments: I appreciate the work being done here, and certainly the estimations are cumbersome and can benefit from faster implementations. It would have been nice to show that this approach enables tackling problems that were previous not possible though, although this is quite intuitive as well. I am in favor of acceptance provided that the authors put some more efforts into acknowledging past efforts.


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: PARALLEL POWER POSTERIOR ANALYSES FOR FAST COMPUTATION OF MARGINAL LIKELIHOODS IN PHYLOGENETICS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Is power posterior implemented by tools other than RevBayes? If so, are those implementations parallelized? How do they compare in terms of performance?
2. Raw data not shared (datasets, scripts/command lines, outputs)
Experimental design: 1. How was parallelization implemented technically? MPI, OpenMP, hybrid?
2. How many sites does Muroidea dataset have? This value is crucial for the scalability of site-based likelihood parallelization (as in Aberer 2014).
3. Please provide hardware details for Gordon cluster (CPU model, number of cores). If multiple cluster nodes were used in some configurations (e.g. for 32/64 CPUs), it probably had impact on respective performance improvements (e.g. higher communication overhead, but higher memory bandwidth etc.). So please clarify/discuss this.
4. In Fig.6 (speedups w/o pre-burnin), how expected speedup values were computed? E.g., with 64 CPUs the expected speedup is only ~50x, why?
Validity of the findings: 1. Number of iterations in pre-burnin and each power analysis are both set to 10,000. Is this common setup in practice? Since pre-burnin represents the sequential bottleneck, changing the ratio between number of iterations in both phases will substantially change scalability of parallelization.
2. Since combined parallelization yields best results (and used by default in RevBayes), it would make sense to focus on combined speedups in reporting (abstract, plots). Currently, it is not clear whether 11.4x speedup on 16 CPUs in abstract refers to combined parallelization or not?
Additional comments: 1. L219: If I/O operations do indeed represent a bottleneck for parallelization, this problem can be by alleviated by standard approaches such as async/non-blocking I/O (=thread does not have to wait until write operation is completed)
2. With combined parallelization scheme, the optimal layout will probably depend on the i) number of sites (bottleneck for likelihood parallelization) and ii) number of steps (K) and iterations (bottleneck for power parallization). So using this information will likely yield a better prediction than N=sqrt(M).

Minor:
- tables 1 and 2 belong together, maybe just merge this info into one table. Also, please indicate best runtime per row with bold/color font.
- please consider using color to make plots more readable (PeerJ is an online journal, so no need to optimize for print)


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: PARALLEL POWER POSTERIOR ANALYSES FOR FAST COMPUTATION OF MARGINAL LIKELIHOODS IN PHYLOGENETICS
Review round: 2
Reviewer: 1

Output:
Basic reporting: This revised version of the manuscript is much improved in terms of acknowledging past efforts, and overall looks very nice. I thank and commend the authors for responding in detail to my questions and comments.
Experimental design: No further comments.
Validity of the findings: The authors focus on the performance improvements and provide references pertaining to the accuracy of these methods. As the authors state, they do not offer a novel method but instead a much needed improvement in performance of an existing marginal likelihood estimation method.
Additional comments: No further comments.


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

Input:
Title: PARALLEL POWER POSTERIOR ANALYSES FOR FAST COMPUTATION OF MARGINAL LIKELIHOODS IN PHYLOGENETICS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Thanks for addressing my comments.
Experimental design: Thanks for addressing my comments.
Validity of the findings: Thanks for addressing my 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: GENETIC STRUCTURE OF MARINE AND LAKE FORMS OF PACIFIC HERRING CLUPEA PALLASII
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This is good article that sheds light on herring freshwater ecological forms. Appropriate biological experiments and bioinformatics analysis were used and all findings were supported by relevant statistics results. I believe this article should be accepted as it is.


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

Input:
Title: GENETIC STRUCTURE OF MARINE AND LAKE FORMS OF PACIFIC HERRING CLUPEA PALLASII
Review round: 1
Reviewer: 2

Output:
Basic reporting: Some minor issues use of appropriate references, some details on filtering of data unclear
Experimental design: Some methodological details unclear, concern about inadequate sample size for analyses used and no tests of significance
Validity of the findings: Some issues with overinterpretation of observed patterns of population structure, difficult to assess validity of findings fully without greater communication of significance/confidence intervals
Additional comments: The authors present an investigation of population structure in marine and lake forms of Pacific Herring across the Northwest Pacific, using a large panel of ddRAD loci. They identify moderate population structure in their dataset, suggesting regional splits in populations of this species, supported by previous analyses using smaller panels of molecular markers. I found these results to be interesting, and likely good candidates for further exploration using larger groups of genotyped individuals, using either RAD sequencing or whole genome resequencing. However, I have some concerns about this manuscript in its current form:

1. The present results are based on limited number of individuals (54, - 4 to 5 per population). This study appears to use primarily population genetics tools (FST, PCA, DAPC), in a phylogeographic framework (sequencing a limited number of individuals from representative populations). This combination of approaches can limit the inferences possible from population genetic data – it is not clear if any of the observed patterns of differentiation are actually significant. I suggest presenting results from differentiation analyses more fully, with significance of assessed FSTs (pvalules) and confidence intervals of these values, which can be provided by StAMPP (stamppfst function). Similarily, the DAPC results indicate population structure, but the number of inferred clusters, which clusters are supported by information criteria, and whether the membership there matches geographic clustering, should also be estimated. Last, an AMOVA approach was used for estimating FST – I would suggest using this method and including hierarchical information based on geography or expected lineage to determine the proportion of variance attributable to some level of geographic or evolutionary separation.
2. I think emphasis on any adaptive component of speciation or population structure cannot be adequately addressed given the data. Geographic separation and glacial history also account for separation between populations, and should be included in the discussion.
Minor comments below:
Line 68: This is not the correct reference here – Baird 2008 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003376
Line 70: ddRAD reference is Peterson 2012. - https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037135
Line 71: These methods have not reduced the cost of sequencing, but have made available surveys of genomic variation in  non-model organisms. Suggest revising.
Line 72: no just aquatic, or animals. Suggest revising to “non-model organisms”
Line 102: revise as Franchini et al. (2017)

Line 114 : 117 : No mention of removal of PCR duplicates here – for ddRAD this can be challenging as there is not secondary shear site to suggest identical reads. Maybe additional filtering based on HWE deviation (potentially challenging with low sample number) or depth cutoffs (remove high depth sites, maybe beyond a given threshold e.g greater than 3sd mean coverage.)
Line 117: Suggest clarifying – more analyses than DAPC were carried out
Line 118: Reference needed here


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: GENETIC STRUCTURE OF MARINE AND LAKE FORMS OF PACIFIC HERRING CLUPEA PALLASII
Review round: 1
Reviewer: 3

Output:
Basic reporting: Basic reporting:

The manuscript by Orlova et al. investigates the population structure of different ecotypes (lake vs. marine) of Pacific herring using ddRAD sequencing data. The authors find evidence of genetic divergence between ecotypes and between herring spawning in the Kara Sea. I enjoyed learning about this very interesting study system! Overall, professional English is used throughout, raw data have been shared on NCBI, and  sufficient context for the research in included. The paper would be further strengthened if some of the some of the following issues are addressed:

1.	The terms “freshwater” and “lake” herring are used interchangeably to describe herring that spawn and overwinter in small bays and brackish lagoons (line 59). This terminology is a bit confusing for the reader; for example, in my first reading of the paper I was under the impression that the lake ecotypes were in landlocked freshwater lakes and it was only until I saw Figure 1 that I realized that the “lake” ecotypes were collected from coastal Kamchatka and the Sea of Japan. It would be helpful if there was a short description of the “lake” vs. marine study sites in the methods section to help the reader visualize and understand the differences between these environments. If you have photos of the lake environments, it might also be good to include them as a figure (or in the supplement) to further illustrate this point. If there are no large differences between the lake and marine environments (or if they form a continuous gradient of habitat rather than a hard barrier to migration), then I might even suggest using different terminology to identify the two environments and ecotypes. The following terms could be used instead: lagoon (or low-salinity or resident or brackish) vs. migratory herring. Additionally, I recommend using different symbols in the figures (map, DAPC) to differentiate between the two herring ecotypes.

