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

Input:
Title: THE COMPARATIVE POPULATION GENETICS OF NEISSERIA MENINGITIDIS AND NEISSERIA GONORRHOEAE
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: I really enjoyed reading this paper and was very happy to see these questions asked for bacteria, especially for species I find very interesting.

I have a couple of concerns, and I apologize if any of these concerns are a result of something going completely over my head.

What first caught my eye was how r^2 decays suspiciously fast with the estimated genetic distance between SNPs. This is also likely why the estimated tract lengths in Table 2 are suspiciously small, since estimating bacterial recombination parameters depends on the rate at which r^2 decays in addition to the r^2 value it asymptotes to. My prior to what Neisseria data may look like include Budroni et al. 2011 (PNAS) for N. meningitidis and Arnold et al. 2018 (Genetics) for N. gonorrhoeae. There’s nothing wrong with proving previous studies wrong, but a key methodological difference is that the previous authors either used whole-genome alignments with Mauve (Budroni et al 2011) or also found core genes like you do, but kept track of their positions using a known, contiguous reference genome (Arnold et al 2018). Alternatively, your analyses are done on alignments generated from concatenating core genes (Concatenated how, by the way? Did you attempt to preserve synteny or concatenate them in random orders?). Thus, distances between SNPs may be systematically underestimated if the accessory genes present between core genes are not accounted for. If there is a recent insertion of a rare accessory gene in-between two core genes, this is likely not an issue for you, since most of the evolutionary history of the flanking core genes is spent adjacent to one another just as you have put them in your concatenated alignments. However, there could be intermediate- and high-frequency accessory genes that could cause problems here. These insertions between your core genes could also be very large, making SNPs that are adjacent to one another in your concatenated alignment have linkage dynamics that resemble the genome-wide background, which would make r^2 decay overly quickly with distance.

Inaccurate estimates of distances between SNPs could also affect your ability to detect linked selection, and perhaps why so little of it was detected in N. gonorrhoeae. It may also make patterns even stronger for N. meningitidis.

Given the low sample sizes of genomes here, it could be feasible to generate alignments from progressiveMauve that should preserve synteny. The pangenome tool “Roary” may also make some attempt to keep synteny in the alignments it outputs, but I’m not entirely sure how reliable they are. In any case, I have a hunch this could be an issue, but happy to be proven wrong.

Moreover, while it is acknowledged that clonalFrameML may not be able to detect all interspecies admixture events (since detection of these events is biased towards either longer recombination tracts and/or ones that are from more diverged donor species), I’m not exactly sure how to feel about the analyses in Figure 2+3. For instance, is there any way to get an idea of how much interspecies admixture made it past the clonalFrameML detection step? Your Figure 5 shows that your use of concatenated alignments may have made it more problematic by artificially truncating recombination segments, but even with perfect alignments this could still be a potential issue. Can only linked selection produce negative correlation between piN/piS vs piS, such that even if there were interspecies admixture, this is still evidence? While interspecies admixture can certainly explain the correlation between piN and piS, can it also contribute to the linked selection signal? It seems this could be the case if the donor population was larger and had more efficient purifying selection, making admixture tract lengths introduce more synonymous than nonsynonymous polymorphisms. Nonetheless, I just don’t have the best intuition for this so commenting in the discussion how much this could drive results would be helpful. I understand detecting *all* interspecies recombination is a very difficult task and on ongoing challenge for the field.

Lastly, these two species could have different population structures, resulting in N. meningitidis having larger Ne. To me, this is never really presented as a serious alternative hypothesis in the paper, and all analyses are done basically treating both species as single populations. This is probably valid for N. gonorrhoeae, but may not be for N. meningitidis? In particular, I’m thinking of Smith et al. 1993. I’m not suggesting that a thorough analysis of population structure be done, but it would at least be useful to mention. They do both have values of Tajima’s D close to zero, but it’s hard to interpret if this means no structure or if its from a balance of factors that create positive and negative values of D that, genome-wide, average out to zero (e.g. structure with purifying selection). However, perhaps structure still does not drive lower piN/piS in N. meningitidis, suggesting it truly has larger Ne even if some structure is present.

Minor comments:

For the paragraph starting on line 133, could you please provide more motivation behind why you used different sets of synonymous polymorphisms to ranks gene and to get averages across a group? I kind of see what you did there but am left wondering why you did it.

Line 198 – I would personally appreciate if it could be emphasized that this is a difference in the population mutation rate (2Nu), not a difference in the physical mutation rate (u). This, however, can be argued as a difference in style.

Figure 1 – the two colors are not labeled

Paragraph starting on line 235 – It’s mentioned later in the discussion that a simple model of gene conversion (or bacterial homologous recombination) can also cause linkage metrics to asymptote to non-zero values (i.e. your drift explanation), since recombination doesn’t linearly increase with distance between SNPs such that it’s expected even in the absence of population structure. I think this should also get mentioned here, since it was on my mind until it was eventually mentioned in the discussion.

Line 279 – after you give the slopes for Ng and Nm, there are numbers in parentheses that follow the slopes. What are these numbers? P-values?

Paragraph starting on line 299 – For this analysis do you remove genes that have experienced interspecies admixture? Wouldn’t this deflate levels of divergence? Although why it would tend to deflate dN more than dS (making dN/dS < piN/piS) is not exactly clear to me, and perhaps it shoudn’t. Nonetheless, it isn’t immediately clear to me how to interpret these results in the presence of interspecies admixture.

Line 341 – g in “NG” should be lowercase

Line 367 – or differences in population structure

Line 434 – should “refs” be populated with numbers referring to 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: THE COMPARATIVE POPULATION GENETICS OF NEISSERIA MENINGITIDIS AND NEISSERIA GONORRHOEAE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript reads like a verbal presentation and not a scientific paper, the wording is ambiguous and vague.  For example, throughout the manuscript statements such as: “We investigate…” should be replaced with a statement of fact such as “X was investigated…”; “We detected…” should read “Using method Y [thing] was detected”; “The two species share a modest amount of variation…” what does ‘modest’ mean?

The authors do not appear to have done a through literature review on the population structure of N. menigitidis (Nm) or N. gonorrhoeae (Ng), there is no discussion or mention of lineages, typing, population structure or implications of these in a comparative analysis; see the 2011 ASM - Clinical Microbiology Review by Unemo and Dillion, 2014 ESCMID - Clinical Microbiology and Infection by Read, 2016 Future Microbiology by Watkins, Maiden, Gupta.

There are four references cited in the manuscript that do not appear in the reference section: Ohta 1972, 1977, 1992, and Welch 2008.

The results section contains information that is better suited to the introduction or discussion.

The genomes used were not referenced properly, many have been published.

Figure 4 is not referenced in the manuscript. All tables and figures are lacking full descriptive information and informative labels.
Experimental design: The genomic comparative analysis of the two pathogenic Neisseria species is useful in understanding their relationship within the genus and the techniques used in this manuscript are suitable for this type of analysis. However, the manuscript does not present what problem the analysis is meant to address, what the gap in knowledge is, or what the hypothesis is regarding the problem or gap.
Validity of the findings: The manuscript does not contain any information on how the sub-set of genomes 'from Genbank' were chosen or how the quality of their assembly was assessed.  The NCBI (Genbank) lists >90 Nm and >20 Ng genomes filed as ‘complete’ however some of the genomes chosen for this analysis are actually poor assemblies.  How was the data set assessed?

No conclusions were presented; what is the added value of this analysis to the field?
Additional comments: The analysis confuses meningococcal colonization (carriage) with invasive infection (disease), for example: Nm asymptomatic carriage rates are presented but the analysis uses Nm invasive disease genomes, which have a different prevalence. The analysis should be redone using a more appropriate data set or correct prevalence rates.

There are inconsistencies through out the paper; for example: the introduction states a 8-25% rate of carriage, yet in the discussion (citing the same 2004 and 2005 papers) states the rate is 5-20%.  Additionally, there are more current publications of infection and carriage rates; for example see the WHO 2018 Meningococcal meningitis report and the CDC Meningococcal Disease web page.


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

Input:
Title: THE COMPARATIVE POPULATION GENETICS OF NEISSERIA MENINGITIDIS AND NEISSERIA GONORRHOEAE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The overall structure of the manuscript is fine, with all results presented in an adequate order, but the style of writing, lack of properly formatted equations and the style of referring to previous works and techniques, makes it very hard to read. This readability issue may have affected the clarity in the exposition of novel results.

A main issue in this article is that it is not entirely clear how the reported results will contribute to the literature in Neisseria or the knowledge of processes affecting diversity of bacteria. For the most part, results are presented as an exploration of some population genetic measures and comparison of these measures between two species of Neisseria. The authors might want to discuss more throughly how their results in Neisseria compare to those from other bacteria and how they lead to novel findings about the strength of the evolutionary processes acting on these bacteria.
Experimental design: The experimental design of this work, per se, does not require deep revisions, unless the authors could figure out a way to either independently estimate the effective population size for the two species under analysis or to separate its effect from those of selection.

