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: GEOGRAPHIC POPULATION GENETIC STRUCTURE AND DIVERSITY OF SOPHORA MOORCROFTIANA BASED ON GENOTYPING-BY-SEQUENCING (GBS)
Review round: 2
Reviewer: 1

Output:
Basic reporting: No further comments
Experimental design: Unfortunately, the authors have not seem to make any change relative to my major concern about the calculations of diversity statistics. Although the authors claim that they changed the parameter “-max-missing” of VCFTools from 80% to 60%, Table 2 is exactly the same as in the original version. This indicates that no alternative filtering was really tried to solve the technical issue reflected in high heterozygosities. Also, the authors seem to misunderstand this parameter. In the methods thext they write “the SNP was called at least 60% of individuals” but according to the documentation of VCFTools it would be exactly the opposite. The consequence of this is that there could be an important number of SNPs with missing data in every individual of the population.

Based on the new supplementary table with SNP statistics per sample, I infer that the total number of SNPs in the VCF used to calculate the diversity statistics is about 400.000. In all GBS experiments that I have analyzed, this corresponds to the number of raw variants and normally contains a large number of false positives, either because each false positive heterozygous call in at least one sample becomes an new false positive SNP or (most likely in this case) repetitive regions contribute a lot of SNPs that are heterozygous in most of the samples and skew population statistics. The percentage of heterozygous SNPs per sample (around 70%) seems to confirm this. Unfortunately, the distribution of minor allele frequency and observed heterozygosity that I requested for the entire set of samples is not provided. These distributions are needed to see the trend in the dataset of SNPs and identify possible technical issues.

To make it clearer, this is more or less the filtering procedure that the authors should apply to the data:

1. Minimum genotype quality 30
2. Distance between variants of 300bp. This is to keep only one SNP per tag and indierctly remove some of the issues with repetitive regions.
2. At least 80% of the samples genotyped (in VCFTools it would be -max-missing 20%)
3. MAF above 0.05

Then, distributions (not only averages) of MAF and observed heterozygosity should be provided for both the entire population of 225 samples and the 4 main groups: (1) populations 1-6, 2) populations 7 and 8, 3) populations 9,10 and 11, and 4) populations 12, 14 and 15). If the authors insist on calculating and making inferences on diversity statistics for each of the 15 populations, then they need to perform additional filtering to keep only SNPs genotyped in 14 out of 15 individuals of each population to try to make inferences based on somewhat reliable numbers.

The authors mention in the rebuttal that “the amount of data in the physical file was too large. If we adjust the parameters too small, it will make the calculation too difficult”. This does not make any sense. A VCF with 400.000 SNPs genotyped in 225 individuals should have for sure less than 100Gb. The procedure described above can be done in a normal laptop and there are plenty of open source software tools useful to perform these filtering steps.

The authors also write in the rebuttal that “However, the previous analysis was done with the help of others. Even though I have tried to perfect it , I am so sorry that I couldn’t finish it within the bound of my abilities”. Whoever performed the bioinformatic analysis in the first place should be able to perform another round of analysis, especially taking into account that important technical issues need to be solved.

As minor comment, the text should not contain qualitative statements about population statistics such as “the Ho is less valuable because it is affected by inbreeding and other evolutionary processes” or “Fst is an excellent measure of genetic differentiation”. Population statistics are just different sources of information of diversity and they just need to be properly calculated to make accurate inferences. For example, the claim that “the populations conforms to Hardy-Weinberg Equilibrium” would not even make sense if the observed heterozygosity is not calculated because HWE is tested as the difference between observed and expected heterozygosity. Also, from a strictly statistical point of view, HWE is not really accepted because that is equivalent to accept the null hypothesis.
Validity of the findings: No further comments
Additional comments: Although I generally like the study and it is clear to me that the authors know the ecology of the species and the sampling is appropriate to draw some of the conclusions, I can not accept the manuscript if a proper bioinformatics analysis is not performed. If the authors are currently lacking bioinformatics support, they should collaborate with a bioinformatician and make proper acknowledgement of his/her contribution to be able to bring this study to a good end.


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: GEOGRAPHIC POPULATION GENETIC STRUCTURE AND DIVERSITY OF SOPHORA MOORCROFTIANA BASED ON GENOTYPING-BY-SEQUENCING (GBS)
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comments
Validity of the findings: no comments
Additional comments: The manuscript has been much improved by the authors. I have only few minor suggestions in the attached file


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

Input:
Title: GEOGRAPHIC POPULATION GENETIC STRUCTURE AND DIVERSITY OF SOPHORA MOORCROFTIANA BASED ON GENOTYPING-BY-SEQUENCING (GBS)
Review round: 3
Reviewer: 1

Output:
Basic reporting: A. Important concerns

There are some typo problems (e.g. line 127 ultilized). Although the English language is understanding; you should give your manuscript to a native English speaker colleague for correction.

B. Minor concerns

Snps data analysis subsection line 157, It is not a python script but a shell command called “cat” that you used to combine the paired-end reads.
Experimental design: No comment
Validity of the findings: 1. Admixture and structure analysis require unlinked SNPs data, you reported 400. 000 snps with not mention if they are linked or unlinked. I do think there linked snps among and therefore the analysis is biased. Furthermore, it seems like you don’t understand stacks program and parameters that is the reason why you used vcftools for filter purpose whereas everything is done with stacks, you should carefully read stacks manual (http://catchenlab.life.illinois.edu/stacks/manual/). However, here are my recommendations of some stacks parameters that you should pay attention for population structure analysis (http://catchenlab.life.illinois.edu/stacks/comp/populations.php):

-- write-single-snp: restrict data analysis to only the first SNP per locus and therefore snps are not linked.
You should try as well --write-random-snp (restrict data analysis to one random SNP per locus) to see this affect the result.
-p minimum number of populations a locus must be present in to process a locus
-R: minimum percentage of individuals across populations required to process a locus
-r minimum percentage of individuals in a population required to process a locus for that population
--structure: output results in Structure format.
As well you can generate many output files see the file output options ((http://catchenlab.life.illinois.edu/stacks/comp/populations.php)

To choose the best stacks parameters you should read this papers: https://besjournals.onlinelibrary.wiley.com/doi/full/10.1111/2041-210X.12775

2. Although you used Sophora davidii as outgroup species to avoid ascertainment bias this may not cluster well individuals of the targeted species if the outgroup species are genetically distant of S. moorcroftiana. Therefore, I suggest that you build an unrooted tree  as well.
Additional comments: This is a great and pionner article for the S. moorcroftiana species. However, I warmly suggest that you should take carefully the recommendation regarding the data analysis (population structure and phylogenetic analysis) which is the weakness part of this manuscript.


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

Input:
Title: GEOGRAPHIC POPULATION GENETIC STRUCTURE AND DIVERSITY OF SOPHORA MOORCROFTIANA BASED ON GENOTYPING-BY-SEQUENCING (GBS)
Review round: 3
Reviewer: 2

Output:
Basic reporting: I have no major comments. I found a few typos and small fixes that the author can make:

Line 96: Scrub
Line 121: Ultilized
Line 147: The command "cat" is not really a Python script. You can just say "bash command cat". About the script, cat itself would not join two compressed files. The command is either:
cat <SAMPLE>_1.fastq <SAMPLE>_2.fastq > <SAMPLE>.fastq

or

zcat <SAMPLE>_1.fastq.gz <SAMPLE>_2.fastq.gz | gzip -c > <SAMPLE>.fastq.gz

Line 192: Populaions
Experimental design: This time the authors addressed my previous comments and updated the analysis. The analysis looks fine now. I do not have any further comments
Validity of the findings: No comment
Additional comments: I am glad to see that the authors could improve the analysis as suggested and that it was helpful.