2.	In Table 1 please provide the date of sampling at each location. Also in the methods section, clarify whether these fish were collected from active spawning events. Might differences in spawn timing also contribute to the deep genetic divergence of lake and marine herring? Here is a paper that explores the role of temporal isolation in Pacific herring that perhaps might be useful:

Petrou EL, Fuentes-Pardo AP, Rogers LA, Orobko M, Tarpey C, Jiménez-Hidalgo I, Moss ML, Yang D, Pitcher TJ, Sandell T, et al. 2021. Functional genetic diversity in an exploited marine species and its relevance to fisheries management. Proceedings of the Royal Society B: Biological Sciences 288:20202398.

3.	Line 81: There is some really nice genomic research on adaptation to low salinity environments in Baltic Sea herring. You should cite that body of research in the introduction and/or in the discussion to provide a richer context for your work. Here are some key papers:

Martinez Barrio A, Lamichhaney S, Fan G, Rafati N, Pettersson M, Zhang H, Dainat J, Ekman D, Höppner M, Jern P, et al. 2016. The genetic basis for ecological adaptation of the Atlantic herring revealed by genome sequencing. eLife 5:e12081.

Pettersson ME, Rochus CM, Han F, Chen J, Hill J, Wallerman O, Fan G, Hong X, Xu Q, Zhang H, et al. 2019. A chromosome-level assembly of the Atlantic herring genome—detection of a supergene and other signals of selection. Genome Research.

4.	In the results section the subheaders (3.1, 3.2, etc.) do not always correspond to the content below them. I recommend removing or renaming the subheaders and re-organizing the results section to improve flow. The sections reporting pairwise population FST could be significantly shortened and the results would be better reported in a table in the main manuscript (rather than as Supplemental Table 2).
Experimental design: Experimental design:
This article is within the aims and scope of the journal and the research question is well-defined. Please see the following comments for suggestions for improvement:
1.	Line 67: I am not sure I understand what you mean by “ the status of lake form herring is still under debate”? Is that because previous studies used relatively few DNA markers? It would be good to clarify this point a little bit more, and I think it will help with the transition to the next paragraph.
2.	Line 102: please name the digestion enzymes you used in ddRAD library preparation. I also suggest re-writing this sentence as: “… constructed using the method described by Franchini et al. (2017), and using modifications described in Nedoluzhko et al. (2020).
3.	Line 105: this sentence about the bioanalyzer chips should come before the previous sentence about the S2 flow cell.
4.	Line 115: Which variant calling model did you use in bcftools? (ex: --consensus-caller)
6.	Line 142-144: The way this sentence is written makes me think that both lake AND marine herring were collected from Bloshoy Vilyuy, Ainskoe, and Nerpiche. But I don’t think that is the case? Please check this sentence for meaning.
7.	In the results section, please provide some information about the average read depth per SNP, percent missing data per sample, percent missing data per SNP, and the number of SNPs with a global minor allele frequency above 5% (or thereabouts). I imagine that sequencing samples on an S2 flow cell results in low amounts of missing data but it would be good to report these metrics. If you have a vcf file, it is easy to produce these summary statistics using the program VCFtools.
8.	Line 146: You should point the reader to Supplementary Table 2 here. Also this table caption has a typo in the species name (should be Clupea pallasii), and some of the population names do not match the Figure 1 map.
9.	Line 163: Are these pairwise FST values? Clarify to what marine specimens they are being compared.
10.	Line 191: Can you clarify what the data from Ainskoe Lake were? Were those dated ancient herring bones?
11.	Line 199: Are the Chukchi Sea samples Scva and Sk12? I recommend labelling the Chukchi Sea in Figure 1 (map).
12.	Line 220: This is an interesting section!
13.	Line 222: The standard term is “genomic islands of divergence”
14.	Line 225: “Fast evolution constructor from precast bricks” is a bit awkward. It would be better to say something like:  “standing genetic variation enabled the rapid local adaptation to low-salinity environments”.
15.	Figure 2A and 2B: Would it be possible to keep the color scheme consistent for each population between these two plots and also plot the lake herring with a different character? That would help showcase the differentiation between lake and marine ecotypes.
Validity of the findings: Validity of the findings
The number of specimens collected from each sampling location is very small (N=5). I think it would be good to address the fact that very small sample sizes are used and there is likely sampling error around estimates of population-specific allele frequencies and FST.
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: GENETIC STRUCTURE OF MARINE AND LAKE FORMS OF PACIFIC HERRING CLUPEA PALLASII
Review round: 2
Reviewer: 1

Output:
Basic reporting: No major issues here
Experimental design: Design appropriate, limited in sample size, but does not preclude accurate results meriting follow-up with larger datasets.
Validity of the findings: As above: small sample size, but large marker panel, and correspondence to previous datasets.
Additional comments: The authors present a revised manuscript exploring population structure in Pacific Herring populations. They have made several corrections and clarified sections to address some reviewer comments, but I still find some sections require additional minor revisions, outlined below:

In addressing the sample size limitations of the present study in their response, the authors assert that when sample sizes of markers are sufficiently large, a small number of sequenced individuals (n =4 – 6) maybe be sufficient. Some mention of this limitation, and discussion and an appropriate reference are still required here (e.g. https://doi.org/10.1371/journal.pone.0042649).

Section titles: Genetic diversity vs differentiation. This study relies on differentiation metrics (PCA, DAPC, FST), rather than diversity metrics (pi, heterozygosity). Correcting section titles is suggested.

Line 154: Which genotyping quality parameter and value was specified here? Depth or other?

Line 227: Unless a linear model was used and produced a specific correlation coefficient that was significant, I’d pick a less statistical term like: “is similar to”, “matches”, or “corresponds to”

Line 237: Genetically distinct?

Line 239: Not sure it needs to be strict natural selection, drift post divergence barrier may be implicated too

Line 242: I’d maybe drop this line, or move to a more general reference (e.g. Nosil 2012 Ecological Speciation book), these “islands” are identified across many species (e.g. sunflowers, stick insect, littorinid snails).

Line 260: Sympatric speciation is not generally associated with weak selection, the strength of selection needs to exceed rate of migration. I’d remove “weak” here.