Since the above is hard, I think this work covers all that can be extracted from this data but, as mentioned above, should undergo extensive review for better clarity. Before such revision, accessing the relevance of this work is hard.
Validity of the findings: Although well performed, It is not clear whether these results actually add something, even as replication. Other works in the same field either (i) introduced novel analysis techniques and models or (ii) compared and inferred general evolutionary processes acting on the organisms studied. I cannot identify such features in the current manuscript.
Additional comments: I believe this work will be publishable after extensive review of its writing style, as stated above.
Since these revisions should affect only style and presentation, I consider it to be minor revisions.

More detailed corrections, specific questions and suggestions that the authors should address were added as comments in the attached PDF.


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

Input:
Title: THE COMPARATIVE POPULATION GENETICS OF NEISSERIA MENINGITIDIS AND NEISSERIA GONORRHOEAE
Review round: 2
Reviewer: 1

Output:
Basic reporting: I appreciate the authors responses to my previous comments as well as addressing concerns of the other reviewers. My main concern was about the discrepancy between the LD decay curves and how they compare with previous findings, although I agree this completely depends on the samples used. While I think the science is sound, I do think the writing could be improved in numerous places to make it more professional. There are many instances of syntax errors and fewer instances of sentences that are not clear. I give some examples below.

Line 93 – strange use of comma, also based on how the previous paragraphs are set up, while this paragraph starts off detailing the “second question”, I’m not quite sure what the first was.

Line 107 – confusing sentence structure of sentence starting on this line, and “may be” should probably be “maybe” based on the context.

Line 119 – random, not “randomly”

The names of the strains used and the previous studies they came from should be put into a table, not listed in line since there are quite many.

Line 143 – there’s a notification of an invalid citation, this should be fixed

Line 160 – sentence starting on this line is awkward, can probably remove “, in the strains affected” since this is implied from the first part

Line 170 – sentence on this line not correct, which Microsoft Word also marked with a green squiggly line underneath. I suggest going back through the manuscript and checking all places Word suggests changing sentence structure, as I agree with it on some occasions.

Line 174 – I think there should be a comma after “positive”

Line 176 – it is never mentioned how these bi-allelic loci are simulated, what program? And what frequencies? Later on, on Line 179, it’s mentioned that the expected value of r^2 is independent of allele frequencies which does not make sense to me, as VanLiere and Rosenberg (2008; Theoretical Population Biology) show this can’t be true. The maximum value of r^2 depends on allele frequencies. So, this must either be explained further, but I suppose you could also delete this sentence because it’s also not clear to me what this information is used for in the manuscript.

Line 213 – sentence starting on this line should be restructured

Line 236 – “they introduced” should be changed to “they are introduced”

Line 248 – “recombination initiates” isn’t followed by a noun. What does it initiate?

Line 340 – should be “spread of an advantageous mutation”

Line 357 – again, awkward sentence here for same reason as above, where “may be” should probably be “maybe”

Line 392 – the e in Ne is not subscripted as before, please go through and change this at other locations if necessary too.

Line 472 – Ng should be followed by a comma, which Word identified with a green squiggly line.

Line 493 – I think you have the citations wrong here, I think Budroni et al studied Nm, not Ng.

Line 503 – Again, this sentence is improperly formatted and Word flagged it as well.

Line 518 – It’s not clear to me what the difference between “genomes of various eukaryotes” and “between various eukaryotic species” is, please clarify

Line 566 – missing comma

Line 579 – I do not understand what you mean here, you use “differences” twice, and these referring to distinct things? Unclear.
Experimental design: I have no comments on the experimental design.
Validity of the findings: I have no comments on the validity of the findings, just how they are presented (see above).
Additional comments: No additional comments


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

Input:
Title: THE COMPARATIVE POPULATION GENETICS OF NEISSERIA MENINGITIDIS AND NEISSERIA GONORRHOEAE
Review round: 2
Reviewer: 2

Output:
Basic reporting: Over all the writing style has not been amended to follow scientific writing standards and still affects the clarity and potential impact of the manuscript.

Line 20, 124 - numerals under 10 should be spelled out
Line 26 - ‘LD’ should be defined
Line 53 -  last sentence is missing a full stop
Line 74 -  ‘SNPs’ should be defined
Line 100-102 -  this sentence needs to be written more clearly, or expalined better
Line 115 - change ‘randomly’ to ‘random’
Line 139 - remove ‘(!!!INVALID CITATION!!! ;)’
Line 161, 190, 192-3, 195, 197, 219, ect. - replace ‘is’ with ‘was’
Line 190-193, 219-229,241-243, 262-266, etc. - this is not a result, it is a discussion point
Line 206-211 - this is not a result, and should be in the methods section
Line 257 - please check the validity of the percentage ‘-15.5%’
Line 346-354 - correct abbreviation inconsistency of fixation index
Line 349, 353 - please indicate what the numbers in parenthesis refer to
Experimental design: n/c
Validity of the findings: Each Nm lineage has it's own distinct molecular epidemiology, the manuscript should address the impact of using multiple Nm lineages on the results. For example:
Line 22 - while recombination is present in Nm, the diversity is just as likely to be a result of the multiple lineages used in the study
Line 200-201 - please clarify: does ‘polymorphism’ refer to the gene diversity in both species, or that the two species sharing common alleles of certain genes?
Line 418-419 - while the Ng strains can be considered ‘very closely related’ the Nm strains are from different lineages and are not ‘very closely related’ to each other
 - The Nm strains used in this study represent multiple distinct evolutionary lineages, each with a different fitness potential and epidemiology which can be highly invasive or asymptomatically cleared by the host. Conversely, Ng is invasive, pathogenic, highly clonal, and does not form distinct lineages.
Line 440 - please reference the comment that the Ng infection rate is lower than Nm; the comment does not represent official reported figures from the ECDC and CDC
- Nm in the USA was 0.12 cases per 100,000 in 2016 (www.cdc.gov), and in the EU/EEA 0.6 cases per 100,000 in 2016 (ecdc.europa.eu)
- Ng in the USA was 171.9 cases per 100,000 in 2017 (www.cdc.gov); while in 2017 in Europe rates varied from 1 to 75 cases per 100,000 population depending on the country and in total 89,000 confirmed Ng diagnoses in 2017 (ecdc.europa.eu)
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: THE COMPARATIVE POPULATION GENETICS OF NEISSERIA MENINGITIDIS AND NEISSERIA GONORRHOEAE
Review round: 2
Reviewer: 3

Output:
Basic reporting: I believe the article now describes, with enough clarity, its results and conclusions.
The goals of comparing population genetics parameters between Nm and Ng is achieved and, although if it seems to me a preliminary incursion into this topic, I believe it is worth publishing.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: OPTICAL MICROSCOPIC STUDY OF SURFACE MORPHOLOGY AND FILTERING EFFICIENCY OF FACE MASKS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: No comment
Additional comments: This manuscript represents the microscopic characterization of the study of surface morphology and filtering efficiency of face masks. The authors tried to quantitatively describe the filtering efficiency of the face mask in connection with washing and drying. The topic is interesting and I recommend for publication in PeerJ with minor revisions as listed below:
(1) Authors expressed pore size in the micrometer scale in their results and compared with PM2.5 and PM10 size. I suggest defining PM2.5 and PM10 size in the micrometer scale so that readers can easily understand the comparison.
(2) % is needed in the y-axis of Figure 3a and x-axis of Figure 7


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: OPTICAL MICROSCOPIC STUDY OF SURFACE MORPHOLOGY AND FILTERING EFFICIENCY OF FACE MASKS
Review round: 1
Reviewer: 2

Output:
Basic reporting: See attached review.
Experimental design: See attached review.
Validity of the findings: See attached review.
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: OPTICAL MICROSCOPIC STUDY OF SURFACE MORPHOLOGY AND FILTERING EFFICIENCY OF FACE MASKS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall, this is an interesting study associated with an important subject related to human health – how well do cloth masks often worn by citizens of countries with high air pollution protect them from inhaling airborne particulates?  The introduction provides a good rationale for conducting the study and is well supported by scientific literature. The methods are somewhat well described. Additional points are made below. The Discussion needs to be carefully reviewed for results that should be moved to the Results section as mentioned below. The supporting figures were designed well graphically and enhance the outcomes of this study.
Experimental design: Numbers are line numbers in the manuscript

124: If this method for determining filter efficiency is novel then that should be stated. If it is derived from another study, then that study should be cited.

As described, this method is severely limited by not applying active air flow through the mask to replicate breathing through it. If only a passive transfer of particles, primarily by gravity settling of particles through the mask, is measured, then the results obtained would represent a best-case estimate of capture efficiency. This should be discussed in the Discussion as a study limitation.