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: GEOGRAPHIC POPULATION GENETIC STRUCTURE AND DIVERSITY OF SOPHORA MOORCROFTIANA BASED ON GENOTYPING-BY-SEQUENCING (GBS)
Review round: 4
Reviewer: 1

Output:
Basic reporting: You should rename the additional file python to shell or bash script.
Experimental design: The authors addressed all my concerns and the results look more accurate now.
Validity of the findings: No comment
Additional comments: You improved a lot your manuscript and it looks fine now.


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: GEOGRAPHIC POPULATION GENETIC STRUCTURE AND DIVERSITY OF SOPHORA MOORCROFTIANA BASED ON GENOTYPING-BY-SEQUENCING (GBS)
Review round: 4
Reviewer: 2

Output:
Basic reporting: I have no further comments.
Experimental design: I have no further comments.
Validity of the findings: I have no further comments.
Additional comments: I have no further comments. I am glad to see that the manuscript went through editorial review to improve the english quality of the paper


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: GEOGRAPHIC POPULATION GENETIC STRUCTURE AND DIVERSITY OF SOPHORA MOORCROFTIANA BASED ON GENOTYPING-BY-SEQUENCING (GBS)
Review round: 4
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Authors' responses and changes in the manuscript are satisfactory and I have no further comments. In my opinion, the manuscript can be accepted


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 BAYESIAN CONFIDENCE INTERVALS FOR MEASURING THE DIFFERENCE BETWEEN DISPERSIONS OF RAINFALL IN THAILAND
Review round: 1
Reviewer: 1

Output:
Basic reporting: WELL REPORTED
Experimental design: RELIABLE
Validity of the findings: STATISTICALLY OK.
Additional comments: Review of article titled "The Bayesian confidence intervals for measuring the difference between dispersion's of rainfall in Thailand". The review congratulates the authors for pointing out a firm understanding of what sorts of inferences confidence interval theory does, and does not, allow which is critical to illustrate the variability in precipitation. However, there exists caution against relying upon confidence interval theory to justify interval estimates, and suggest that other theories of interval estimation should be used instead.
The following are some of the points the authors need to address for possible acceptance of the manuscript.
1. The authors nowhere provide descriptive statistics of rainfall data used in the study.
2. Confidence interval evaluation by any method can be unreliable for small samples (<10000) as in your situation. Can authors justify?
3. Likelihoods, confidence intervals, and Bayesian credible intervals should be plotted.
4. Discuss on dependence of the interval on small variations in the data and the large amount of computing required to obtain the interval estimates.


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 BAYESIAN CONFIDENCE INTERVALS FOR MEASURING THE DIFFERENCE BETWEEN DISPERSIONS OF RAINFALL IN THAILAND
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a nicely written paper with a clear message. It compares different approaches for confidence interval estiamtion in the case of difference of two coefficients of variation. The Bayesian approach using Jeffreys' prior as well as the Fiducial Generalized CI comes out beneficial.
Experimental design: not applicable here.
Validity of the findings: The findings are real and substantiated.
Additional comments: There are a few points I wish to make.

l108: " since the coverage probabilities do not cover the target in a few cases" does not  make sense to me. CIs corver the target or not, but coverage probabilities?

l126 and what follows: it would be good to say how the coefficient of variation is actually defined. In fact, on line 65 it is said that  the CV "is the proportion of the standard deviation to the mean". I do not think that this is right.

(10) what is the Fisher information here? What does | | mean here? Determinant or absolute value or both? Can you motivate why this is called left-invariant Jeffreys' prior?

Fig1 -3 are quite small and you will score more sales here if you produce better graphs. Clearly, make the bench mark line visible. It would be good to incorporate some text into the figures to make these plots more illustrative.


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 BAYESIAN CONFIDENCE INTERVALS FOR MEASURING THE DIFFERENCE BETWEEN DISPERSIONS OF RAINFALL IN THAILAND
Review round: 2
Reviewer: 1

Output:
Basic reporting: I am convinced by the revised version of the manuscript. It's now of acceptable form.
Experimental design: Relevant and satisfactory
Validity of the findings: Reliable and satisfactory
Additional comments: I recommend for acceptance of the manuscript in current form.


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

Input:
Title: THE BAYESIAN CONFIDENCE INTERVALS FOR MEASURING THE DIFFERENCE BETWEEN DISPERSIONS OF RAINFALL IN THAILAND
Review round: 2
Reviewer: 2

Output:
Basic reporting: Revision is fine.
Experimental design: No issues here.
Validity of the findings: Ok.
Additional comments: Thank you for a careful revision.


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: VARIABLE ABSORPTION OF MUTATIONAL TRENDS BY PRION-FORMING DOMAINS DURING SACCHAROMYCETES EVOLUTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The investigation touches on the very interesting topic of the evolution of amyloid and prion-forming domains. Nevertheless, number issues can be improved before publication.

1. Abbreviations for yeast prion names (PSI, PIN, SWI, etc) should be written in italic ([/PSI/+], [/PIN/+], [/SWI/+]). The yeast prion forming nucleoporin was originally called Nup100 but not Nu100 (Halfmann R et al. 2012. Prion formation by a yeast GLFG nucleoporin. Prion 6: 391–399.).

2. I would recommend simplifying the phrase in the introduction, lines 86-90.
"It is discovered that these protein regions have a spectrum of behavior, variably absorbing mutation biases that are observable in the proteome as a whole, evidenced in the numbers of prion-like proteins, the % guanidine and cytidine (GC%) in the DNA, and the proportions of poly-asparagine and poly-glutamine."

3. Tables 4-5 referenced on line 169 are absent.

4. Couple mistakes or missed references were found.
line 38. The articles demonstrating the role of Sup35 in translation termination should be mentioned:
Stansfield et al. 1995. The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae. EMBO J 14: 4365–4373.
Zhouravleva et al. 1995. Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3. EMBO J 14: 4065–4072.
line 53. The reference Alberti et al., 2009 might be added to the list of algorithms (Alberti et al. 2009. A systematic survey identifies prions and illuminates sequence features of prionogenic proteins. Cell 137: 146–58).

5. The tables in the supplementary are hard to follow because they are presented as images. Also, I'd suggest presenting protein names in a separate column, which will contain the only name of the protein but not merged UniprotID, protein name, and organism.

6.  I'd suggest adding two new columns ("Weighted correlation" and "Ascoidea rubescens excluded") with "Yes/No" values in tables 1 and 2 because it is a little bit difficult to catch the difference between 2-4 values in one cell in tables 1-2.

7. The main idea of Figure 4, as I understand, is a demonstration that lysine frequency within prion forming domains is lower than in the proteome. From this point of view, the boxplot or barplot is more informative. Also, the demonstration that this fact is specific for NQ-rich domains will increase the impact of the paper. If the lysine frequency in non-NQ-rich region is comparable to the same parameter across proteome, corresponding speculation can be made.