Line 287: Low sample size (4-6 individuals) and RAD data (incomplete sampling) precludes a full assessment, rewording suggested here (e.g. “the most detailed genomic investigation to date”)


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: GENETIC STRUCTURE OF MARINE AND LAKE FORMS OF PACIFIC HERRING CLUPEA PALLASII
Review round: 2
Reviewer: 2

Output:
Basic reporting: There are several errors in the figures and text that should be addressed before publication:

•	In the downloaded materials, I did not see any figure captions. Are these missing from the manuscript?

•	In the abstract and the first paragraph of the introduction, the authors should add that the species range of Pacific herring also extends to the Northeastern Pacific Ocean (there are a multitude of spawning locations in North America).

•	In all of the Figures: In Figure 1, the Lake populations are depicted in blue and the marine populations are depicted in red. In figures 2 +3, the ellipses that encompass these ecotypes have the opposite color scheme, causing confusion for the reader. Please adjust figures so they have consistent color scheme.

•	Figures 2 A and B: There is an error in the color scheme and the legend. In Figure 2A, I think that the Kara Sea individuals are in the upper right-hand corner, but this color is missing from the legend in the right. Additionally, according to the methods section there should not be Kara Sea individuals in Figure 2B, but the legend makes it seem like there are. Please fix these labeling errors.

Line 146 (tracked changes document): should be "bootstrap replicates"

Lines 183 - 185 (describing the results of the PCA): these lines do not actually convey any PCA results as written. Re-write so you describe the major patterns observed in the PCA.

Line 186: This should be in the methods section. Remove it from the results.

Line 192 : I don’t understand how the FST values are reported here. Clarify that these are the maximum pairwise FST values observed between a particular lake and marine population, and direct readers to Table 2.
Experimental design: The experimental design was adequate for the questions asked. But there is one thing that should be changed in the introduction:

Line 87: You do not test whether any genes are associated with low-salinity environments in this study, so you should remove the line where you state that your objective is to describe genes that are involved in adaptation to freshwater.
Validity of the findings: no comment
Additional comments: In the abstract and the first paragraph of the introduction, the authors should add that the species range of Pacific herring also extends to the Northeastern Pacific Ocean (there are a multitude of spawning locations in North America).

Line 87: You do not test whether any genes are associated with low-salinity environments in this study, so I would remove the line where you state that your objective is to describe genes that are involved in adaptation to freshwater.

Line 146: should read bootstrap replicates

Lines 183 - 185 (describing the results of the PCA): these lines do not actually convey any PCA results as written. Re-write so you describe the major patterns observed in the PCA.

Line 186: This should be in the methods section. Remove it from the results.


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

Input:
Title: DISTRIBUTION OF CLONES AMONG HOSTS FOR THE LIZARD MALARIA PARASITE PLASMODIUM MEXICANUM
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clarity of the manuscript. This is an interesting article, unfortunately, the strengths of the manuscript are currently obscured by the writing style.  The author has often used long sentences, and sometime hard to follow, as many unanswered questions discussed.  I think that the same information could be presented in a more condensed form. The manuscripts reads as a graduate thesis, with lots of redundancy.

Background. The author has presented an elaborative background covering most of the augments on the MOI. However, the majority of cited data are conventional and relatively old, with exception one published in 2018, all references are almost a decade old.
There is a lack of references to some approaches addressing factors influencing the distribution of malaria parasites in nature. For example, a large body of literature on the human malaria parasites P. falciparum and P. vivax have shown spatial heterogeneity has a profound effect on predictions of MOI and parasite prevalence. The author has not considered these findings, to argued the approach taken in the present study.

Structure. The article is prepared in standard format and relevant data is presented in a well analysed forms in tables and figures, adequately addressing the research question making a coherent story - supported by appropriate data. However, the elaborative writing style, makes some sections read redundant. Thus the author should may consider shortening some sections (e.g introduction and discussion).
Figures. The figures are clear and well labelled containing essential data addressing the research question.
Raw data. It would be of great help to interested readers to have the microsatellites data for the examined isolates

Self-contained with relevant results. The manuscript contains appropriate material and results addressing the research question.
Experimental design: Research question. Aim and research question are well defined, and the study design and approach used suit the research question. However, as mentioned above, I am not sure how novel is the research question?
Association between MOI and parasite prevalence (surrogate measure of transmission) has been addressed extensively, many studies have reported a clear association among natural infection of the human malaria parasites (P. falciparum and P. vivax) (as mentioned in the intro). Nonetheless, the relationship between MOI and parasite prevalence is not simple, this has been attributed to many epidemiological and biological factors (as stated by the author).    Many studies have clearly demonstrated the impact of spatial crusting of transmission on relationship between MOI and parasite prevalence (e.g. Bousema T, et al., PLoS medicine; 9(1):e1001165). Karl Sl;, PLOS ONE DOI:10.1371).  Heterogeneity is maintained in all transmission setting, its not easy to identify in high transmission settings.  The author has not considered this point in the study design, and has not adequately bring it in the discussion.
The author has listed challenges to estimates of MOI in nature, however it is not clear how the design of the current study overcomes the above limitations.
Analysis of distribution of P. mexicanum clones is interesting, and adds to existing models for analysis of MOI.   This the manuscript, should be restructured to make the model of MOI as a major theme. The titles should be amended to reflect the main focus of the manuscript.

Investigation performed and methods. The methodology section contains adequate information on data collection and analysis. However, there it is not clear if the author has used existing genotype data for the examined microsatellites or whether the author has generated this data. Thus, some restructuring can help, e.g. sub-section on “microsatellites analysis”.   Line 263. All infections in this study that had available dried, frozen blood dots? Are the samples stored dried on filter papers? What type? Has the long term storage effect on MOI been verified? Any cut-off peak height been applied for detection of multiple clones per locus, to remove spurious fragments, to follow on this, Line 420 - 22 “the minor clone may be missed due to the relative weakness of the electrophoresis signal, ….that falls in the stutter” .  These sentences are not clear.
In addition, the sub-section “Individual sites for testing goodness of fit”, can be added to the analysis?
There is some overlap, lines 229-33 in section “MOI at high vs. low prevalence sites” better fit in section “MOI at high and low prevalence”.
The analysis and interpretation of the data are adequate,
Line 339, S3 table has come prior to S2, rearrange the order of table S3 and S2.
As mentioned above, It would be of help to present the row, on microsatellites and parasite isolates analysed.

The discussion is limited in scope; the author has adequately compared her data to previous data on P. maxicanum and some studies of the human malaria parasites.  However, limitations in approach were not adequately presented.
Validity of the findings: The author has presented an extensive data, complied over many years, summarized/analysed in clear tables and figures in line with the research question. However, I am not sure about the novelty of the research question. It would therefore be of benefit to clarify how the approach taken in the present study extends existing body of knowledge.
The above is reflected in the conclusion, line 456  “What these data do provide is guidance for
shaping future hypotheses and insight into whether commonly held assumptions about MOI are supported by data”,  line 461 “The use of MOI as a proxy for transmission or prevalence (or vice versa) warrants substantial caution” and “Third, this study provides a reasonable hypothesis for why high vs. low prevalence sites may not differ in MOI”

The 1st para of conclusion can easily be taken out without affecting the main points raised in this section.