Likewise, more description is needed regarding the placement of the assembly “at twenty feet above the ground”. Were they placed in an open box or a shelf? Was there any attempt to make the measurements during the same time of day? And, most importantly, the same weather (especially wind) conditions? Were they sampled one-mask-at-a-time or were multiple masks sampled at the same time? These details should be described as they are factors that can greatly influence the amount collected.

143: The magnification used to make these counts should be mentioned, along with a statement of the smallest particle size measurable at that magnification. This detail is later provided in the Discussion (lines 273-278) and should be moved to either the Methods here, or as a Result.

159: the statement that 80% used different types of cloth masks is not supported in Fig 1B which only separates by cloth vs surgical masks. Refer to figure 1B at the end of the first sentence (155) rather than here to fix this problem.

250-251 These values for pore size constitute a result and should have been given in the Results section (line 167 section). Here should just be a discussion of those results and their implications on filtering qualities, and with respect to the results of any other studies that characterized surface qualities.

253 The same is true for the results presented here.

269 This discussion should include a comparison between the efficiency values found here and those found by other researchers, especially that of Rengasamy et al. 2010 who show penetration values as high as the efficiency values shown here, which is a consequence of using a system that provides air flow through the masks versus one that does not (this study).
Validity of the findings: 291 Results not presented in the Results section should not be brought up here in the Discussion. State them in Results and discuss them here.

329 A conclusion is provided here: “Findings of this study suggest that cloth mask are not effective.” However there is no basis stated for what is “effective” versus what is not “effective”. For example, somewhere in the Introduction or Methods a statement should be made as to the relationship between mask efficiency and an assessment of effectiveness.
Additional comments: A major issue with this paper is the lack of using a system that pulls air through the masks to simulate human breathing through them. The passive process used is apparently novel and therefore not comparable to results found in other studies. It also provides very high estimates of efficiency considering that a particle has to fall (or waft) through the mask pores rather than be pulled through. The concern, then, is that the results presented here provide a much more favorable indication of the ability of these masks to block the passage of particles through them than would occur when worn (and assuming a good fit against the face, which wasn’t discussed). If those results remain, the authors must provide a thorough comparison of these results with those of others with the warning that these results do not represent the efficiency when breathed through. Instead the results are just a general comparison of relative performance between mask types.

As such, it would be preferred that the plot associated with Figure 3A not be shown. Instead, the other figures can be retained (including Fig 5) so that the point of this paper is to demonstrate how cloth masks compare structurally to surgical masks, and how they compare before and after stretching and washing.


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: OPTICAL MICROSCOPIC STUDY OF SURFACE MORPHOLOGY AND FILTERING EFFICIENCY OF FACE MASKS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors addressed my comments in the revised version of the manuscript. Therefore, I recommend the paper for publication in this journal.


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: OPTICAL MICROSCOPIC STUDY OF SURFACE MORPHOLOGY AND FILTERING EFFICIENCY OF FACE MASKS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have satisfactorily addressed my concerns and improved the manuscript with clarification and suggested changes.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: VALIDATION OF ENDOGENOUS REFERENCE GENES IN RAT CEREBRAL CORTEX FOR RT-QPCR ANALYSES IN DEVELOPMENTAL TOXICITY STUDIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: English is clear with only small editorial suggestions
1.	Line 145, add space after P/N 4303859)…
2.	Line 177, add and after this problem…
3.	Line 195, add the after upregulation in…
4.	Line 203, add space after Ct method…

Tables and figures
1.	Statistical significance denotations in the figure legends are missing.
2.	In figure 1 the Rps18 and Rpl13a as well as RPS18 and Ubc alone (not in combination) is not plotted.
3.	The numbers in figure 1 do not match with the numbers in table 3.
Experimental design: Methods
1.	There is no information on how many animals were used other than in table 2. Add to material and methods and figure legends.
Validity of the findings: 1.	The testing of only two genes for the proof-of-principle gives limited evidence. I would recommend using at least 3 genes of each different expression pattern after exposure (up-regulated, down-regulated, and unchanged) to conclude that more than one reference gene should be used. This could also provide some guidance for the minimum fold-change to consider significant in this experimental setting and add value to the manuscript.
2.	I would like to see these genes of interest after all the exposures; PTU, OMC and DE-71 as they might very well show different results and my not support the conclusion statement that these reference genes are to recommend following in utero exposure to TH disruptors.
3.	As the authors state, several assumptions are made when performing RT-qPCR (Nolan et al, 2006) and at least some of these should be addressed in this manuscript. At least the RNA quality should be provided and a serial dilution of the samples to assess PCR efficiency and presence of inhibitors.
4.	I would also suggest to compare different methods than 2-ΔΔCT for quantification (e.g. Rao et al, 2014 and Pfaffl 2001) as this would add additional value to the manuscript.
Additional comments: This manuscript describes the evaluation of 10 different endogenous reference genes in rat brain after exposure to three putative TH disrupting chemicals. All selected genes were considered stable when analyzed by three different algorithms. The expression of two genes of interest (likely dysregulated after exposure) was assessed using the different reference genes. Pvalb was significantly downregulated regardless of which reference gene was used while Dio2 was only significantly upregulated when three out of the ten reference genes were used for normalization.
The manuscript has in general scientific and methodological soundness. However, it provides no new information and conclusion from the limited amount of genes of interest makes the evidence for the overall conclusion weak. In addition, several quality assurance methods should be added to understand the reliability of the results. Therefore, I would recommend major revision before considered for publication.


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

Input:
Title: VALIDATION OF ENDOGENOUS REFERENCE GENES IN RAT CEREBRAL CORTEX FOR RT-QPCR ANALYSES IN DEVELOPMENTAL TOXICITY STUDIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Ramhøj et al, have studied validation of reference genes for appropriate normalization of qRT-PCR. The study has potentially interesting data, but the manuscript requires improvement in organization and clarity.

-Abstract is poorly written. Its more like a summary of results. No effort has been taken to underline the problem, in-addition authors have not addressed why 10 genes mentioned were chosen.
-Similar studies have been done in same context they have not been cited.
Experimental design: - Technical samples in triplicates is standard not duplicates as in this study.
- Since only duplicates were used statistical values could be misleading.
-I would like, that authors should clarify the technical and biological replicates.
-The raw data in excel sheet, beta actin is spelled wrong.
Validity of the findings: - Box plots should be used for all samples in figure 2, it give a better understanding of the distribution of the values.
- A plot comparing all 10 RG’s to each other should be included.
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: EVALUATION OF EXTRACELLULAR POLYMERIC SUBSTANCES EXTRACTED FROM WASTE ACTIVATED SLUDGE AS A RENEWABLE CORROSION INHIBITOR
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors report a study on the use of EPS extracted from WAS as a corrosion inhibitor. The background was well articulated and research gaps are clearly identified. The manuscript is well written and organized. The hypothesis is well defined and experimental design and analysis were used to test the hypothesis. The results are new and interesting and support the hypothesis.
Experimental design: The research is novel and original. The experimental design was well used to plan the experiments. and the results were analyzed to support the hypothesis. The methodology is appropriate and comprehensive. The research question is interesting.
Validity of the findings: The hypothesis is well supported by the experimental results. Data are robust. Conclusions are valid.
Additional comments: Overall, this study is well planned and executed. The results are interesting and advance our knowledge on the new application of EPS from sludge for corrosion inhibition. Specific comments are listed below:

1. The centrifuge rpm should be converted into centrifugation force ( x g), as different types of centrifuges (different diameters) at the same rpm will have different centrifugation force (x g). The changes should be made in a number of places, such as lines 131 and 138 etc.
2. Line 266, "maximum" should be "maximal".
3. The quantity of EPS extracted from WAS seems very low, as compared to a number of EPS studies. The typical EPS amount is from tens of mg EPS/g MLSS to hundred mg EPS/gMLSS. Please provide an explanation of the low EPS extraction efficiency.
4. Fig 3, FTIR spectra. Please label the typical FTIR peaks with wave numbers.