8. The author attached the sequences of S. cerevisiae proteins, which were analyzed. The addition of ortholog sequences can increase the interest to the paper.
Experimental design: The choice of correlation method (Pearson correlation) for calculations should be explained and it is essential to check that the data are normally distributed. Otherwise, I would advise using the Spearman correlation coefficient.
Validity of the findings: The author demonstrated that NQ-rich proteins behave as a whole proteome in terms of accumulation of asparagine and glutamine and claimed that these proteins adsorb the general trend. This observation seems to be expected, however, it has not been published yet. At the same time revealed correlations raise a new question about the specificity of this feature for NQ-rich proteins/domains only. If the same results can be obtained for a randomly chosen yeast protein the conclusion about the "absorption of general mutational trend" may be preliminary.
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: VARIABLE ABSORPTION OF MUTATIONAL TRENDS BY PRION-FORMING DOMAINS DURING SACCHAROMYCETES EVOLUTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a concise and well-written manuscript which will be of interest to, and a resource for, researchers working on prion proteins, yeast biology, intrinsically disordered proteins and computational biology more broadly. The study builds on work on prion phenomena from the author's laboratory over the past decade. The overall conclusion of the paper is that prion-forming domains in a number of yeast proteins (from species in the Saccharomycetes class) "have a spectrum of behaviour, variably absorbing mutation biases that are observable in the proteome as a whole".
*Suggestion #1: It might help, at the outset of the introduction, to have a brief sentence to highlight that Saccharomycetes is a class of fungi that includes species such as S. cerevisiae, given that yeast prions are usually (at least initially) associated first with proteins in this species.
*Suggestion #2: Would it be possible to have the subsection headings in the Results section be more descriptive, to include the essence of the findings in each subsection?
Experimental design: The methodology and analysis are clear. Data file S1 provides the prion-forming domains used in the manuscript in FASTA format.
*Suggestion #3: If possible, it might be useful to convert the formats of Tables S1/S2/S3 into text-based or Word document tables, so that the text, UniProt IDs, etc., could be easily searched.
*Suggestion #4: In Figures 1 and 2, it might help to include "Ure2p" in the x-axis labels, and, in the figure legends, to mention (and perhaps also mark on the figures) the outlier in panels A, B and D.
*Suggestion #5: Eight measures of compositional bias are stated starting from line 110. It might help to explain in a few additional sentences, why, in the context of (yeast) prion research and protein structure disorder, these measures were selected and if other measures could also be used and why they might not have been relevant to this study.
Validity of the findings: The underlying data has been provided and the conclusions and methodology link back to the original question and scope of the paper. The findings have also been presented with reference to relevant prior publications. For example, citing the reference An et al. 2016, the author states in the Introduction that "A large population of yeast-prion-like proteins emerged en masse early in Saccharomycetes evolution, as a result of mutational trends to form more polyasparagine runs, thus providing an evolutionary 'test set' from which several prion-forming domains seem to have developed". In the Results section, when discussing this theme, the author writes that "during the surge in formation of prion-like regions during Saccharomycetes evolution, the degree of N-bias in the individual prion-former Ure2p also increased in correlation with the general trend as it panned out across various sub-clades". These linkages are quite helpful. However, I think there could be some further commentary and speculation added to the Discussion section in terms of the broader context of the findings and what steps could be taken in subsequent studies (please see next section of comments).
Additional comments: In addition to the suggestions above, I would like to suggest the following three points to be considered for possible addition to the Discussion section:
*Suggestion #6: Given that there is now considerable interest in the prion-like behavior of alpha-synuclein, which is thought to be a disordered protein (perhaps notwithstanding its plasma membrane interactive domains), there might be some interest and relevance in mentioning this in the Discussion.
*Suggestion #7: Somewhat similar to the above, might there be some interest in mentioning the possibility of applying aspects of the methodology of this paper to computationally investigating polyglutamine tracts in Huntington's disease and other neurodegenerative conditions?
*Suggestion #8: Given that the aim of the manuscript is centered on prion-forming domains and disordered regions, it might be helpful to provide some speculation as to whether one could use the study of selection pressures on the sequences of these regions to understand their *structure* a bit better. Just as an example, let us briefly consider this scenario: Protein X is a prion-former and, (i) is intrinsically disordered, and (ii) is capable of forming amyloids. If we say that protein X, because of its intrinsic disorder, is "unfolded" in the cell, does this mean that *each* protein X in the cell is unfolded in a random and *unique* way and therefore different from another protein X in its structure? Or, do we mean that all protein X's in the cell are "unfolded" in the same way? I think the latter scenario is more likely, because: (i) If each protein X was different structurally from another protein X in its "random structure", then how can they undergo a homologous amyloid-forming process, a process which implies aggregation based on some new and shared acquired templating structure (this might also be true in the case of selective heterologous amyloid deposits, e.g., in the case of tau protein and Abeta)? (ii) The amino acid sequence of all protein X's in the cell are the same; and (iii) the cellular microenvironment that these proteins are exposed to is also the same. A relevant recent paper to this topic is: Shida et al., "Short disordered protein segment regulates cross-species transmission of a yeast prion", Nat Chem Biol 2020.


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

Input:
Title: VARIABLE ABSORPTION OF MUTATIONAL TRENDS BY PRION-FORMING DOMAINS DURING SACCHAROMYCETES EVOLUTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: All my comments were addressed, thank you. Two last advises.
1. Please, add the full legend on fig.4B, including all three colors, or hide the legend on the plot.
2. The square brackets in the prion names is usually writtern in general font and "+" is in superscript.
Experimental design: All my comments were addressed, thank you.
Validity of the findings: All my comments were addressed, thank you.
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: VARIABLE ABSORPTION OF MUTATIONAL TRENDS BY PRION-FORMING DOMAINS DURING SACCHAROMYCETES EVOLUTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: The suggestions in the previous review have been implemented in the revised version.
Experimental design: The suggestions in the previous review have been implemented in the revised version.
Validity of the findings: The suggestions in the previous review have been implemented in the revised version.
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: RECENT POPULATION EXPANSION OF LONGTAIL TUNA THUNNUS TONGGOL (BLEEKER, 1851) INFERRED FROM THE MITOCHONDRIAL DNA MARKERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: MS entitled "Recent population expansion of longtail tuna Thunnus tonggol (Bleeker, 1851) inferred from the mitochondrial DNA D-loop marker" tries to identify the population structure of Thunnus tonggol in Malaysian waters . The methodology followed is fine and the work needs reporting. However some more concrete  analysis like Bayesian analysis need to be done to determine the time of expansion of the population, rather than making hypothetical assumptions as in Line 263-274.
Experimental design: Experimental designs meet the general standards, though the article don't try to address the spatial and temporal variations.
Validity of the findings: Conclusions are well stated, linked to original research question.

MS concludes saying that the population is panmictic. However, Pairwise Fst values, show they are significantly different at least in 22 cases (See table 3).Author may justify this, before coming to a blanket conclusion like " panmictic".

Also improve the discussion, by considering the significant difference in pairwise Fst of populations.
Additional comments: More concrete  analysis like Bayesian analysis must be be done to determine the time of expansion of the population, rather than making hypothetical assumptions as in Line 263-274.

Significant difference in pairwise Fst should be taken care and reexamine if there is any fine scale structuring among populations?

Result of AMOVA Fst and its significant level should be given in result and explained


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: RECENT POPULATION EXPANSION OF LONGTAIL TUNA THUNNUS TONGGOL (BLEEKER, 1851) INFERRED FROM THE MITOCHONDRIAL DNA MARKERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript was well structured, however, some statements are not clear and the overall English used can be improved.

Some sentences may make reader confuse and some terms used were inappropriate.

Lines 60-61: What does "unique pattern of distribution" means? What is the relevant of distribution pattern with standing in tuna fisheries globally?

Lines 67-68: The catches is increase or decrease? (291264 tonnes in 2007; 237124 tonnes in 2016)

Line 104: What does “to establishing baseline data and stock delineation pattern” means?

Line145: interpreted?

Line 169: investigate relationship or correlation?

Line188: obtained from?

Line 228: explored or assessed?

Literature review is sufficient, however, some explanations are not clear.

Line 75: This statement is not relevant in this study.

Lines 77-87: What do authors would like to express in this paragraph by mentioning different Thunnus species population studies?

Line 91: to describe population…Regarding what?

Lines 92-95: Should rewrite this statement because not all mtDNA region are highly variable. It is also contained some conserve region as well.