Lines 478-85. The third point in the conclusion. There are many speculations here, parasite prevalence is not a sensitive epidemiological index, EIR provides a more meaningful transmission intensity index.  Many previous work has established that MOI is a nonlinear function of parasite prevalence.
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: DISTRIBUTION OF CLONES AMONG HOSTS FOR THE LIZARD MALARIA PARASITE PLASMODIUM MEXICANUM
Review round: 1
Reviewer: 2

Output:
Basic reporting: The data can be presented in a more readable way. It is not clear how close were the sampled locations, representing them on a map would be very helpful. In addition, I suggest further describing how the low prevalence locations were grouped and what were the ranges defining low versus high prevalence. Is figure 1 showing the distribution by pooling the data across all sites? How do the fitted distributions look for each side individually?

To ensure reproducibility of the study, the code and the data should be provided as well.
Experimental design: While the research question that the manuscript addresses is of great importance, this question has been addressed in many previous studies and I am not convinced that the data and analysis presented in this manuscript stands to the conclusions that the author wants to draw. I think that by looking at the MOI in lizard populations, once cannot draw general conclusions about malaria in human hosts. There is a fundamental difference in the disease biology in humans compared to lizards, not to mention the different vectors, the intervention effects and other aspects of human malaria which affects this relationship. Furthermore, it is widely accepted that transmission intensity is linked to MOI, especially at large spatial scales and here are just a few studies that have shown that:
https://pubmed.ncbi.nlm.nih.gov/29325146/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3537863/
Validity of the findings: While I agree that the relationship between MOI and prevalence is complex and should be further investigated, the author tries to claim that MOI and transmission intensity are not linked just because the analysis of one particular data set did not support it. Drawing these types of conclusions and extrapolations need to be done with particular caution. I suggest a rewrite of the manuscript, focusing on the presented data and the results, without extrapolating these results to human malaria.
Additional comments: The manuscript focuses on dissecting the relationship between the observed multiplicity of infection (MOI) and inherent malaria transmission. To address this matter, the author studies the MOI from historical samples collected between 1995-2010 from lizard hosts infected with Plasmodium mexicanum and the relationship of the MOI with the observed prevalence in the lizard populations sampled across several locations in California between 1980-2010. To determine the MOI, the observed allele diversity at 4 microsatellite loci is assessed.

While the research question that the manuscript addresses is of great importance, this question has been addressed in many previous studies and I am not convinced that the data and analysis presented in this manuscript stands to the conclusions that the author wants to draw. I think that by looking at the MOI in lizard populations, once cannot draw general conclusions about malaria in human hosts. There is a fundamental difference in the disease biology in humans compared to lizards, not to mention the different vectors, the intervention effects and other aspects of human malaria which affects this relationship. Furthermore, it is widely accepted that transmission intensity is linked to MOI, especially at large spatial scales and here are just a few studies that have shown that:
https://pubmed.ncbi.nlm.nih.gov/29325146/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3537863/

While I agree that the relationship between MOI and prevalence is complex and should be further investigated, the author tries to claim that MOI and transmission intensity are not linked just because the analysis of one particular data set did not support it. Drawing these types of conclusions and extrapolations need to be done with particular caution. I suggest a rewrite of the manuscript, focusing on the presented data and the results, without extrapolating these results to human malaria.

Furthermore, the way the statistical analysis of the data is done and presented needs further improvement. While Poisson and Negative Binomial distributions are often used to represent count data, and the assumptions of many models, they are not a "gold standard" and the observed data does not necessarily have to follow them. It is unclear why the author emphasizes that these distributions are expected to reproduce the data. For example, I would have considered power law distributions which are more widely used for describing genomic properties.

An important limitation of the study: the author assumes that MOI can be estimated by taking the maximum number of observed alleles at any of the 4 microsatellite loci. This number is a lower bound of the true MOI and thus it is not surprising that there is little to no difference between low and high prevalence sites. A more robust measure of MOI, potentially incorporating the variation observed at all the 4 loci would be more adequate here.

The data can be presented in a more readable way. It is not clear how close were the sampled locations, representing them on a map would be very helpful. In addition, I suggest further describing how the low prevalence locations were grouped and what were the ranges defining low versus high prevalence. Is figure 1 showing the distribution by pooling the data across all sites? How do the fitted distributions look for each side individually?

To ensure reproducibility of the study, the code and the data should be provided as well.


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: DISTRIBUTION OF CLONES AMONG HOSTS FOR THE LIZARD MALARIA PARASITE PLASMODIUM MEXICANUM
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thank you addressing my review 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: DISTRIBUTION OF CLONES AMONG HOSTS FOR THE LIZARD MALARIA PARASITE PLASMODIUM MEXICANUM
Review round: 2
Reviewer: 2

Output:
Basic reporting: Exposition is clear and concise
Experimental design: The methods and experimental design are relevant
Validity of the findings: I have many reservations about findings, explained in my letter
Additional comments: The paper aims to assess distribution patterns of MOI (multiplicity of infection) using field data for lizard malaria, and 4-locus microsatellite analysis.
The issue of malaria MOI distribution and its relation to transmission (intensity and prevalence) is wrought with complexities and controversies, brought up by the author and the reviewers. Just quantifying diversity poses a challenge; the proposed 4-locus microsatellite could be a reasonable approach. The next big question is what MOI pattern one should expect, and its relation to transmission intensity (EIR) and parasite diversity in host population.
Beyond qualitative inference (‘higher EIR implies higher MOI’ and prevalence), I don’t know any direct way (mathematical or other) to assess it. Fitting data by itself has limited value, in my view without an explanation.
In that sense, MOI could be contrasted to another commonly used ‘Poisson’ / ‘Negative binomial’ distribution describing parasite burden in population. Those could be derived by conventional statistical (event-count) methods, mentioned by the author. However, burden distribution does not translate directly to MOI, for multiple reasons, some mentioned by the author. I also have reservation about lack of correlation between ‘infection prevalence’ and MOI, and what conclusions to draw. Besides, ‘prevalence’ has limited values in the context of malaria.
Overall, I share the previous comments, and defer it to their judgement.


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: DISTRIBUTION OF CLONES AMONG HOSTS FOR THE LIZARD MALARIA PARASITE PLASMODIUM MEXICANUM
Review round: 2
Reviewer: 3

Output:
Basic reporting: The manuscript is overall well written and the quality of the language is sufficient. However, parts of the methods section are difficult to understand and seem contradictory at first read. I had to go over it several times to understand the methodology. This can be improved by adding more structure with subheading to the methods section.

The references are appropriate and in context. However, there could be more references to statistical models concerning MOI. While Hill and Babiker 1995 and Schneider and Escalante 2014 are appropriate, there are more statistical methods to estimate MOI, e.g., Hastings and Smith 2008 Malaria J. Particularly, COIL (Galinski et al 2015, Malaria J) and McCOIL (Chang et al, 2013, PLoS Comp. biol.) should be mentioned. A good summary is found in Schneider 2018 (PLoS One, https://doi.org/10.1371/journal.pone.0194148).

The article structure and quality of the figures are  appropriate. The raw data is shared on a repository, including the R scripts used.

The article is self-contained, however the hypothesis itself needs some adjustments (see detailed comments).
Experimental design: The ms fits within the scope of the journal.

The research question per se is important and interesting. However, it is not completely meaningful. See specific comments.