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: EVALUATION OF EXTRACELLULAR POLYMERIC SUBSTANCES EXTRACTED FROM WASTE ACTIVATED SLUDGE AS A RENEWABLE CORROSION INHIBITOR
Review round: 1
Reviewer: 2

Output:
Basic reporting: - English is generally good
- Insufficient background material was provided. I have added that the authors go back and conduct a more thorough review of the relevant literature
- graphs need improvement, tables need to be revised with respect to significant figures
- results are relevant to hypothesis
Experimental design: - relevant to journal
- the methods are described in detail.
Validity of the findings: - The study is interesting. However, the authors seem unaware of the existing literature in their area.
- The logic for conducting some of the experiments will need to be explained better.
- The authors will need to do a better job in comparing their results with the existing literature and putting their work in context
Additional comments: Line 64: I think at some point in the document it should be mentioned that many wastewater treatment facilities have the means to recover energy from WAS through processes such as anaerobic digestion (biogas) and incineration (heat recovery).
Line 88: The authors claim that there is no literature on the use of EPS as a corrosion inhibitor. Here are some examples that have not been cited in this paper:
Su,W.; Tang, B.; Fu, F.; Huang, S.; Zhao, S.; Bin, L. A new insight into resource recovery of excess sewage
sludge: Feasibility of extracting mixed amino acids as an environment-friendly corrosion inhibitor for
industrial pickling. J. Hazard. Mater. 2014, 279, 38–45.
 Line 93: slight modification… vague wording
Line 114: all samples were collected during summer season, what variabilities in the EPS composition are expected in the winter months?
Line 119: the wording of WAS solid is confusing, are we speaking here about the WAS TSS and VSS content? Line 150-157: this seems like a tedious method to measure TSS, also why are you not calling it TSS?
Line 133: how did you measure metabolic activity to make sure it was terminated?
Line 141: solvent extraction is a process and not a substance. Fix the writing here.
Line 158: So there are many papers on the use of amino acids on corrosion inhibition. The extraction method includes heating to 80 degrees. Do the authors expect that this would alter the amino acids?
Zhang, D.Q.; Cai, Q.R.; He, X.M.; Gao, L.X.; Zhou, G.D. Inhibition effect of some amino acids on copper
corrosion in HCl solution. Mater. Chem. Phys. 2008, 112, 353–358.
24. Gece, G.; Bilgiç, S. A theoretical study on the inhibition efficiencies of some amino acids as corrosion
inhibitors of nickel. Corros. Sci. 2010, 52, 3435–3443.
25. Khaled, K. Corrosion control of copper in nitric acid solutions using some amino acids—A combined
experimental and theoretical study. Corros. Sci. 2010, 52, 3225–3234.
26. Eddy, N.O. Experimental and theoretical studies on some amino acids and their potential activity as inhibitors
for the corrosion of mild steel, part 2. J. Adv. Res. 2011, 2, 35–47.
General comment: quality of graphs produced needs improvement before they can be published. Also, the curves can not be distinguished in black and white print. I suggest the authors use a combination of shades and dashed curves so that the curves can be reviewed properly.
General comment: I think the authors will need to do a more in-depth review of the relevant literature to strengthen their discussion and include a comprehensive review of the literature as it pertains to their subject and findings.
293-296: proper analysis of the FTIR results was not provided. This is a key component of the paper as it discusses the functional groups contributing to the corrosion inhibiting properties of EPS.
General comment: there has been no discussion on the oxidation-reduction activities in the biofilm and how this can impact the corrosion inhibition as a result of biofilm formation.
Line 226: what is the mechanism of the functional groups contributing to corrosion inhibition, this needs to be discussed.
Line 235: the “WAS samples” basically include supernatant from centrifuged WAS that was freeze-thawed a few times. Why were the authors thinking that this would have any potential corrosion inhibition in the first place? The logic is not clear to me. Centrifuging WAS samples and taking the supernatant will give you water with minimal carbon. Were there any COD tests conducted to determine the differences in the carbon content of supernatant collected from WAS samples and EPS samples? This needs to be included as the main corrosion inhibitor is the amino acids (which this discussion is also missing from the paper).
266-273: this part is missing comparisons with other studies and literature values. I think the authors should think a bit more about what the EPS composition is and do a literature search on the corrosion inhibiting properties of EPS constituents, then they will be able to find a breadth of literature for comparing their data.
275: WAS is not grown…
General comment: the paper can use some discussion on the various methods of EPS extraction and how that impacts the EPS composition and functional groups and also what the expected impacts are on the corrosion inhibition of the extracted EPS.
General comment: significant figures in tables vs. variability in samples and measurement method. 7.11 vs. just reporting as 7. This needs to be thought through and fixed in all tables


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

Input:
Title: COMPARISON OF SOIL BACTERIAL COMMUNITY AND FUNCTIONAL CHARACTERISTICS FOLLOWING AFFORESTATION IN THE SEMI-ARID AREAS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	Although I, like the authors, am not a native English speaker, and I don't like being constantly criticized for the poor English writing, I have to ask the authors here to examine carefully the wording and, more importantly, the logic of writing. I would like to correct all of them, but there are too many ‘tiny flaws’, so I would like to ask the authors to check the whole manuscript carefully before the next submission.
2.	Populus ×canadensis is nomencaltured by Moench. Therefore, Moench should not be italic.
3.	The figure legend in Fig. 3 is too brief to understand what the author is presenting. What are those numbers? The “Veen” diagram should be revised as the “Venn” diagram.
4.	Fig. 4 and 5 should be combined as one.
5.	Fig. 6: Explain what the numbers of the color scale are.
6.	Fig. 7: Please redraw the figure so that the numbers on the coordinates are at the bottom and left of the graph.
7.	Fig. 9A and B should be typesetted as consistently as possible.
8.	Figure 10 is too complex to read. Firstly, figure legend should be written more clearly and completely. Secondly, blue and red already indicate positive and negative correlation, so there is no need to use ellipse circle to express it. Third, for the part of the number, authors should present the “significance” (P-value) rather than the correlation coefficient (which has been expressed in color). And it would be easier to compare numbers if they could be placed on color squares rather than diagonally.
Experimental design: 1.	The sample sizes for soil property microbial 16S rRNA metagenome should be added. In L151, authors described that there are 9 plots with 10-15 soil cones per plots, so there should be at least 90 soil samples. However, Table 1 and 2 only showed three samples (n=3) in each plantation forest. Are those soil samples pooled together for physiochemical property analyses and microbial metagenome sequencing, or analyzing and sequencing independently but pooled data for analyses? For the former, many statistics are meaningless because of insufficient sample size; for the latter, all data should be re-analyzed separately.
2.	A similar question as above, how many samples were used for the correlation analyses?
3.	Authors want to compare the changes in soil properties and soil bacteria after three-different tree plantations. However, the lack of control group (e.g. pre-afforestation or non-afforestation soil data) makes it difficult to eliminate the influence of other factors, so it is difficult to confirm that the differences of soil properties and soil microbiomes are caused by different afforestation tree species. The author needs to make a more convincing argument in the article.
4.	Some statistic suggestions:
(1)	L211, L223, and Table 3 & 4: Using nonparametric correlation analysis, such as the Spearman, to replace the parametric Pearson correlation, unless you can prove that the data fit the assumption of a normal distribution (e.g. by Shapiro-Wilk normality test). In fact, I would encourage the authors to re-analyze all independent factors at the same time with GLM, rather than test the correlation of a single factor to a single response individually. If necessary, even consider multicollinearity and model selection. Using individual independent variables to test correlation without considering other factors is more suitable for the analysis of results under a controlled experimental environment rather than under natural conditions.
(2)	L214, L221, L225: Authors used three kinds of ordination analyses to interpret relationships among planted tree species, edaphic factors, and microbial diversity. However, such analyses are messy and would be confusing. According to the research questions, I suggest using the constraint ordination analyses (CCA, RDA, etc.) to replace the unconstraint ordination analyses (NMDS, PCA, etc.).
(3)	As for the testing of tree spp effect on edaphic characteristics, the PCA can just represent the result of “vector fitting” instead of testing the effect. When you say that you want to test the effect of X on Y, we would like to see can those dependent variables (Ys) be explained significantly by the independent factor (X) and how many proportions of variations can be explained. The vector fitting cannot tell you these stories. I suggest authors performing the MANOVA or PERMANOVA if you want to see how these tree spp. affect the entire soil environment, or you can use logistic regression to test the tree spp. effect on the influence of every single edaphic factor.
(4)	Another analytical suggestion is that when you want to test the effect of an independent variable (such as a soil factor) on relative abundance (such as the RA of soil bacteria or functional groups), you need to consider the interference of richness. This is because RA is usually influenced by richness. Therefore, it is necessary to condition on "number of functional groups within soil samples" when testing the effect of edaphic factors on RAs of functional groups.
Validity of the findings: 1.	As I mentioned above, the insufficient sample size is a big problem. This leads to a lack of confidence in the outcome. For example, the conclusion “the influence of afforestation is greater on richness than on diversity (?) of soil microbes” (L369-371) is inconvincible because of the sample size problem. I can't offer any other suggestions here except to ask for an increase in the sample size unless authors can provide evidence to convince readers (and reviewers) that such sampling is representative.
2.	The hypotheses are cliched despite clearly. I have some suggestions on these hypotheses:
(1)	The first hypothesis is not a hypothesis. The comparison is a kind of methodology but a hypothesis. Unless the authors meant that these forests harbored different soil bacterial diversities and community structures. This hypothesis must be rewritten.
(2)	The second and third hypotheses have already been common senses actually. I do not mean that these hypotheses cannot be tested here, but strongly suggest to provide more background information why these hypotheses should be tested in the Three North Shelterbelt afforestation areas. Is it expected to have different results from other studies, or is the change in microbial diversity and ecological function in the Three North Shelterbelt afforestation areas more important than other places and must be tested?
(3)	I also suggest that some relevant literatures should be supplemented to strengthen the foundation of these hypotheses, for example, PeerJ 7:e6147 (2019), PeerJ 6:e6042 (2018), PeerJ 6:e5747 (2018), etc. In addition to the introduction of Three North Shelterbelt afforestation, these literatures on how the composition and ecological function of soil microorganisms have been altered should also serve as a basis for hypothesis-building.
(4)	Finally, I suggest you cite your own paper PeerJ 6:e6251 (2019) as a basis to arise these hypotheses.
3.	No result can validate the third hypothesis directly. The results only present 1) soil properties are different between broadleaf forest and pine forest, and 2) soil bacterial composition and the corresponding functions are different among different plantation forests. However, a conclusion was made that the soil bacterial diversity is influenced by the plantation tree species through altering the soil properties. This conclusion was just addressed in L446-450 actually, which is a discussion or deduction rather than a consequence from the investigation, analysis, or statistical evidence. I strongly recommend a more conservative conclusion.
Additional comments: I still have some minor comments in addition to those of the above:
1.	How to define the top 50 genera of soil bacteria? Are the top 50 means those of total soil samples or of each soil sample?
2.	L239-240: Please explain it.
3.	L241: How many explanatory proportions in PC1 and PC2?
4.	L246-248: The first-two axes plot of PCA can only show the separation of PC plantation from the other two, but cannot directly imply the “great impact” of forest type on soil properties. The interpretation is suggested to be more conservative.
5.	L282: P>0.05
6.	L340: I do not understand this sentence. Please explain it or rephrase it.
7.	L369-370: Richness is also an index of alpha-diversity.