Line 96: Inappropriate reference was used (Fliss et al., 2000). Please look for the mutation rate for fish not human. Mutation rate mtDNA in fish is not up to 10-17 folds higher than nuclear DNA.

Line 102: Why need to include phylogenetic relationship of T. tonggol from Taiwan waters and the northwest coast of India in this study? How this phylogenetic relationship will relate to the present study?

Tables 2 and 3:
1) What is the purpose to include the ϴs value but no explanation or discussion about it?
2) Please standardize the decimal number of values. e.g. 1 ->1.000; 0.25 -> 0.0250

Figure 1:
1) Should include the longitudes and latitudes.
2) Location of India and Taiwan population were not plotted in the map.

References:
Some references were not cited in text: lines 339, 342, 397 and 438.
Experimental design: No issue for ethical statement and field work permit. The English writing for this statement can be improved.

Identification, preservation and storage of specimen were not explained.

Analyses solely based on single mtDNA gene marker will provide limited information. More gene markers should be included to product more robust result. Single gene marker is unable to reflect the true phylogenetic relationship among the samples. In present study, the constructed tree is considered as gene tree not phylogenetic tree. Please use the term correctly. The constructed tree also should be provided to enable reader to confirm the results explained by the authors.

Additional Minimum Spanning Network construction should be conducted in which it can better explain the relationship among the haplotypes compare to the gene tree.

AMOVA result also need to be presented such as FCT value, population/group partitions and etc..

Other comments:

Line 126: fin or fin tissue?

Line 127: PCR should be written in full sentence.

Line 131: No description about Mytaq. Is that a Premixed solution?

Line 134: The purpose to visualize on agarose gel not explain.

Line 138: This paragraph explains about data editing/verification, not data analyses.

Lines 150-154: Refer comment for line 102 and experimental design.

Lines 166-167: Genetic divergence within population and between haplotypes was calculated respectively using MEGA 6.  (Results not shown)

Line 171: What is linear distance?
Validity of the findings: As mentioned, information provided by single gene markers are limited and not robust. Additional analyses as suggested is needed.

Interpretation of results not clear and still need to be improved.

Explanation of some results  and discussions are confused and will pointed out below.

Lines 190-191: The majorities (92.2%) of haplotypes are private to single locality indicating some degrees of population structured or lack of gene flow. This is contradicts to the panmictic population as claimed by the authors. Network analysis may help to clarify this issue.

Lines 199, 202: The constructed tree is need to be presented.

Line 198: What does “single cluster with unsupported clades (< 90%)” means?

Line: 203: AMOVA result is need to be presented? How about the FCT value?

Line 208: According to Table 3, some comparisons (especially comparison of BT with others [>0.05]; TG, PR..) show significant moderate FST value, which indicating existence of population structuring. Why authors claim low differentiation? Refer the comment for lines 190-191 also. Panmictic population seems like inappropriate.

Line 211: Does the average of distance 1217.6km means anything in this analysis?

Line 219: The statement is not correct based on the above comments.

Line 230: All of the nucleotide diversity values are around 0.02; ranged from low to moderate was not observed.

Line 242: Refer the comment for line 219.

Line 249: It is not uncommon that Taiwan is closely related to Malaysia populations as they are located at South China Sea as well and distinct from India's  population. However, the population from west Malaysia (KP, PR and SB) are considered part of Indian Ocean. So, if these population were mixed with the South China Sea populations, then the suggested genetic partition is seens not related to the Sunda Shelf barrier.

Line 264-274: The authors try to explain and relate the demographic pattern with the paleogeographical changes of Indo-Malay region. However, the explanation was not clear/well. What is the consequence to the demography changes when the limited population distribution occurred during lower sea level? How the submergence of Sunda shelf have fueled the population expansion?
Population expansion time estimation may clarify whether the demography changes was related to the cyclical glaciation event or not.

Line 281: The statement “deemed as a single stock unit and was found to be highly diversified” is contradicts.

Line 282: Based on what results the authors claimed that “the species’ vulnerability to extinctions may be low”?

The overall conclusion are not well stated.
Additional comments: There is still room for improvement especially in data analyses and interpretation.


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: RECENT POPULATION EXPANSION OF LONGTAIL TUNA THUNNUS TONGGOL (BLEEKER, 1851) INFERRED FROM THE MITOCHONDRIAL DNA MARKERS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This work is a valuable effort for improving the knowledge on the highly exploited species.
The amount of work to produce and analyze the dataset is evident.
I consider this manuscript properly structured and presented, but the data analysis should be improved and integrated with multiple and recent analytical methods to have comprehensive and robust results to be assessed and discussed.
The dataset is constituted by a single mtDNA marker and this limitation should be accounted for and results should be discussed with consequent awareness.
Experimental design: This work is a valuable effort for improving the knowledge on the highly exploited species.
The amount of work to produce and analyze the dataset is evident.
I consider this manuscript properly structured and presented, but the data analysis should be improved and integrated with multiple and recent analytical methods to have comprehensive and robust results to be assessed and discussed.
The dataset is constituted by a single mtDNA marker and this limitation should be accounted for and results should be discussed with consequent awareness.
Validity of the findings: This work is a valuable effort for improving the knowledge on the highly exploited species.
The amount of work to produce and analyze the dataset is evident.
I consider this manuscript properly structured and presented, but the data analysis should be improved and integrated with multiple and recent analytical methods to have comprehensive and robust results to be assessed and discussed.
The dataset is constituted by a single mtDNA marker and this limitation should be accounted for and results should be discussed with consequent awareness.
Additional comments: I attach a  pdf file where you will find more punctual remarks inserted as 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: RECENT POPULATION EXPANSION OF LONGTAIL TUNA THUNNUS TONGGOL (BLEEKER, 1851) INFERRED FROM THE MITOCHONDRIAL DNA MARKERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript seems like not ready for submission as there are still a lot of mistake, not well-prepared data (table’s caption, decimal number, low resolution of figure), weak data explanations (will be pointed out below) and not organised references. Please check in detail before submitting.

Literature review is sufficient. However, some sentences may make reader confuse and some terms used were inappropriate.

Line 66: author claimed, “decrease in its catch from 291,264 tonnes to 67 237,124 tonnes (FAO, 2018b) from 2007 to 2016” but in line 78 author claimed, “steady increases in fisheries catches over the years”. So, what author try to explain? Increasing in demand, I think.

Line 98: Inappropriate reference was used (Haag-Liautard et al., 2008). This reference is about mutation rate in Drosophila melanogaster. Please look for the mutation rate for fish.

Line 103: The term “COMPREHENSIVE” doesn’t sound suitable without including nuclear gene markers in this study.

Lines 105-111: It is not necessary to include this explanation.

Lines 116-121: This sentence need to be rewritten to make the statement clear.

Lines 122-129: Based on my understanding, the main objective of this study is to conduct population study of T. tonggol in Malaysian water and further investigate the phylogeography of T. tonggol in Indo-Pacific region. So, please rewrite this paragraph to make the objectives of this study clearer.

Line 126: Use the term “Provided by…” instead of “contributed by…”; “further investigate” instead of “expand the analysis”.
Experimental design: No issue for ethical statement and field work permit.

Research question is relevant and meaningful. However there are room to be improved in data analyses.

Line 138: “sample sizes ranged from 13 to 23” but based on Table 2, the sample size ranged from 12 to 24. And the average of 18.9 individuals per sampling location is meaningless as you can not get 18.9 individual.

Line 139: Following the identification keys as described in Lim et al., 2018, not “using fish identification books”.

Line 156: Genomics DNA extraction or isolation is more appropriate in this context.

Line 167: Any reason why 32 cycles of amplification and final hold at 4oC for D-loop while 35 cycles and final hold at 10 oC in ND5?