The methodology is sound, and with the information provided at repositories the results are replicable.
Validity of the findings: The investigation is sufficiently novel. All underlying data is provided at a repository, and the methodology is statistically sound.
The conclusions are not always clear, which can be fixed.
Additional comments: The author studies empirically the distribution of multiplicity of infection (MOI) in malaria. MOI is recognized to be an important quantity in malaria which correlates with transmission intensity and is believed to interfere with disease severity.
Claiming that statistical methods to estimate MOI usually assume that MOI follows a Poisson or negative binomial distribution, the author fits these distributions to a dataset of Plasmodium mexicanum. The author shows that the mentioned distributions do not fit the empirical data well, while the zero-inflated Poisson and negative binomial distributions do fit well. The author further shows that there is no apparent difference between MOI in areas of low and high transmission.
While the topic itself is important I have a number of points of criticism, which require changes in the manuscript.

General comments:
1. The definition of MOI in the literature is ambiguous. In the empirical literature, it is usually defined as the number of distinct “haplotypes” in an infection. In the theoretical literature, it is defined as the number of haplotypes (not necessarily different) infecting a host, which is well summarized in Table 1. In the empirical literature, MOI is fuzzily defined: namely, each parasite is an individual and hence its own haplotype, although what is meant is actually the number of distinct haplotypes (characterized by a handful of genetic markers) that infected the host. This is then often estimated at the maximum number of alleles found in a sample from the infected host across several markers. Sometimes it is also defined as the average number of alleles across several markers.    In the theoretical literature, MOI is an unobservable quantity that allows being infected multiple times with the same parasite haplotype. These ambiguities in the definition of MOI should be discussed carefully in the introduction.

2. In the manuscript, it is claimed that MOI is assumed to follow a negative binomial or Poisson distribution in the theoretical literature. While this is true, the article uses a different definition of MOI, namely the maximum number of alleles across several markers. Even if MOI follows a Poisson or negative binomial distribution, the maximum number of distinct alleles across several markers will no longer follow these distributions - and there is no statistical model available that would claim so. This is the logical mistake made in this article. In a revision, this should be discussed carefully.  Furthermore, rather than fitting Poisson or negative binomial distribution to the empirical data, MOI has to be derived by a statistical model along with allele/haplotype frequencies. From these estimates, the distribution of the maximum number of alleles across the considered markers has to be derived and compared to the empirical data. These issues MUST be resolved in a revised version of the article. Otherwise, the results just state that empirical data does not fit a distribution, which no statistical model claims it does.

3. In practice, in human malaria, where MOI is considered to be important, typically (as claimed in the ms) only data from infected hosts is available. Hence, MOI (given samples descend from infected hosts) follows a conditional or zero-truncated distribution. What the article does not make clear explicitly is that conditioning the Poisson and zero-inflated Poisson (or negative binomial and zero-inflated negative binomial distribution) yields the same conditional distribution. Considering this, in the manuscript, it is not made clear why it is important to consider an unconditional model. While the article shows that the zero-inflated distribution fits empirical data well when also considering uninfected hosts, it actually gives evidence that assuming a conditional Poisson or negative binomial model, when considering only infected hosts, is appropriate. While this is stated in the conclusions it is not made as explicit in the abstract and introduction.

4. A problem that arises in the study design is the use of Plasmodium mexicanum data. In humans, MOI as the number of super- or co-infections during one disease episode makes sense, as infections are cured by chemotherapy. However, in lizards, malaria will be chronic. Therefore, MOI is the number of parasites that infect the lizard from birth to the time of sample collection. This is very different. Intuitively, MOI should hence be higher even in areas of low transmission in P. mexicanum, in agreement with the fact that MOI does not significantly differ between low and high transmission areas. This needs to be addressed in more detail.

5. I found the method section starting from L183 rather confusing and needed to go over it several times to understand the study design. Particularly, it was unclear to me which samples were analyzed for which type of analysis.

Specific comments:

1. The methods are not always clear. For instance, fitting the Poisson distribution with the function glm() in R. The GLM used here is actually a Poisson regression with just an intercept kept in the design matrix. Thus, fitting a glm() is just equivalent to ML fit of the observed count data to the desired distributions. In the case of the Poisson distribution, the Poisson parameter is estimated to be the sample size, divided by the number of events. This should be made clear explicitly.


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: CONCENTRATIONS OF MG, CA, FE, CU, ZN, P AND ANTHROPOMETRIC AND BIOCHEMICAL PARAMETERS IN ADULTS WITH CHRONIC HEART FAILURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Manuscript Number:  No #59941
Title: “Concentrations of Mg, Ca, Fe, Cu, Zn, P and anthropometric and biochemical parameters in men and women with chronic heart failure”
I appreciate the opportunity to review the manuscript.
Recommendation: The article is interesting and brings an important perspective on the subject into the available literature. The article discusses the very important problem of chronic heart failure. In my opinion, the study is of scientific relevance, it is interesting and should be published in PeerJ.
The heart failure is one of the biggest challenges facing health care systems in Poland and in the world, both in terms of morbidity, mortality, burden resulting from disability and inability to work, as well as the use of system resources and health services.
Over 1.2 million people in Poland suffer from heart failure (data for 2018), and the mortality rate among them has been very high for years. About 40% of patients die within 5 years of diagnosis. The incidence of heart failure has slightly decreased in the last 5 years and is now around 125,000. new cases every year.
Research methods (inductively coupled plasma optical spectrometry (ICP-OES) is appropriate, and the description of the method is detailed (sample preparation, digestion method, parameters of measurement, precision of the method used to measure the concentration of elements studied). Certified reference materials have been used for the validation of the methods.
	Strong points of the paper: topicality of the subject, the social utility, especially because heart failure is one of the biggest challenges facing health care systems in Poland and in the world.
Comments on the paper: The Title reflects the content. The Abstract is well written.
Introduction: The introduction orients the reader to the problem of the heart failure.
Materials and Methods: The study was conducted using modern and well-chosen methods, which guarantee the reliability of the results obtained. Statistical methods were properly selected.
Results: Tables are clear and easy to follow.
Discussion: The paper is clear and succinct.
Conclusions: The conclusions drawn from the study are appropriate.
I have a few small corrections:
1) In the text of the manuscript I noticed a gaps between the tables, this should be corrected technically.
2) Poland should be in all affiliations, not Polska.
3) Please note that there is serum throughout the text
Experimental design: Materials and Methods: The study was conducted using modern and well-chosen methods, which guarantee the reliability of the results obtained. Statistical methods were properly selected.
Results: Tables are clear and easy to follow.
Discussion: The paper is clear and succinct.
Conclusions: The conclusions drawn from the study are appropriate.
Validity of the findings: No comment
Additional comments: I have a few small corrections:
1) In the text of the manuscript I noticed a gaps between the tables, this should be corrected technically.
2) Poland should be in all affiliations, not Polska.
3) Please note that there is serum throughout the text