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: COMPARISON OF SOIL BACTERIAL COMMUNITY AND FUNCTIONAL CHARACTERISTICS FOLLOWING AFFORESTATION IN THE SEMI-ARID AREAS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In manuscript entitled “Consistent variations of soil bacterial community and function
characteristics following afforestation in the semi-arid areas”, the authors investigated the changes of the soil bacterial community diversity, structure, and function under different plantation forests, which is relevance for “Peer J”. This study is valuable and the results of this study are crucial for the assessment of ecological restoration in Chinese plantations. However, the manuscript has many problems should to be considered.
My main concerns about the article are as follows:
1.	Line73-74, the statement of “ ‘Three North’ Shelterbelt Development Program” should be corrected as “ ‘Three North Shelterbelt Development Program’ ”.
2.	L86, the ‘Three North’ Shelterbelt is not the first time you refer in the text, so please use the short term. Also note the same problem as follows.
3.	L94-95, “Compared with the physical and chemical properties of soil, soil microorganisms are more sensitive and rapid to the change of soil quality” is there any published work that shows that this is true for the study region? I think this would be more convincing with an appropriate citation from the literature.
4.	L99, the statement of “What is more” should be corrected as “In addition”.
5.	A section in the introduction on the silvics of and soil characteristics preferred by Pinus sylvestris (PS), Populus ×canadensis Moench (PC), Pinus tabuliformis (PT) would have helped the reader not familiar with the species. The soil description of the sites was adequate.
6.	L136-137, Please, use classes based on international taxonomy classifications.
7.	L304-306, CCA like multiple regression show an increase in the variance explained just by adding more variables. Many studies with CCA with similar number of points and variables show very high % of explained variance, but usually these results are spurious. So, how this axis could be interpreted? is showing any kind of gradient?
8.	The manuscript contains many grammatical errors that need to be corrected, so a native English speaker review and revise the manuscript would be useful.


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: COMPARISON OF SOIL BACTERIAL COMMUNITY AND FUNCTIONAL CHARACTERISTICS FOLLOWING AFFORESTATION IN THE SEMI-ARID AREAS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall, this paper has a professional English writing style, some places still need further editing, for example: line 35, replace “under” with “in”; Line 38-39, should not use “increased” because this is not a manipulation study like nutrient addition, please use “had higher soil pH, DOC…and lower C/N ratio”; Line 39, maybe write it out for DOC, dissolve organic carbon; Line 43, when you decided use C or carbon? Above authors had C/N but here authors had carbon and nitrogen, please be consistent; Line 45-46, this sentence is not very informative, it can be applied to anywhere, maybe delete it for Abstract; Line 51, if authors want this paper to be searched broadly, maybe use “semi-arid area” instead of “the semi-arid areas of Northwest Liaoning”; Line 125, no need for that “,”; Line 136, inconsistent font for those coordinates, that maybe why there is a big space there; Line 155, typo, “r”; Line 158, extra space; Line 229, 250, 254, 292, can just say "three plantation forests"; authors can use "different" in the Discussions section; Line 238, again, better not use "increase or decrease", using "had higher or lower.."; Line 328-330, odd space, please choose left align; Line 351, "were higher than those in the PC"; Line 354-355, long sentence, break it down; Line 361, avoid using "anyway", this is only for oral, should not use it in scientific writing. Probably say, "in summary" or "overall"; Line 457, "especially for?";

The first paragraph of Introduction made this paper fall into a limited pool of audiences. I suggested to start with discussing desertification issues globally, more general. And put the description of Chinese program in 1950s and 1978s in the last paragraph or second last paragraph with sentences like “the special program in China gives us an advantage of studying the benefit of afforestation…”

Line 80, this sentence is not correct: “increased soil organic carbon sequestration in both above ground biomass and soil”, aboveground biomass is not about soil organic carbon

Line 90, this first sentence sounds like the authors will be talking about soil characteristics but then the next few sentences are all about soil microorganism. So the topic sentence of this paragraph is confusion. I suggested starting the paragraph with the second sentence

Line 95, is “soil quality” the same thing as “soil characteristics”? and in line 97 “soil properties” and in line 102 “soil parameters” are those the same thing? Authors may avoid using too many different words to describe a similar thing

Line 104, what is “vegetation”? vague, coverage? Vegetation type?

Line 121, “16S rRNA high-throughput gene sequencing technique” can be omitted here, this belongs to Method section

Line 126, it is hard to make this judgement because authors only have 1 replicate for broadleaf (Populus Canadensis Moench) even if using NMDS

Line 127, better to use “difference” instead of “change”.

Line 130, if authors want to discuss the point of selecting tree species, should give more details about what tree species were commonly planted in the Introduction, only those three species reported here?

Figure 1, change panel B to Pinus sylvestris, panel C to Pinus tabuliformis, etc to be consistent with the order of description in the text

Figure 4, please indicate what "a, b" indicates in the figure caption.

Figure 4 and Figure 5, what is "PA"? typo? should be "PC"?

Table 3 and Table 4, two digits would be enough
Experimental design: Authors need some references in the section of site description, particular about soil type and climate.

What are those same conditions? Climate? Vague

Line 150, no up limit? For the plot distance

The section of “Measurement of the soil properties” missing description of quality control (reference materials, recovery, etc).

The section of “statistical analysis” should be separated into two paragraph, maybe at line 219
Validity of the findings: Line 239, why this sentence is here? "This work is guided on “Observation Methodology for Long-term Forest Ecosystem Research” of National Standards of the People's Republic of China (GB/T 33027-2016)." seems not relevant to the results

Line 262-263, this first sentence is not necessary, belong to method; choosing another topic sentence

Line 266, "Venn diagrams were used to compare.." method sentence, not belong to results

Line 304-308, those three sentences are method to me. "a clear correlation" what does that mean? Please choose a better topic sentence and directly describe about results. instead of saying using what method to plot what and in what figure.

Line 347, again, better not using "change", using "had difference" instead. Because technically, those three forest types were all manipulation plots, authors don't have a control with nothing planted to be compared with.

Line 356, what is the afforestation time in this study?

Line 454, in the conclusion, authors may want to write out the full name of PS, PT and PC to make it readable with itself.

Line 458, another paragraph from here?

Line 462, again, it is hard to make this conclusion because of only 1 replicate for broadleaf.
Additional comments: This paper describes variations in soil physical parameters, and soil bacterial community and function at three plantation forests in the semi-arid areas in China. This paper has important values for the studies of afforestation as it reports the basic soil characteristics. Please revise the paper based on the comments listed above.


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: COMPARISON OF SOIL BACTERIAL COMMUNITY AND FUNCTIONAL CHARACTERISTICS FOLLOWING AFFORESTATION IN THE SEMI-ARID AREAS
Review round: 2
Reviewer: 1

Output:
Basic reporting: This article has been revised according to some of the suggestions provided by three reviewers. However, I still have serious concerns about the statistical analyses which are the basis to make conclusions of the causes or effects on the soil bacterial diversity. Authors have hardly answered any questions about statistics positively. So some similar questions and comments are given this time. In addition, there are still multiple typos. Authors should carefully check the article before submission.