Line 172: Are you sure the main purpose to visualized on agarose gels is to estimate the size of DNA fragment?

Line 176: Please don’t use symbol (&) in sub-title.

Line 198-202: What does the author try to explain? Inclusion of Taiwan and India sequences in phylogenetic tree or haplotype network?

Line 199: “courtesy of researchers…”?

Line 203: Why use Tamura-Nei model?

Line 238: Why use GTR model? Why use different model for same dataset in different analyses (tree construction, Pairwaise FST and Bayesian analyses)?

Line 239: The analysis was run for 108 iterations with a pre-burn-in of 107. Are you sure? After burn-in only left 10 runs?

Please use suitable term in explanation e.g.:
Line 186: “inspected”?
Line 196: “to view”?
Line 197: “phylogenetic network”?
and much more in the whole manuscript.
Validity of the findings: Weak data interpretation.

Line 267: Table2: label (location) for first data was missing. Decimal number not consistent for all data.

Lines 273 and 289: 1) Are you sure these are the unrooted trees?
2) What is the purpose to include the outgroup in the constructed tree?
3) Why there is no bootstrap value? I’m doubt that there is no single clade having high bootstrap value.

Lines 277 and 299: 1)The constructed tree (Fig. 3) didn’t not shows that the T. tonggol from Adaman and India differentiated from the rest as the haplotypes 55, 56, 59, 4 and 8 from this region are clustered together with the others in the main clade which is polytomy. Only two haplotypes (9and 11) were in different clade and no strong bootstrap value to support.
	2) Many haplotypes were missing/ not included in the tree.
	3) What do you mean “All positions with less than 50% site coverage were eliminated”?
	4) “Molecular Phylogenetic analysis” is not suitable as only one gene was used.

Lines 280, 283 and 299: Fig 4A is haplotype network for D-loop or ND5, same for Fig 4B. The resolution of the figure is too low until the label can’t to be read.

Line 316: Pairwise Fst value for Bintulu are consider moderate as there are more than 0.05. For tables 3 and 4, number of replications is 1000 or 10000 as stated in materials and methods? The decimal number should be consistent for all data.

Line 320: Any explanation for Table 6?

Lines 355-357: Which result that you referred? Pairwise Fst or genetic distance? For pairwise Fst, it tells the gene flow between populations or population structuring but not the genetic differentiation between population. Genetic differentiation between sequences from Taiwan and Malaysia is around 2% (not lack of genetic differentiation). Please make sure you know how to interpret the pairwise Fst and genetic distance before making the statement.

A lot of the pairwise Fst value and genetic distance for Malaysia populations in Table 7 are different from Table 3. Please check.

Line 373: Based on Table 2, all Tajima’s D value for D-loop are not significant. Any explanation?

Lines 379 and 384: Based on the Bayesian skyline plot, the expansion time occurred at around 3(not 5) kya for D-loop and 2kya for ND5. Why author suggest that “a recent expansion that could reasonably have followed the cyclic climatic oscillations defined by the late Pleistocene era (~110 – 15 kya)” which was happened more earlier? (expansion time not in the Pleistocene time range).

Lines 389: Any explanation for the huge differentiation of the expansion time between Bayesian result and the calculation using tau value (5 kya; 2kya vs 55kya;323kya) although both analyses using same mutation rate. Which result are more reliable?

Lines 402-403: Fig 4A illustrated complex reticulation while Fig 4B illustrated star-like pattern. Please make sure Fig 4A is D-loop or ND5 and also for Fig4B.

Lines 423-424: This statement is not clear.

Line 434: What does “high haplotypes” means?

Lines 451-452: Any other better explanation for population structuring of BT population as both gene markers reveals nearly same result.

Line 466: This statement is contradicting to the discussion of this paragraph.

Line 471: Refer the comment for lines 379 and 384.

Lines 494-506: In the first sentence, author want to relate the changes in geological features and climate event in the past with the demographic pattern observed. However, further discussion does not relate the changes of sea level with population demographic pattern but relate to the population structuring. It is confusing.

Lines 503-506: It is farfetched by relating the short period of LGM with low population structuring. Low population structuring should be related to the ocean currents, high dispersal ability of T. tonggol and the rising of sea level which promote gene flow as discussed in the other paragraphs.

Lines 510 -515: Based on the expansion time discussed in line 488, it was happened far more earlier than the LGM. So, the expansion of T.tonggol in the present study seems not related to the rising of sea level after LGM.

Lines 519 -512: The explanation of the hypothesis was not clear. What does “intermediate and parallel genetic distance” means?
Additional comments: Week data interpretation.

Some fundamental knowledge should be improved such as how to differentiate rooted and unrooted tree and how to interpret tree; don’t confuse with genetic distances, pairwise Fst value, population structuring, population demography, etc…

Please check carefully and well prepared before submitting manuscript.

References
A lot of references are not organised and sorted accordingly.


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: RECENT POPULATION EXPANSION OF LONGTAIL TUNA THUNNUS TONGGOL (BLEEKER, 1851) INFERRED FROM THE MITOCHONDRIAL DNA MARKERS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The manuscript was well structured; Literature review is sufficient, well referenced & relevant.

Figures are relevant and high quality, however, some mistakes were found.

Abstract:

Line 28: In this study, past demographic history (population expansion) may promote gene flow between populations but this is just deduced from the demography history’s result, however, not certainly. So, the term “attributed to” is not appropriate. Low genetic differentiation may cause by many other factors such as no barrier, high dispersal ability, recent gene flow, etc… (Refer to lines 419 -432). Please revise this statement.
Line 31: AMOVA is mainly for testing whether there is obvious different grouping/partition as author stated and not for testing gene flow among study sites. Pairwise FST value is more appropriate for this statement.

Introduction:

Line 58: The status of subgenus Neothunnus is ambiguous. Any references? Please check.
Experimental design: No issue for ethical statement and field work permit.

Research question is relevant and meaningful.

Should be more careful and detail in describing methodology.

Line 140: The sampling sites can be easily divided into 4 geographical regions. Is it necessary or any reason to follow Akib et al., 2015?

Line 141: Please state how many sequences from Indian water.

Line 193: Your model selection is based on the model test and the model availability of the selected program (e.g. Beast/Arlequin). If the best model not available, use second or third best model. Why follow Kunal et al., 2014 as their dataset is different from yours.

Line 197: “Malaysian haplotypes”? Please use proper term.

Line 200: Based on the explanation in lines192 and 193, I guess you are using weights genetic distance for pairwise FST (population comparison setting in Arlequin [compute distance matrix]). So, the use of the term “phi (Ф)ST” is more appropriate instead of FST.

Line 212: Make sure you are fully understood about FST (Wright’s fixation index). FST is describes variation between individuals within populations ?? Or FST describes differentiation between population. Don’t simply links the AMOVA result in Arlequin with the Wright’s fixation index. Please refer to Arlequin ver 3.5.2’s manual section 8.2.1.2.

Line 226: How you get 315 sequences in total as sequence from Genbank is 157 plus 152 from Malaysian haplotypes?

Line 238: The mutation rate of 2% per million years for ND5 is per site or per gene?

Lines 252- 254: Population in equilibrium will shows multimodal distribution, but multimodal not necessary indicate demographic equilibrium, it can be indicating other situations as well. Same for population with expansion will show unimodal……. Be careful in writing statement.
Validity of the findings: Data presentation and interpretation need more careful and to be improved.

Line 279: The phylogenetic gene tree in Fig 2A doesn’t match with the supplementary 1. Please state clearly different type of trees produced e.g. scaled tree or not scaled tree, etc…
- Hap 218 and Hap 231 is clustered in the same clade with “All region and Hap167, 253” in supp.1, but in Fig. 2A there are in polytomy status.
- Some branches with no taxa label.
- Species name Thunnus orientalis not Iltalic.