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

Input:
Title: CONCENTRATIONS OF MG, CA, FE, CU, ZN, P AND ANTHROPOMETRIC AND BIOCHEMICAL PARAMETERS IN ADULTS WITH CHRONIC HEART FAILURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Authors may consider the possibility of replacing “men” and “women” with just the word “Adults” in order to shorten the length of the title and render it more understandable to the general readership
	The abstract of the manuscript is well-written in good English and conforms to the Peer J Journal standards
	I humbly suggest that, the researchers consider the possibility of including a brief discussing of the neurohormonal basis of the pathophysiology of chronic heart failure. This approach will enable most readers to better understand the relevance of the study objectives and the relationship between the risk factors under investigation and heart failure as a chronic disease
	Although the researchers have attempted to discuss the relationship between the individual risk factors under investigation and chronic heart failure, the underlying physiochemical mechanisms that explains and establish these relationships remain unclear (For example: how will hypo or hypercalcemia affect prognosis in patients with chronic heart failure). These mechanisms should be briefly but explicitly described with proper references.
	Chronic heart failure classifications have been adequately discussed. However, there is no mention on how these patients were diagnosed with heart failure and whether diagnosis was conducted and or confirmed by a residency trained and board-certified cardiologist. This approach will give the general readership the opportunity to assess the validity of such diagnosis
	Generally, the language is clear and unambiguous, literature review was thorough and extensive and properly referenced.
	Figures are well labelled, very relevant and accurately described.
	The structure of the entire manuscript conforms to PeerJ Standards
Experimental design: The research objectives are clearly defined and concordant with the scope of the Journal
	The researchers have clearly stated and discussed how their results will definitely fill a knowledge gap within the scientific context
	The research question is also well defined, relevant but not that all meaningful
	The authors exercised due diligence in their investigative procedures and have exhibited some level of technical and ethical standards
	The use of only signs and symptoms to diagnose heart failure is not clinically prudent and constitute a scientifically unacceptable approach. The use of signs and symptoms plus electrocardiographic findings for the diagnosis of heart failure will be more appropriate.
	The researchers have also clearly indicated in their manuscripts that, study participants were divided into two groups. However, there is no mention of how these participants were selected and how they were assigned to each group. This approach has the great potential to create various sources of selection and allocation bias
	The conclusions are based on the study results. However, since this study can somehow be considered as a “me too” study, the findings more or less constitute a confirmation of existing knowledge on the topic
	Finally, the last sentence in the conclusion section “Changes in the serum concentrations of macro- and micronutrients may significantly affect the severity of HF in patients with chronic heart Failure “needs a little bit more of clarity
	Humble suggestion for consideration: “Abnormalities in the serum concentrations of macro-and micronutrients may significantly affect the prognosis in patients with chronic heart failure “.
Validity of the findings: Although the findings are can be considered relatively valid and relevant and very well supported by literature with a great potential to significantly expand the scope of the current literature on the topic, these findings do not meaningfully add any new knowledge to the already available knowledge on the topic. This is because the current topic under review has been exhaustedly studied and many findings reported in the literature as evidenced by the quality and quantity of citations used under the references
	All underlying data have been sufficiently made available and statistical methods and approaches are considered valid and appropriate based on the nature and type of data collected
Additional comments: No comments


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

Input:
Title: PLGA-PEG-PLGA HYDROGEL WITH NEP1-40 PROMOTES THE FUNCTIONAL RECOVERY OF BRACHIAL PLEXUS ROOT AVULSION IN ADULT RATS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1-There were only a few spelling mistakes which could easily be corrected.
2-All the in-text citaitons have to be edited acording to journal's instructions.
3- Some parts of the introduction needs proper citations.
4- All the figures were uploaded in pdf format and their resolutions are very low. The author guidelines of the journal clearly indicates PNG and JPEG are accepted formats for figures and they should be 300 dpi in resubmission.
5-Some parts of the materials and methods section is actually belongs to results and discussion section. Reshaping is needed in this matter.
6-Discussion part should be improved.
Experimental design: 1-The reseach question is well defined and answered.
2-Methods seems suffiecient. A few comments were made in this context which can be seen in the uploaded file.
Validity of the findings: I would like to re-evaluate this part after authors upload necessary images as supplementary documents.
Additional comments: Dear Author,
I’ve finished reviewing your manuscript on behalf of PeerJ. It was a pleasure for me to review this article. I believe that your manuscript could benefit some editing especially by improving the discussion part. When I checked the figures I saw that they were all in pdf format and the quality was very low. I also could not see any pictures in the supplementary files despite you’ve menioned it in the manuscript. I would kindly like to re-evaluate this manuscript after you make necessary corrections and upload proper images. You can see my detailed comments at the end of the attached file.
Best Regards


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: PLGA-PEG-PLGA HYDROGEL WITH NEP1-40 PROMOTES THE FUNCTIONAL RECOVERY OF BRACHIAL PLEXUS ROOT AVULSION IN ADULT RATS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript has a professional language and quality of the English and editing is good.

Literature references are included and sufficient background is provided.

Raw data is shared, article structure, figures and tables are good. But, Figures 5E, 5J, 6I and 6J may be bigger for easier reading.

The manuscript has relevant results to its hypotheses.
Experimental design: The study is in within aims and scope of the journal.

Research question is well defined and the method used is meaningful to fill the knowledge gap. It can be helpful to include in vitro literature in the references.

Investigation performed has good technical and ethical standard.

Methods are described with sufficient detail to replicate.
Validity of the findings: All data have been provided and they are well controlled and statistically sound. The authors may comment on the reason why the healthy side of the animals were not included as controls instead of a seperate control group. And the authors may also comment why they chose 6 weeks time point postoperative. They should include references for this time point and comment on neural regeneration regarding this time point.

Conclusions are well stated and linked to the research question. Authors may also include discussion on other behavioural methods for assessment and why they chose TGT method.
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: PLGA-PEG-PLGA HYDROGEL WITH NEP1-40 PROMOTES THE FUNCTIONAL RECOVERY OF BRACHIAL PLEXUS ROOT AVULSION IN ADULT RATS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is clear and unambiguous. And professional English is used throughout. Literature references, background, and context are sufficient. Please revise/edit the following:

1. Line 80 needs to be revised as it looks like there is a missing statement. The line is inconsistent with the previous statement.
Experimental design: The experimental design is good and the research question is well defined. The investigation performed is well versed. I would like to see the following added/revised:

1. In line 177, include details or reference articles on the rationale justifying choosing reduced treatment time (14 days)
Validity of the findings: The release profiles have been well studied and documented. Conclusions are well stated and address the question raised. I suggest the authors could share more details on the concentration of NEP1-40 in the hydrogel would be appreciated- please also include a rationale, or cite references as to your decision to choose this concentration. The concentration and drug release profiles can be addressed in the conclusion, material preparation.
Additional comments: It is a well-detailed article that needs minor revisions. There is novelty in the study conducted, and the impact is well defined. I commend the authors for their study design and data set shared. The manuscript is clearly written in professional, unambiguous language. The minor revisions stated in the review need to be visited before Acceptance


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

Input:
Title: PLGA-PEG-PLGA HYDROGEL WITH NEP1-40 PROMOTES THE FUNCTIONAL RECOVERY OF BRACHIAL PLEXUS ROOT AVULSION IN ADULT RATS
Review round: 1
Reviewer: 4

Output:
Basic reporting: Line 56, “associated protein-related ligands” should be “receptor”.

Line 59 should cite paper Nature. 2002 May 30;417(6888):547-51, Nogo-66 receptor antagonist peptide promotes axonal regeneration.

Line 62-64, cite reference specifically after each marker.