L132: “However, variations in the soil bacterial communities, which are crucial for the assessment of ecological restoration in Chinese plantations, have never been studied.” This sentence is incorrect. I can easily search for lots of papers about the community diversity of soil bacteria in China, and several of them are related to the afforestation. For example, Zhao et al. (2018) Eur J Soil Sci, 69: 370-379; Liu et al. (2018) Plant Soil 423: 327; Yang et al. (2018) European Journal of Soil Biology 85: 73-78; Wu et al. (2019) PeerJ 7: e6147.
Experimental design: A question raised in the experimental design in last comments, authors responded that they emphasize the differential bacterial community between forest types and focus on the soil characteristics and high-throughput sequencing (to estimate the bacterial community compositions). They also declared that the use of LEfSe analysis to determine the “classified bacterial taxa with significant abundance differences”. However, this response did not answer the question of no control group and how to avoid other confounding effects in addition to the forest types.

If authors want to identify the bacterial taxa with significant abundance differences between forest types, why not just simply comparing the abundances of sampled bacterial taxa with more direct and intuitive statistical methods? why choosing the LEfSe?
Validity of the findings: In statistics, the authors seemed not to understand what I meant. For example, in ordination analysis, I emphasize that unconstrained ordination analysis can only explain "vector fit" rather than "effect", in other words, it cannot tell any causal relationship by any unconstrained ordination analysis. So, if authors want to test some “effect”, please use the ordination analysis.

As for the MANOVA and/PERMANOVA, the tested effect is categorical data (e.g. tree species, which is the independent factor) and the response is edaphic characteristics (this is the dependent factor, i.e. the outcome or response), and the dependent factor is comprised of “multiple variables”, so the “multivariate” analysis of variance (MANOVA) or the permutational multivariate analysis of variance (PERMANOVA) is suggested. Strictly speaking, your experiment has not done any treatment. If you have other treatments (such as varying degrees of watering) beyond this research question (effect of planting species on edaphic characters), you need to set the watering factor as the random effect. If you are not familiar with relevant statistics, I strongly recommend consulting experts in statistics.

Another statistical question concerns the conditional factor. I asked the authors to condition on the richness when testing the effect of edaphic factors on RAs of functional groups of soil bacteria, instead of asking how to estimate the richness. The reason I ask this question is simple. If the total numbers of bacteria are the same in two communities, but there are only 10 bacteria in community A and 10,000 bacteria in community B, the relative abundance of the same bacteria in two communities will inevitably different by the differential bacterial richness. Therefore, the bacterial richness in each soil sample must be taken into account in estimating the effect of edaphic effects on microbial RA. Ignoring it may directly affect the results of estimates.
Additional comments: I don't think it's very meaningful to evaluate the results of this study until the suggestions of these statistical analyses have been substantially answered and revised.


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: COMPARISON OF SOIL BACTERIAL COMMUNITY AND FUNCTIONAL CHARACTERISTICS FOLLOWING AFFORESTATION IN THE SEMI-ARID AREAS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have intensively modified the original manuscript following reviewers' comments and suggestions. They have also answered, explained and clarified the reviewers' questions and concerns. The improvement is significant and mostly satisfactory.


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: COMPARISON OF SOIL BACTERIAL COMMUNITY AND FUNCTIONAL CHARACTERISTICS FOLLOWING AFFORESTATION IN THE SEMI-ARID AREAS
Review round: 2
Reviewer: 3

Output:
Basic reporting: Line 78, The first sentence is not relevant.Omit it

Line 94, "...of the forested areas"

Line 168, "soil corns"? should it be "soil cores"?

Line 173, this is a laboratory analysis, so C/N should not be here. It is calculated by mathematics.

Line 278-279, Authors declared "no significant differences.." but with a p value less than 0.01? And for p values throughout the results section, I prefer to see the specific p values if there are. For example, if one p value is 0.02, authors should write "0.02" instead of "< 0.05". If one p value is 0.23, authors should write "0.23" instead of "> 0.05".

Line 313, the first sentence is method, no need to be here

Line 320, ",and the other..."

Line 332 "In total..." This sentence is not necessary, omit it

Line 334-335, would two digits for r values be enough?

Line 394, I am confused by this sentence: "The Simpson index of PC was the highest, while, there were no significant differences among different plantation forests (P < 0.01)" why saying no significant differences but the p value is less than 0.01?

Line 415, "previous findings that have established that a high availability of carbon has been associated with a high.." this sentence is bad, revise it

Line 424, "..considered as a key..."

Line 497-499, no funding source for this study?
Experimental design: None
Validity of the findings: None
Additional comments: Authors have revised this paper intensively based on reviewers' comments. I really appreciated that effort! I only have some minor suggestions on some sentences (see above).


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: COMPARISON OF SOIL BACTERIAL COMMUNITY AND FUNCTIONAL CHARACTERISTICS FOLLOWING AFFORESTATION IN THE SEMI-ARID AREAS
Review round: 3
Reviewer: 1

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: In fact, what I am most concerned about is the correctness of statistical methods and the interpretation of statistical results, because the inferences of this marker-based community ecological process almost depend on statistical results. I do not intend to argue with the authors about the statistical method. The importance of discriminating key microbial taxa by LEfSe has been repeatedly emphasized in rebuttal letter. I am sorry that I am not familiar with this method. However, as far as I understand, LEfSe is an application of linear discriminant analysis (LDA), while LDA can be regarded as a combination of ANOVA and regression analysis, and the fundamental assumption of LDA is the normal distribution of independent factors. If the independent variables cannot be assumed to be normally distributed, the use of ANOVA and/or regression analysis is more appropriate than using LDA (or LEfSe). I strongly recommend referring to the article “Paulson et al. (2013) Nature Methods 10(12):1200–1202” for the identification of taxa with key differences between communities. As for other statistical suggestions, if the authors are not so familiar with the principles and applications of these analyses (unconstrained and constrained ordination analysis, MANOVA, linear regression, linear mixed-effects model, etc.), I suggest that the description and discussion of their results should be conservative, especially avoiding the discussion of causality. In principle, I affirm the authors’ efforts and patience in data processing and research results in this study, although I think this data should deserve better interpretation. So I don't have any further critical comments, except for a few minor ones.

1.	L382-385: This inference is too arbitrary and suggests weakening or deleting it.
2.	L397: There are differences in soil composition and microbial diversity among different forest lands. However, due to the failure to detect before and after afforestation, it is difficult to verify whether the difference comes from the difference in geographical distribution or is really caused by afforestation. It is, therefore, necessary to avoid such causal inferences.
3.	L428-432: As mentioned above, such an inference is too arbitrary. It is not difficult to find a positive or negative correlation between some specific microbial taxa and the content of some soil components in a large number of microbial community dataset, but there is not necessarily a causal relationship between them. It is suggested that inferences should be more conservative when false positives cannot be ruled out or the causal relationship cannot be evident.
4.	L430: “Available P” should be “available P”.
5.	L441-446, L471-473, L483-485: These inferences should be more conservative and avoid the inference of causality.
6.	L475-476: Change to “The soil bacterial community compositions, diversity, and functions are different among plantation types, …”. L478-479 should also be modified in this way.
7.	For the same reason, I don't think the experimental design and analytical method of this study can answer the causal relationship. At most, it is the correlation between the soil microbial composition/diversity/function and the afforestation tree species. Therefore, I suggest that the title of this article should be modified appropriately.


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: COMPARISON OF SOIL BACTERIAL COMMUNITY AND FUNCTIONAL CHARACTERISTICS FOLLOWING AFFORESTATION IN THE SEMI-ARID AREAS
Review round: 3
Reviewer: 2

Output:
Basic reporting: None
Experimental design: None
Validity of the findings: None
Additional comments: I think this paper is well revised, and I don't have any further comments. I suggest to accept this paper 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: COMPARISON OF SOIL BACTERIAL COMMUNITY AND FUNCTIONAL CHARACTERISTICS FOLLOWING AFFORESTATION IN THE SEMI-ARID AREAS
Review round: 4
Reviewer: 1

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: Overall, I'm satisfied with this version. In addition to some minor details that still need to be corrected, I would recommend accepting this article.

1.	Please check the scientific name carefully, especially spaces before and after the “’x” between the generic name and species name of hybrid species Populus × canadensis.
2.	Fig. 9: there are two redundant correlation coefficient values between TN and TN and between TN and C/N.
3.	L346-349: Please check the space carefully. There seem to be two spaces between some words.