Line 279: Some taxa with no label and species name not Italic.

Lines 282-283: The Fig.3 is very messy and hard to “read”, should be re-arranged. The color version (previous submission) of haplotype network will be better. The length of the line should be proportion to the number of mutation steps. e.g. line with one mutation should be shorter than the line with more mutations.

Line 363: The overall tau value should based on the value generated by DnaSP or Arlequin by consider all samples as one single population dataset, and should not based on the mean value as shown in Table 2.

Line 364: Different gene markers show different estimated expansion time don’t mean the expansion time was happened from 65,438 to 323,217 years before present. Please revise the statement.

Lines 364-367: Any explanation for the huge differentiation of the expansion time between the calculation using tau value and Bayesian result (e.g. ND5; 65438 [based on tau value] vs 150000 [BSP]) although both analyses using same mutation rate. Which result are more reliable?

Lines 425-426: Even though plankton bloom will enrich the food resources, but why creating an optimal spawning ground for the species since T. tonggol is not plankton feeder? This statement is confusing.

Line 437: According to the Fig. 2A, Supplementary 1 and pairwise comparisons FST (Table 3), there are also another possible break between ECIO and WCIO though the moderate pairwise FST value is not significant.

Lines 444-448: You just mentioned other tuna species (Akib et al., 2015) also have same pattern, yet no explanation why different tuna species can have the same pattern. What are the factors that cause different species having same pattern?

Line 469: It doesn’t make sense that the population keep expanding from 65438 to 32217 years ago. Please refer to the comment for line 364.

Line 470: Which result indicates that Indian region with subsequent population retention?

Line 472: Refer to the comment for line 469.

Lines 472-475: You should have better explanation or more support information if you want to explain that the cyclical glaciation during Pleistocene have contributed to the population expansion but not just possibly because of increasing climatic conformity only.
Line 476: You can not simply state that LGP has no influence. Many occurrences can be happened during LGP that help in maintaining the large population size. You need to prove or explain this statement, not just make a statement in just one sentence.
Additional comments: Data presentation and interpretation need more careful and to be improved.


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

Input:
Title: RECENT POPULATION EXPANSION OF LONGTAIL TUNA THUNNUS TONGGOL (BLEEKER, 1851) INFERRED FROM THE MITOCHONDRIAL DNA MARKERS
Review round: 4
Reviewer: 1

Output:
Basic reporting: This revision is almost ready for publication, however, a few issues need to be addressed especially on the population expansion time.
Experimental design: no comment
Validity of the findings: Figure 3: Please follow the editor team’s advice whether the color or pattern figures to be presented.

Supplementary 1: “Unscaled” ML tree shows……
The last sentence “Several haplotypes…..” Do you mean “Individuals for several haplotypes distributed in more than one regions.”?

Lines 356 and 462: It is not between 67,865 to 284,252 years. It should be 67,865 and 284,252 years ago respectively inferred by ND5 and D-loop genes.

Line 359 : For BSP, continuous expansion started from 150,000 years ago and more recent expansion around 100,000 years ago based ND5.

For expansion time, it can be summarized in this way:

- ND5 able to detect more recent expansion time around 70,000 years ago (based on tau value) and 100,000 (based on BSP).

-D-loop, 2 expansion events occurred:
  1st: around 200,000 (BSP) or 284,000 years ago (tau value)
  2nd: around 950,000 years ago (BSP)

In general, both markers able to detect population expansion occurred around 200,000 years ago (ND5, started before 150,000 years ago).
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: IMPACT OF PROXIMAL CAVITIES AND PRIMARY MOLAR ABSENCE ON SPACE IN THE DENTAL ARCHES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Use of English language is clear and unambiguous, but authors should kindly rephrase line 33 and 34 in the abstract session.

The study title is clear and reflects the content in the proposal. The title, aims and objectives form a meaningful whole

The concepts are well defined, and the study is well conceptualized

The literature review is relevant adequate and recent.
Experimental design: Authors should kindly indicate why the choice of space analysis using photographs of casts rather than direct measurement/assessment of space from the casts.
Validity of the findings: •	Authors should kindly include in limitation session that radiographic assessment/analysis of space was not done
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: IMPACT OF PROXIMAL CAVITIES AND PRIMARY MOLAR ABSENCE ON SPACE IN THE DENTAL ARCHES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Improvement is required in overall writing as the text is not aptly able to convey the message.
Experimental design: The authors have done a cross-sectional study - which seems to be done in continuation to already done a clinical trial. But clear explanation of the same is somewhat obscure.
Validity of the findings: It seems OK.
Additional comments: Overall clarity is missing in the manuscript. It seems you have done a cross-sectional study - which seems to be done in continuation to already done a clinical trial. But clear explanation of the same is somewhat obscure.


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

Input:
Title: IDENTIFICATION OF OLFACTORY GENES OF A FORENSICALLY IMPORTANT BLOW FLY, ALDRICHINA GRAHAMI (DIPTERA: CALLIPHORIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are sections (discussion and  conclusions) that are not at the expected level of english and should be reviewed.
REferences are provided however the authors often use references that discuss previous work - the authors should use the original reference in these intances - the authors need to check many of their references for this.
Experimental design: Authors should provide further context on the behaviour and ecology of this organism to more fully explain how understanding the oflactory system will make a meaningful contribution to the forensic investigations.
Validity of the findings: Discussion and conclusions is very speculative and not particularly easy to understand. More general statements should be made and the english needs to be improved

There are far too many figures in the main body of the text - authors should include some in the supplementary information and only keep the most relevant ones that are discussed in more depth.
Additional comments: Overall i think the authors need to provide more context about the organism and its behaviour rather than rehash already well known and reviewed information about insect chemosensory systems.
I have provided specific comments below - these are not comprehensive and the authors should have the manuscript edited by a native english speaker.

Abstract
What impact will having the transcriptome and genes identified have in the field of research?
Line 20 – add ‘an’ between important role

Intro
Line 62-62 – what is meant by the behaviours are related to gender in insects? What is known about behaviours with gender differences in A. grahami??
More info on the ecology and behaviours of A. grahami would provide better context for this paper. Many many papers summarise the olfactory system of insects. Consider reducing this part and increasing the info provided about the organism you are studying instead.
Line 66 – tentacles and lower lip whiskers are not insect olfactory tissues!!!
Line 68 – the collective noun for sensillum is sensilla not sensillums
Line 76-78 – this sentence is a bit clunky, consider rewording
Line 95 – remove ‘other’

Results
Line 257 – ‘9 genes’ – what genes are these?
Line 260- 271 upregulated in what? Female?
Line 270 – how did you verify the expression – perhaps state this in this sentence
Line 279 – what does ‘were screened out’ mean? This section is difficult to understand
Line 305 – 306 what does ‘relative domain for further analysis’ mean?
Line 310 – this is unclear – are you saying there are 2 orco genes in your transcriptome? This is highly unusual
Line 336-337 – this is unclear – it reads like the male specific genes are enriched in female heads – rewrite this sentence in the context of the previous sentence.
Line 338-345 – the English is a little clunky in this section – please review
Line 346 – what do the authors mean by olfactory transduction genes? Fig 11 is olfactory receptors??