Line 175-179, please delete repetition.

Line 297 “in order to display the cells more intuitively, parts with obvious cells are only put there (the original figures could be seen in the supplementary file).” I don’t see the original pictures in supplementary file.

Fig.5A-D picture resolution is too low, but it looks like NEP1-40+hydrogel have less survived motoneurons than NEPI40. Also, E and J are hard to read.

Fig. 6 Lacks mice number in legend.

Paragraph starting from line 55 introduces Nogo signaling, but is poorly organized. Nomenclature should be clear and consistent. Please consider rewrite this paragraph.

Please discuss potential hydrogel toxicity if any or cite relative reference about its safety.

Authors mention “In our experiment, we used the drug NEP1-40, with the dosage and time (14 days) referring to relevant literature [1-3]” twice, please specify the dosage.

Please provide reason why female rats are selected.
Experimental design: Nogo has 3 variants. Is NgR1 expressed in the BPA injury site? Authors can cite a reference.

As authors mentioned, there are couple biomarkers are associated with Nogo signaling and are indicative of functional improve, such as GAP-43, MAP-2, APP. It would be necessary to look at these signaling in vivo to confirm NEP1-40’s protective effect from the mechanistic perspective.

Lacks a real control group which have no injury. Recovery by treatments need to be compared to no injury to get an idea how much is recovered.
Validity of the findings: For comparison between multiple groups (>2 groups), what statistic was performed, for example, on way or two way ANOVA followed by post hoc tests, please specify in Method. Students t-test is not appropriate.

Fig.4A, statistics of comparison between 4 groups at each time point can be added to the figure.
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: PLGA-PEG-PLGA HYDROGEL WITH NEP1-40 PROMOTES THE FUNCTIONAL RECOVERY OF BRACHIAL PLEXUS ROOT AVULSION IN ADULT RATS
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1- The english language is clear.
2- Literature references and article structure is sufficiently edited after referree recommendations.
3- In text citations are properly corrected.
4- Newly uploaded figures are satisfactory.
5- Authors moved parts of the manuscript to the related section and made an effort to enrich the discussion part.
6- Authors should mention which picture represents which group in figure legends. (For ex in figure 5; CHAT (+) cells in control group (A), blank hydrogel group (B),.....)
Experimental design: It is not essential but it could be better if authors could give catalog numbers and dilutions of their primary and secondary antibodies. It could help other scientsists to design their studies and also would make the article matching with the repeataility principle for scientific research.

It would be better if authors could add calatog number for anti-Chat primary ab and rabbit abti-Goat secondary ab (and dilution) in this manner.
Validity of the findings: Supplementary data is well provided.
Results are well stated.
Additional comments: Dear author;
First of all I would like to offer my best wishes regarding your efforts on COVID-19 pandemic. I finished re-reviewing your manuscript on behalf of PeerJ and decided to recommend minor revisions. You can see my remaining concerns in the report.

Best Regards.


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: PLGA-PEG-PLGA HYDROGEL WITH NEP1-40 PROMOTES THE FUNCTIONAL RECOVERY OF BRACHIAL PLEXUS ROOT AVULSION IN ADULT RATS
Review round: 2
Reviewer: 2

Output:
Basic reporting: All revisions are done by the authors, after first round of review. So, not other comment.
Experimental design: All revisions are done by the authors, after first round of review. So, not other comment.
Validity of the findings: All revisions are done by the authors, after first round of review. So, not other 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: PLGA-PEG-PLGA HYDROGEL WITH NEP1-40 PROMOTES THE FUNCTIONAL RECOVERY OF BRACHIAL PLEXUS ROOT AVULSION IN ADULT RATS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The article is clear and unambiguous. And professional English is used throughout. Literature references, background, and context are sufficient. Image corrections have been made. The suggestions and questions raised by the reviewers have been sufficiently addressed and answered.
Experimental design: The article is clear and unambiguous. And professional English is used throughout. Literature references, background, and context are sufficient. The rationale for the 14-day period has been addressed.
Validity of the findings: The release profiles have been well studied and documented. Conclusions are well stated and address the question raised. The rationale for choosing concentration and dosage has been addressed by the authors.
Additional comments: It is a well-detailed article and major revisions and changes have been addressed. There is novelty in the study conducted, and the impact is well defined. I commend the authors for their study design and data set shared. The manuscript is clearly written in professional, unambiguous language.


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: PLGA-PEG-PLGA HYDROGEL WITH NEP1-40 PROMOTES THE FUNCTIONAL RECOVERY OF BRACHIAL PLEXUS ROOT AVULSION IN ADULT RATS
Review round: 2
Reviewer: 4

Output:
Basic reporting: 1. Comment: Paragraph starting from line 55 introduces Nogo signaling, but is poorly organized. Nomenclature should be clear and consistent. Please consider rewrite this paragraph.

Response:
We are so sorry for the carelessness, and have conducted a whole modification regarding the problems you pointed out.

Second comment: I cannot localize any changes in this paragraph (the third paragraph of Introduction) in terms of reorganization/ any rewriting.

2. Comment: Authors mention “In our experiment, we used the drug NEP1-40, with the dosage and time (14 days) referring to relevant literature” twice, please specify the dosage.

Response: Thanks for your question. Please see the file attached here.
[1] Jing, Xu, Jian, et al. Comparison of RNAi NgR and NEP1-40 in Acting on Axonal Regeneration After Spinal Cord Injury in Rat Models. [J]. Molecular neurobiology, 2017.

Second comment: Thanks for the clarification. Please add your dose and administration route in method as this is a key information.

3. Comment: Please provide reason why female rats are selected.

Response: Thanks for your question. The main reasons are the following two:
(1) Female rats are less aggressive and have no sense of territory, so they are less aggressive to each other. Because male rats have territorial awareness, they may attack each other, causing the experiment to fail. (2) After brachial plexus injury, some rats will be aggressive or eat limbs that do not feel sensation due to loss of sensation on one side. Females are less aggressive, so they are less likely to eat their own limbs.

Second comment: Thank you for providing the reason. Please include this reason briefly in method when introducing the animal models.
Experimental design: 1. Comment: As authors mentioned, there are couple biomarkers are associated with Nogo signaling and are indicative of functional improve, such as GAP-43, MAP-2, APP. It would be necessary to look at these signaling in vivo to confirm NEP1-40’s protective effect from the mechanistic perspective.

Response: Thanks for your advice. We have conducted in-depth research on the basis of existing research to explore the mechanism and molecular pathways of NEP1-40. We will report on related research in the follow-up, hoping to better explain the mechanism of NEP1-40.

Second comment: Thank you. Examination of molecular biomarkers In vivo are usually necessary to confirm the biomechanical function recovery, in addition to histology level examination (motor neuron numbers, morphology of motor endplate).
Validity of the findings: 1. For comparison between multiple groups (>2 groups), what statistic was performed, for example, on way or two way ANOVA followed by post hoc tests, please specify in Method. Students t-test is not appropriate.

Response: Thanks for your question. The statistics we use in our manuscript are to first compare blank hydrogel group, NEP1-40 group, and NEPl-40-loaded hydrogel groups with the control group, and then compare NEP1-40 group, and NEPl-40-loaded hydrogel groups with blank hydrogel groups, and finally compare NEP1-40 group with NEPl-40-loaded hydrogel group, so there is no simultaneous comparison between the three groups as you mentioned.