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: FALSE HEAD COMPLEXITY AND EVIDENCE OF PREDATOR ATTACKS IN MALE AND FEMALE HAIRSTREAK BUTTERFLIES (LEPIDOPTERA: THECLINAE: EUMAEINI) FROM MEXICO
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This paper expands upon previous collections-based studies of functional morphology of the false head (FH) wing pattern elements in Lycaenidae butterflies. The study is well executed and is a valuable addition to our understanding of wing pattern evolution in Lycaenidae. I believe, it can be published in its present form.
The study can be used as a template for similar studies of FH (and other attack-deflecting wing pattern elements) that are quite common in Lepidoptera. There are many examples of such elements. For example, Tonner et al. (1993) demonstrated by analyzing wing damage in Stichophthalma camadeva (Nymphalidae) that about a third of butterflies in their sample bore signs of presumed predator attacks in FH region of the wing. In another nymphalid, Archaeoprepona chromus, the FH pattern elements are enhanced by sagittal movements of the hindwings, which are initiated when approaching predator is detected (Sourakov 2015). This behavior is akin to that displayed by the hairstreaks. Authors may want to include this in the Discussion section of their paper.

Tonner, M., Novotny, V., Leps, J., Komarek, S. 1993. False head wing pattern of the Burmese junglequeen butterfly and the deception of avian predators. Biotropica 474-478.

Sourakov, A., 2015. Antipredation and “antimimicry”: wing pattern is supported by behavior in Archaeoprepona chromus (Lepidoptera: Nymphalidae: Preponini). ATL Notes, December 2015 issue: 1-7.


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: FALSE HEAD COMPLEXITY AND EVIDENCE OF PREDATOR ATTACKS IN MALE AND FEMALE HAIRSTREAK BUTTERFLIES (LEPIDOPTERA: THECLINAE: EUMAEINI) FROM MEXICO
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: see the general comments to the authors
Validity of the findings: see the general comments to the authors
Additional comments: The topic is interesting as it tackles a subject which is connected to the subfamily Theclinae. Thecline imagines possess a peculiar wing shape and ventral wing pattern posing the false-head hypothesis. This is briefly but well posed in the introductory part.

The paper is written in a clear and unambiguous language. The manuscript was composed with high care. There are very few typos and all the references-citations are relevant. Figures are in high quality, and all necessary, the legends are informative enough. The descriptions and the data supplied are enough to reproduce the experiment.

The authors examined more than 20 thousand well curated specimens for their experiment. The working hypothesis they posed are simple but important enough for having the further step in understanding the role of the false-head pattern in theclines. The statistical method for me seems to be relevant and well chosen (although I am not an expert in biostatistics).

I have some remarks and notes which are listed below, I ask the authors for taking these notes into consideration.

1.	Title should be more precise: False head complexity and evidence of predator attacks in male and female hairstreak butterflies (Lepidoptera: Theclinae: Eumaeini) in the Neotropical region; the subject of the paper are hairstreaks imagines form Mexico (I think the single copper species should be taken out from the species examined (see later below)
2.	Line 29 – The acronyms of CNIN and MZFC in the abstract are unresolved, it is impossible or difficult to understand them for the reader who is not familiar in the usage museum acronyms.
3.	Lines 53-57 – The elements of the false head are listed. Although I absolutely understood the description but I am sure that readers less familiar in butterfly wing pattern these lines could pose some difficulties. Why you do not give an extra figure for showing these components, or use Fig. 1 to mark them with arrows pointing to the component?
4.	Paragraph starts from line 73 – It is necessary to cite Robbins 1981 only once. Please remark that Robbins analysed only the Neotropical hairstreaks.
5.	Line 103 “belonging tp 49 genera” – Please indicate what kind of nomenclature and taxonomy is used, I guess Robbins 2004 in Lamas.
6.	Line 109 – Again, the false-head components should be shown visually in a figure.
7.	Line 116 – probably at the end of this paragraph a reference should be given to the Supplementary Material.
8.	Line 122 – probably it would be good here to mention that Robbins and his colleagues shown that the male androconia is also the subject of rapid evolution (there are papers on Arcas, Denivia and Thereus).
9.	Lines 122-125  In fact… - This sentence should better fit under the Results or Discussion, and probably it would be necessary to show these results in more detailed manner (for example listing and discussing the genera, the phenomenon….); but this is not essential for the paper.
10.	Line 137: “sex*” – why the asterisk?
11.	Line 148 “results (not shown)” – why these results are not shown? if they are really VERY similar, this should be stated more strongly proving it is not necessary to show the details, but if they are just somewhat similar…
12.	Lines 185-187: “However…” – this statement is not clear enough just like this, more explanation would be welcome.
13.	Lines 190-198: “The shapes and…. induced by the FH)” – This is just a repetition of the introduction.; should be deleted.
14.	Line 204: write Theclinae, instead of the typo “Techlinae”; but probably it would be better to write “Lycaenidae” (see the next entry).
15.	Lines 205 – the references Fischer & Fiedler and Dinesh & Venkatesh do not refer to Theclinae; see above the entry no. 15.
16.	Line 210 – the semi sentence “if this is…” should be not in brackets; it is an important remark.
17.	Line 222 – I would be happy to see here this result also in a Table with the names of the genera.
18.	Line 231 – FH can be supplemented with special behaviour (see Bálint & Hoskins, Lepidoptera Novae 9: 33-36 (2016)
19.	Figure 1 _ probably it would be important also to present an image what show the false head from a different view – most probably more relevant for the paper; this is what I mean:  https://www.butterfliesofamerica.com/L/imagehtmls/LycRio/ADW-Atlides_h_halesus_F_Fruit_Cove_St_Johns_Co_FL_USA_30-X-05_2_i.htm
20.	Figure 2. – please write that the images show the ventral wingsurfaces; please give scales (indicating forewing lengths or giving scale bars); the specimens of Laothus erybathis  seem to be all females (on the basis of wingshape, abdominal tip; and the male have a blue reflector and lacks the V shaped medial pattern  in cell CuA2)
21.	All the following remarks concern to the Supplementary material
-	why the FH component are not independently coded? that would be more easy to check the evaluation? Because of this I do not really understand some of the values
-	for having more clarity it would be better to list the taxa in alphabetical order according to genera, and not according to the collections;
-	Iophanus pyrrhias is not a thecline species, it is a Lycaeninae; should be removed from the list;
-	I suggests the following corrections, changes
1.	Brangas jetus = Brangas getus
2.	Bussa busa = Brevianta busa
3.	Laspis castimonia = Iaspis castimonia
4.	Symbiosis (???) = probably Symbiopsis
5.	Thecla arza = Arzecla arza
6.	Thecla caninius = Gargina caninius
7.	Thecla ligurina = Kolana ligurina
8.	Thecla lyde = Kolana lyde
9.	Thecla phobe = Nicolaea phobe
10.	Thecla seton = Arzecla sethon
11.	Thecla thoria = Gargina thoria


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: FALSE HEAD COMPLEXITY AND EVIDENCE OF PREDATOR ATTACKS IN MALE AND FEMALE HAIRSTREAK BUTTERFLIES (LEPIDOPTERA: THECLINAE: EUMAEINI) FROM MEXICO
Review round: 1
Reviewer: 3

Output:
Basic reporting: English used throughout the manuscript is clear and technically correct (except one point: I suggest more widely used term “[wing] margin” instead of “outline” in lines 26, 55, 110).
Figures are relevant to the content of the article, of sufficient resolution, and appropriately described and labeled. Additionally, I suggest to include scheme (or colour photo) of the hindwing of species with four false head elements numbered and marked with arrows for better understanding.
I thank you for providing the raw data. I suggest to include geographical labels data of the taxa in question in the table (if possible). Also, please check line 11 of the table (delete the species name).
Experimental design: No comment.
Validity of the findings: Discussion of FH elements in morphologically variable species should be included and appropriately stated in conclusion (please see “General comments for the author” section).
Additional comments: I commend the authors for their extensive dataset and solid background based on both publications of predecessors and previous works of the authors of the current manuscript, and I’m generally satisfied with presentation of the data, their interpretations, and conclusions. The research in question fits into the broader field of knowledge devoted to prey-predator interactions.
One point was not considered in the manuscript: how does the intraspecific morphological variation (degree of FH elements development) occuring in some species (e.g., Micandra cyda, Oenomaus ortygnus, Cyanophrys species, etc.), and widely known in Eumaeini in the whole, impact on the statistical analysis and general conclusions? Hindwings of some treated species vary in the number of FH components (eyespots and lines converging towards the posterior end of the hindwings) due to individual variations. I suggest this point to be added in the discussion and conclusion.


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: FRESHWATER RESERVOIR OFFSETS ON RADIOCARBON-DATED DOG BONE FROM THE HEADWATERS OF THE ST. LAWRENCE RIVER, USA
Review round: 1
Reviewer: 1

Output:
Basic reporting: I have made a small number of suggestions for improving the clarity of the English.
Experimental design: I have noted a couple of small mistakes in formulae used, which do not however affect the interpretation.
Validity of the findings: Compared to other research in this field, including my own, the data set seems pretty robust. I have raised a couple of questions about the applicability of the published data used to interpret the new results - which I think the authors will be able to address.
Additional comments: please see the uploaded PDF version of my comments


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

Input:
Title: FRESHWATER RESERVOIR OFFSETS ON RADIOCARBON-DATED DOG BONE FROM THE HEADWATERS OF THE ST. LAWRENCE RIVER, USA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Language: no comments.