Discussion
Overall discussion needs to be edited by an English speaker
Line 351 – discussion does not need to be plural
Line 353 – ‘food resources are in distant’ – grammatically not correct please revise.
Line 360 – ‘through the receptors on lymphatics’ – re word as this is incorrect
Line 364-365 – this is the first mention of spawning – how may these olfactory differences affect location of spawning?
Line 365- 370 – this is clunky – please review the grammar
Line 370 – PBPs probably – this is too strong a statement – use ‘may’ instead of probably
Line 374 – grammar – replace sensitivity with sensitive
Line 375 – why is this relevant here? Do you mean in the advanced decay stage of corpses? This whole section needs to be much clearer
Line 382 – “these genes identify odours…’ this makes no sense
Line 386 – CSPs are not a ‘recent’ discovery
Line 388 – what evidence do the authors have the CSP is ‘less expressed’? You cannot compare the expression levels between transcriptomes of other species.
Line 389-390 – there is no developmental stage data presented in this manuscript???
Line 398 – 399 – how does A. grahami sexually communicate with pheromones? Does the male produce a Pheromone? Why do the authors think highly expressed female ORs are involved in pheromone detection? Could they not be involved with selecting locations for egg laying???
Line 391 – 411 – this whole section is very speculative – how strong are the homologies between the genes? Perhaps the authors should be more conservative in suggesting functions for the particular genes and just point out important odours for the organism and the clusters of ORs that may detect them.


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

Input:
Title: IDENTIFICATION OF OLFACTORY GENES OF A FORENSICALLY IMPORTANT BLOW FLY, ALDRICHINA GRAHAMI (DIPTERA: CALLIPHORIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Basic reporting well writing and scientifically sounding, but one more round of proofreading is required checking the use of some verbs and typos and add some references (ie. Line 85: “are instead of “is” to refers to the OBPs; line 227: add reference to the procedure). In abstract, Methods section authors should explain that the used NGS and DGE to antenna, and q-PCR to compare those with other tissues. Introduction provides sufficient background, except for olfactory transduction genes (lines 35, 330, 346, Fig. 12). Authors should consider to merge Results and Discussion sections in order to avoid the repetition of findings description and also to enrich the discussion going beyond of just state that results were similar to other studies. Authors should improve the discussion providing more robust quantitative comparison.

I couldn’t access to the RAW trancriptome data using the provided link or searching by PRJNA513084 code in NCBI website.
Experimental design: In general, experimental design seems to be appropriate to the objective of the study. However, some details are required to improve this section.
Line 140: Was pork intentionally left to be infested with A. grahami? How long?
Line 141: Were adult individuals kept separately? Were they virgin or mated when antennae were collected?
Line 145: How many antennae from how many individuals did the authors collect to extract the total RNA?
Line 209: What was the criteria to choose rp-49 as reference gene?
Line 210: Did the authors collect 50 antennae per replicate? Did the authors use the same insects to extract RNA sample to perform the NGS sequencing than RNA to perform qRT-PCR?
Line 212: Considering the explanation about the procedures used to obtain the antennal cDNA, do the authors use the same procedure to obtain them from other tissues? Also is important to know if heads and legs included palpi and arolium respectively, because they are well known for having chemo-sensitive functions in many insect species.
Validity of the findings: Conclusions are too general and more details are required
Additional comments: No comments


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

Input:
Title: IDENTIFICATION OF OLFACTORY GENES OF A FORENSICALLY IMPORTANT BLOW FLY, ALDRICHINA GRAHAMI (DIPTERA: CALLIPHORIDAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: the new version of the paper has improved notoriously respect to the original one. It looks better funded, it is more pleasant to read and it is more scientifically sounding. However, still remain some issues that I think author should solve easily.
A second round of English writing checking is suggested because typos were found in the document (i.e. “conclisions” instead of “conclusions”), as well as authors have to check some reiterations of naming the same reference in a single paragraph, first as part of the sentence and then as the reference itself; i.e.: "According to the study of SSSS et al. (SSSS et al 2015)".
Figures and tables were rearranged. Many of them are now part of the supplementary information making the work more clear and concise. However I noted that some figures given in .pdf and .png format don't show similar quality. Also I suggest to improve Figures 5 and 6. Please use a more contrasting color combination in order to makes more notorious differentiation of cluster bootstrap values (such as FigS3 does).
Results are concordant with intended purpose of the work, but please note that ORCo is used to abbreviate Odorant Receptor Co-receptor instead of ORco.
As reviewer I was unable to check the raw data because link sent by authors carries to an NCBI page that note the data are still protected form public viewing. I suggest to the authors upload a version of the information to an open repository such as Figshare, just to let the editor and reviewers to check the information.
Experimental design: No comment
Validity of the findings: In discussion section: Please, beware about “suggest” that some transcripts could detect or could be sensitive to certain chemical compound just because they clustered together with a given reported sequence in a topological analysis. First of all, authors selected and cured an arbitrary group of sequences coming from certain phylogenetically related species before to perform that analysis. In second place, the actual expression and translation of some olfactory and gustatory protein is very dynamic, and the different type of proteins participate as part of a chain of events where OBPs are probably the less selective. Moreover, rearing conditions (food) didn’t emulate field condition (corpse) , so further studies are a must before to suggest transcript-chemical
Additional comments: I have to note that I tried to check the document in two different computers, having the same problems to match the line numbers listed in the rebuttal letter with the line numbers in the version of the document containing the tracked-changes. I also noted that .docx missed many line numbers across the document. Probably it was me, but unfortunately that situation made the reviewing task much more complicated in order to check the modifications and review the entire work.


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

Input:
Title: SLUMBER IN A CELL: HONEYCOMB USED BY HONEY BEES FOR FOOD, BROOD, HEATING… AND SLEEPING
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study categorized the behavior of bees inside comb cells by using a specially designed observation hive that gave a full view of the cell interior. The central findings are that bees often sleep in comb cells, and that this behavior can be reliably detected by inspecting the overall motion and ventilation pattern of the rear end of the bee, which is readily observable in a conventional hive. These findings are of interest to students of bee behavior and sleep, and the methods and data are sound, to the degree that I can evaluate them. However, this evaluation is limited, because the organization and presentation of the work make it difficult to follow. I suggest a major reorganization of the text to better explain the study.

The introduction should start with the central topic of the study, sleep in honeybees, rather than a discussion of comb and hive design. Instead, sleep and the behavior of bees in cells is only introduced in the second paragraph.

The Materials and Methods section needs a plain statement, early on, of the basic design of the experiment. My take on the design, which may be inaccurate in places, is as follows. A small colony of bees was set up in an observation hive that permitted an interior view of some cells. Over a period of two days, the hive was video recorded and simultaneously imaged with a thermal camera. The videos were then scanned and the behavior of bees in the visible cells was classified into one of four categories, based on a variety of criteria. These data were taken as an accurate description of the bees’ behavior. Then, naïve viewers looked at a subset of the videos, which had been masked so that only the posteriors of the bees were visible, as in a normal hive. They classified the bees into the same four categories, and their performance was compared to the accurate ground truth from the interior views.

There are a number of methodological points that I could not determine from the text. Why were some bees marked? Were only marked bees subjected to behavioral categorization? How long was the hive recorded? How were bees selected for behavioral categorization? Was every visible bee observed, or only a subset? Was the hive repeatedly scanned, or were focal areas watched continuously? Were the behaviors observed and classified only after the fact, from videos, or were they watched live and the classifications recorded? The latter is suggested by a reference to “announcing a bee’s behavior” (line 167), but this is not explicitly described anywhere. In short, there needs to be a straightforward and complete description of how the study was conducted.

The section on behaviors of interest would be clearer if it systematically went through the four behavioral categories and explained exactly what observations led to putting a bee in each category.

The statistical analysis is a bit hard to follow, because the nature of the sampling regime is not clear, as noted above. What is the meaning of the “surveys” mentioned at line 240? What exactly is an event? Over what sample were mean percentages of bees in each behavior calculated?