Comment: Thank you. The pair of comparisons authors reposed is typical post hoc test post ANOVA. Unlike the Bonferroni, Tukey, Dunnett and Holm post hoc methods, a set of individual t tests does not correct for multiple comparisons. As a community, we tolerate a 5% false positive rate (reject null even when true), ie. true negative rate=1-0.05=0.95;
for 2 t-tests, true accept rate=0.95*0.95=0.9025;
for 3 t-tests, true accept rate=0.95*0.95*0.95=0.8574;
for 4 t-tests, true accept rate=0.95*0.95*0.95*0.95=0.8145, ie False positive rate=(1-0.8145)=0.1855, which means multiple t-test increase chance of false positive rate, way more than 5%. That's why correction for multiple comparisons is needed, or researchers will need to account biologically for multiple comparisons when they interpret the data.
Additional comments: I would more appreciate if authors could response by specifying the line number of the changes/additions in the revised manuscript.


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

Input:
Title: DEGRADATION OF LIGNOCELLULOSES IN STRAW USING AC-1, A THERMOPHILIC COMPOSITE MICROBIAL SYSTEM
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article introduces the degradation of lignocelluloses in straw using AC-1, a thermophilic composite microbial system. This article introduces the exogenous inoculants. AC-1 on the degradation efficiency of rice straw at different temperature and materials, which provides major theoretical basis for further application of AC-1.
It is interesting study for degrading straw based on exogenous thermophilic composite microbes. However, some details need attention.
Experimental design: Abstract:
1. Line 15-16: the degradation of lignocellulose in straw is problematic, what is the problem? Please state clearly.
2. The experimental design is unclear and the logic is confusing.
3. what is simple and complex cellulose materials?
Introduction:
4. Line 49-50: Please revise, “complex mechanism of lignocellulose” changed to “complex structure of lignocellulose”
Materials & Methods
5. The rice straw is pretreated by NaOH method, please explain why. Are the subsequent series processed with NaoH?
6. Line 79-81: “filter paper broken” refer to enzyme activity or which medium? What is the carbon source?
7. Screening strains and culture degradation experiments are not clear, please revise carefully.
8. Is DGGE at different temperatures or different carbon sources?
Validity of the findings: NONE
Additional comments: Results and discussion
9. Line 118-119: “the content of rice straw” refer to what?
10. Figure 3 is inconsistent with the material method introduced
11. Line 133-134: Genus level should be in italics.
12. The article did not introduce those a thermophilic composite microbial system that are related to temperature or at are related to carbon sources, which can be further explored. The dominant microorganisms at different temperatures or different carbon sources should be further studied. Please discuss the changes in microorganisms in detail. There is no CK treatment in the article.


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: DEGRADATION OF LIGNOCELLULOSES IN STRAW USING AC-1, A THERMOPHILIC COMPOSITE MICROBIAL SYSTEM
Review round: 1
Reviewer: 2

Output:
Basic reporting: The idea of using AC-1 for lignocellulose degradation is necessary because the substrate is resistant to degradation. Because the research site is in a cold climate and is not conducive to the growth of microorganisms necessary for the decomposition of rice straw, the researcher must show references from journals in a reliable database that AC-1 is cultured and capable of degrading lignocellulose at temperatures near 0 °C.
Experimental design: The experimental objectives should be consistent with the experimental scope. The researcher had to demonstrate that, in this investigation, AC-1 could actually decompose lignocellulose from rice straw. The process of preparing AC-1 from pig manure (lines 78-86) should be reliably attributed to either the method itself developed or a derivative of any researcher's research (Show references).
   The information from the introductory section, the researcher wanted to decompose AC-1 with rice straw, which contains the chemical composition of lignocellulose (lignin bonded cellulose), but the experimental design process was seemed likely that the researcher takes AC-1 to decompose commercial compounds of lignocellulose. Researchers should describe whether they wish to directly decompose lignocellulose from rice straw or to experiment with the above constituents for future applications.
Validity of the findings: From the experimental design and the results, the investigator used the GC for analysis which measured the volatile organic acids and assumed that the experimental results were converted to a reduced total lignocellulose content. In this case, the investogators should explain in the experimental design part and show references.
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: CO-CULTURING HYPHOMICROBIUM NITRATIVORANS STRAIN NL23 AND METHYLOPHAGA NITRATIREDUCENTICRESCENS STRAIN JAM1 ALLOWS SUSTAINABLE DENITRIFYING ACTIVITIES UNDER MARINE CONDITIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript by Villemur et al. reports on the co-culture of two methylotrophs that together perform the denitrification pathway using methanol as carbon source. The pure culture profiles of the two microorganisms were compared with the co-culture profile in the presence of nitrate (planktonic and biofilm). Since JAM1 cannot reduce nitrite, the complete denitrification may only occur in the co-cultures, though the authors did not show this. The authors present evidence that co-culture enabled the growth of NL23 strain in the presence of NaCl (in a concentration that precluded growth of the pure culture).

The manuscript is well written, the Figures are explanatory and high quality. In addition, the experimental design to answer the hypotheses that were well made, and I agree with the majority of the conclusion drawn.
The background is provided in the introduction, but some references are missing relative to denitrification genes and denitrification of marine bacteria.
Experimental design: 2. The amount of protein was determined for whole cells (without lysis) or after cell lysis?
3. The authors state that the study is on denitrification, however they have only monitored the expression of nar, nap and nir genes (Table 2). Thus, there is no indication that the pathway is active. Only that cultures can reduce nitrate and nitrate. Therefore, the title and also the text should be changed to take this into account and only mention nitrate and nitrite reduction.
7. Transcriptomic analysis: The primers used in this study are not described. How the values reported in Figure 5 were determined? Authors should report the original/raw data prior the calculations.
Validity of the findings: Thus, these are some of the major concerns:
1. The references to the denitrification pathway (1 to 3) should not be only of nitrate reductases but include references for each enzyme or for the study of the denitrification pathway. They could even refer manuscripts of the denitrification pathway of  other marine bacteria (planktonic growth).
2. The amount of protein was determined for whole cells (without lysis) or after cell lysis?
3. The authors state that the study is on denitrification, however they have only monitored the expression of nar, nap and nir genes (Table 2). Thus, there is no indication that the pathway is active. Only that cultures can reduce nitrate and nitrate. Therefore, the title and also the text should be changed to take this into account and only mention nitrate and nitrite reduction.
4. Line 299 – 300: “Our results 300 showed that these co-cultures failed to perform complete denitrification.” What do the authors mean by complete denitrification? Nitrite reduction?
5. What the authors mean by denitrification rates? How were these measured?
6. What is the composition of the gas that is released?
7. Transcriptomic analysis: The primers used in this study are not described. How the values reported in Figure 5 were determined? Authors should report the original/raw data prior the calculations.
8. Both strain in co-culture (Biofilm or planktonic) are expressing nor and nos genes?
Additional comments: Other comments
1. Substitute “prescribed” in several sentences in the manuscript (Materials & Methods) with “different”. Indicate which concentrations were used in parenthesis.
2. Line 189: what is the meaning of virgin ?
3. Figure 1 should be transfer to Supplementary Materials.