References/background:
In the Introduction (see lines 42-43), the discussion about the suitability of dog diet as proxy for human diet could be expanded. I attach a few references with arguments for (Noe-Nygaard 1988, Clutton-Brock and Noe-Nygaard 1990) and against (Eriksson & Zagorska 2003; Fischer et al. 2007: Fig. 5B and 5C) this approach. Some of these were already referred to in Edwards et al. 2017, which you cite in the Introduction.

Noe-Nygaard N. 1988. 13C-values of dog bones reveal the nature of changes in man's food resources at the mesolithic-neolithic transition, Denmark. Chemical Geology: Isotope Geoscience section 73(1):87-96.
Eriksson G, Zagorska I. 2003. Do dogs eat like humans? Marine stable isotope signals in dog teeth from inland Zvejnieki. In: Larsson L, Kindgren H, Knuttson K, Loefler D, Åkerlund A, editors. Mesolithic on the Move. Papers presented at the Sixth International Conference on the Mesolithic in Europe. Oxford: Oxbow Books. p 160-8.
Fischer A, Olsen J, Richards M, Heinemeier J, Sveinbjörnsdottir ÁE, Bennike P. 2007. Coast-inland mobility and diet in the Danish Mesolithic and Neolithic: evidence from stable isotope values of humans and dogs. Journal of Archaeological Science 34:2125-50.
Juliet Clutton-Brock, Nanna Noe-Nygaard. 1990. New osteological and C-isotope evidence on Mesolithic dogs: Companions to hunters and fishers at Star Carr, Seamer Carr and Kongemose. Journal of Archaeological Science, Volume 17, Issue 6, Pages 643-653.

Structure/figures/tables/raw data:
In Figure 2, there are several instances of one symbol that I do not understand nor find in the caption: A diamond overlain by the blue triangle, which is the symbol for turkey. Of course, this could happen accidentally in cases where e.g. a mammal has exactly the same isotope ratios as a turkey sample – but not so often, I assume. Please check.

In Figure 2 as well as in the caption of Figure 2, please indicate relative to which standard the d13C and d15N values are presented (e.g., “d15N (‰ AIR)”).

From the manuscript in its current form, I do not understand why it is necessary for your FRO analysis to calibrate the radiocarbon ages, e.g. using phase models or KDE plots in Figure 3. You write in line 157 that you use phase and KDE plots to graphically display the modelling results. However, there must be another reason for these calculations – please state it clearly either in the methods section or in the caption of Figure 3. As mentioned later, I think it is best to perform FRO calculations in 14C space, not calendar age space. However, I think it is useful, as you did in Table 3, to model your results with and without DeltaR on the dog bones to show the effect of the FROs.

In Tables 4 and 5, you could explain the abbreviations in the table caption (FRO, FMC, FDC).
Experimental design: Original primary research within Aims and Scope of the journal:
Yes/no comments.

Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap:
Yes/no comments.

Rigorous investigation performed to a high technical & ethical standard:
In the last part of the Results section, lines 198-218, you describe the connection between carbonate alkalinity and dead carbon fraction in fish. Specifically, you mention that a total alkalinity of >90mg/L CaCO3 in the water causes a dead carbon fraction of >0.05 in fish. However, I do not think that we should operate with threshold values like these; instead, increasing concentrations of 14C-free carbonate cause increasing FROs in fish, in a continuum. Later in the discussion, though, we can choose a threshold value to separate relevant from irrelevant FROs.

A minor correction: even though most of the total alkalinity in natural systems is carbonate alkalinity, you should consider to separate the two concepts in the text. The total alkalinity comprises all the bases in the solution, and not all of them contribute with carbon.

Methods described with sufficient detail & information to replicate:
The beginning of the sentence in lines 92-93 is a bit out of context: how have you established that fish accounted for 20-40 % of the dogs’ diet? By the archaeozoological analyses mentioned in the introduction, or by the isotope measurements that you mention later in the article?
Validity of the findings: Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.
No comments.

Data is robust, statistically sound, & controlled.
Yes – although I would not use calibrated radiocarbon ages to draw conclusions about reservoir offsets. Differences in radiocarbon ages, which are caused by carbon originating from different carbon reservoirs, are already visible on the radiocarbon timescale. When the ages are calibrated, an additional uncertainty is added to the ages from the shape and measurement uncertainty of the calibration curve. Therefore, small offsets are less visible on the calendar timescale than on the radiocarbon timescale.
In line 189, you state that you used the criteria by Van Klinken (1999) to assess the quality of the extracted collagen. However, as you use ultrafiltration (which Van Klinken did not use), you might want to consider using other criteria, e.g., lower collagen yields should be acceptable, as using a pretreatment method with ultrafiltration removes some of the sample mass.

In lines 162-165, you describe that you combined multiple dates with OxCal’s R_Combine function. I think it is better to use the Combine function in cases when the carbon derives from different samples. The R_Combine function should be used when the carbon derives from the same carbon reservoir, e.g. several samples from the same bone.
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: FRESHWATER RESERVOIR OFFSETS ON RADIOCARBON-DATED DOG BONE FROM THE HEADWATERS OF THE ST. LAWRENCE RIVER, USA
Review round: 2
Reviewer: 1

Output:
Basic reporting: all good, except for 2 text edits;
line 133 - insert "fish", i.e. "For example, fish isotope values were..."
line 170 - the square root sign should be outside the bracket
Experimental design: all good
Validity of the findings: all good
Additional comments: I have checked all the revisions and rebuttal comments and I am satisfied that the authors have addressed all my concerns


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

Input:
Title: MULTI-GENE INCONGRUENCE CONSISTENT WITH HYBRIDISATION IN CLADOCOPIUM (SYMBIODINIACEAE), AN ECOLOGICALLY IMPORTANT GENUS OF CORAL REEF SYMBIONTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: There were a few inconsistency of language/words, such as:
1) double negative (grammatically not encouraged)
2) there were some inconsistency of citation: "Wilkinson and colleagues" and "Wilkinson et al."

There are few suggestions on sorting some of the contents especially from DISCUSSION to either INTRODUCTION, METHODS or RESULTS.

The manuscript, especially in the METHOD section relied too much on referring the readers to one specific paper published previously. This left a lot of important details out in the manuscript, which caused some confusion.

Are the 27 alignments in this paper publicly available in repositories (i.e. GenBank?) or is it possible for the author(s) to submit the alignments as Supplementary data in nexus format?

(Please see attachment for more details)
Experimental design: The most important issue with the explanation of the study design is that the author relied on referring the readers to a specific paper previously published. This left a lot of details for the readers to find out themselves, which does not serve the purpose of independent study. Details are as below:

1) The study did not mention specifically what type/species hosts of the Cladocopium extracted from (L 143-160)
2)How many specimens in total does the author(s) collected? (L157-159)
3) What are the detailed procedures in amplifying the markers (cob, psbA and ITS2), with at least specify the markers used and methods modified from previous papers. (L161)
4) psbA is a hypervariate region, often impossible to make consensus between forward and reverse sequences, hence only one direction is used for alignments. Is it true for this study? (L166-167)
5) 'METHOD: Incongruence tests' section needs to be separated into more paragraphs for ease of reading and identify the different hypotheses tests. (L170-199)
6) The actin gene came in suddenly without explaining its contribution in this study in detail. Furthermore, the method ended abruptly at the sequencing phase. Perhaps the author can mention "...no further step taken and will be explained in the discussion"? (L288-301, L537)

(Please see attachment for more details)
Validity of the findings: There are a few inconsistency of language in explaining the data when it comes to the words "congruence", "incongruence", "no incongruence". These three are essential in explaining the data, explaining the data using only two out of the three will be much appreciated.

1) May I know what is the standard of ' approaching significance'? Providing citations is much appreciated. (L311-312)
2) The hypotheses should be established clearly in METHODS so readers will have an idea coming to the RESULTS. (L340-342)
3) LIW serves as the control for the post hoc test? (L349-353)
4) Thornhill et al. (2017) mentioned specifically that sexual recombination do happen but in a very low frequency (correct me if I am wrong). Do you think mentioning this will also prove that why most of your 'hybridized' Cladocopium present in a very low frequency? (L503, L570-572, L580-591)
5) The NGS results are important in explaining the hybridization section. However, this part is missing from the RESULTS section. Can you please write something in the results so readers can have the right idea going into the DISCUSSION. Initially, I was skeptical with the hybridization theory without this data in the results, as I was thinking there might be a co-dominant in play. Including the NGS in the RESULTS will be much appreciated. (L509-512)

(Please see attachment for more details)
Additional comments: Please see attachment for more details