Other points:
Line 181: What is JWatcher and how was it used to record ventilatory rates?
Line 254: Why were only 11 bees examined for ventilatory signatures of sleep?
Line 261: What is an event separation?
Figure 7b: What is the difference between the upper and lower plots? What exactly are the events whose separations are plotted? The y-axis would be a bit clearer scaled in seconds, rather than milliseconds.
Experimental design: The description of how the temperature of a bee’s surroundings was measured is not clear. Wouldn’t a circle with a radius of one bee length, centered on the bee’s thorax, include the entire body of the bee? This would  confound her temperature with that of her surroundings.

Lines 268-285: This analysis of temperature differences among behavioral categories is circular, is it not? Temperature was the means by which bees were classified as heaters, so it is not informative to then do a statistical test showing that their temperature is different from the others.
Validity of the findings: No comment
Additional comments: No comment


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

Input:
Title: SLUMBER IN A CELL: HONEYCOMB USED BY HONEY BEES FOR FOOD, BROOD, HEATING… AND SLEEPING
Review round: 1
Reviewer: 2

Output:
Basic reporting: General: The authors used both thermal imaging and detailed video analyses to observe the behavior o bees concealed inside their comb cells, they used vertical comb setting (Lindauer hive) to see what is happening inside the cells. This enabled to compare how the bee behavior is seen in normal view, which reveals only the tip of the bee abdomen, with the actual behavior of the bee inside the cell. The finding show that the frequency of abdominal ventilation movements seen from outside can be used to identify the bee behavior inside the cell. Heating brood cells or sleeping were identified with high level of certainty. The identification of feeding and cleaning behaviors was less accurate without seeing the cell contents. The study shows valuable, unique and beautiful videos and thermal imaging of the different behaviors of honey bees inside cells. The findings of this study will help future investigations of bees’ behavior inside cells, specifically sleep. The Introduction is interesting, relevant and well-written. The hypothesis is well formulated and explained.
Figures and tables:
1.	The resolution of the figures is too low so impossible to zoom in, specifically on the pulses in fig. 6.
2.	The figures showing still images from the movies are redundant, given the movies. The figure annotations can be adapted and added to the beautiful movies, which lack legends and annotations.
3.	Fig. 5: the ticks on the Time axis are distributed equally, but the time gap between the ticks varies.
4.	Fig. 7: the legends are not consistently phrased: sometimes they describe the findings (here) and sometimes describe the plot details (e.g., fig. 6, 8). A: graph should have more ticks and better resolution to demonstrate the 10sc threshold. Please explain the plots in (B) in detail.
5.	Table 1: please increase the spaces between the table lines
Discussion: There are some redundant repetitions on the data shown in Results section; e.g., L. 301, L.309, L.323 etc.
Experimental design: The methodology is not sufficiently explained and some statistics is missing:
Major:
Materials and Methods
1.	The description of the experimental design is not sufficiently well organized. The descriptions of behavioral recordings, thermal measurements and abdominal pulse measurements are mixed. Only after looking at the data sheets, it becomes clearer that these are 3 sets of recordings (is that correct?).
2.	It is ambiguous whether thermal measurements were used to validate every behavioral bout (e.g., L. 170-171, L. 277-278).
3.	The methodology for scoring behaviors is unclear- was the hole hive scan sampled?
Results:
4.	L. 240-244: If the data was obtained by scan sampling, one must make sure that sampling time was equal between day and night. The duration of the behavioral recordings is not reported. According to Fig. 5, recording times varied (6-20 minutes). I suggest  to correct the analysis such that  day/night sampling duration is equal to prevent bias.
5.	L.250-251: there are no statistics backing the claim that ventilatory patterns differed. One could use contingency table comparing event separations above and below 10s between the behaviors.
6.	268-270: the claim is not supported with statistics. The authors could test if the Tdiff is significantly different from zero in each behavior, although the sample sizes are extremely low. In anyway, claims it should be backed with statistics.
Minor:
Materials and Methods
1.	Room temperature is not reported- may affect  the frequency of heating behavior,  for example.
2.	How were observations assigned to night and day? According to sunrise/set times?
3.	The age of the focal bees is not reported. This can affect the distribution of observed behaviors because of the age related division of labor in honey bees.
4.	L. 164-165: claim there is no data on brood tending, but ‘feeding’ definitely qualifies as brood tending.
5.	L.195: to which analyses do the authors refer? Only after comparing the sample sizes, it is possible to understand that it is the abdominal pulses’ analysis.
6.	214-216: results of this analysis are absent.
7.	The terms ‘abdominal pumping’ and ‘abdominal pulses’ are used alternately, causing confusion. Please use a consistent term.
Results
8.    270-271: can you please clarify what does the claim “Tdiff was only statistically significant in heating bees…” mean? Tdiff significantly differed from zero?
9.	272-275: correction for multiple comparisons is required.
10.	294-296: the analysis is not clear: what is reported? Is it the percentage of the most common mistake for each behavior? If so, why should the percentages sum to ~100%?
Validity of the findings: Major:
1. Sample sizes for the thermal measurements and for abdominal ventilation measurements were extremely small- with just 2-9 or 2-11 events per behavioral category, respectively (fig. 6 & 8),. There were no repetitions on the experiment with more colonies. It is hard to reach concrete conclusions based on this data set. For example, the videos show that cleaning bees have high thorax temperature but the analysis shows no difference between cleaning and sleeping bees, is this conclusion valid considering the small sample size? Feeding behavior was characterized based only on 2 behavior bouts. This must be addressed.
2. The only criteria for distinguishing sleep and heating is abdominal ventilation frequency. Therefore, showing differences in that measure between the 2 behaviors is slightly tautological. Please address this point.
3. 216-217: This is not clear: why compare the distributions? One would compare the frequency of each behavior during night and day (using binomial test for example), assuming that the sampling duration was even during night and day.
4. L.319-325: the conclusion that “sleeping was not circadian” does not follow from the data.  This study did not monitor the circadian rhythms of individual bees; it monitored what happens inside given cells. Individual bees may still have a circadian rhythm.  For the same reason, the percentage of sleep occurrence in cells cannot be compared with the time an individual spends sleeping.
5. L.342-352: I agree with the authors on the limitation- The significance of the proportions reported for each behavior is questionable. I think the authors should provide explicit estimation on the direction of expected biases- for example, more empty cells/young bees result in higher frequency of sleep and cleaning. Can we really assume that the colony developed natural demography and division of labor after such short period of time (the experiment lasted only 4 days, L. 146)?
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: SLUMBER IN A CELL: HONEYCOMB USED BY HONEY BEES FOR FOOD, BROOD, HEATING… AND SLEEPING
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: The methods are generally described well and in detail, but a few details should be added:

- Description of the overall organisation of the video/thermal image recordinging sessions over the two "data collection" days (23/24 August): what triggered the start/end/duration of a recording session, what were the rules for switching from the overall view of the entire hive to the close-up record of individual cells/bees and vice versa; how were long cell visits (longer than a continuous recording session) handled? Lines 165-166: were there also record periods when the author was NOT present and did not announce the behaviour?

- Use of the JWatcher software (line 181): please specify how this software was used to record the ventilatory pulses, e.g. via image analysis or via a human observer manually pressing a key when ventilation occurred, and what were the limititations of this method (shortest time between two pulses that could be recored)?
Validity of the findings: No comment
Additional comments: Other suggestions for improvement (not critical):

Line 294: It may be interesting to re-examine the 8.7% misidentification of heating/sleeping and the 18.5% misidentification of cleaning/heating to find out why they were not classified correctly by all persons. This may improve the authors' suggested proxy method and help avoiding/reducing wrong identifications in the future when using this method.

Table 1, category "cleaning": the authors should reconsider if the observed bees that were assigned to this category really  performed only "cleaning" work or also construction work, i.e. massive manipulation of the wax comb: The appearance of cell walls in movies 1-4 suggest that some cell walls apparently have been firmly attached to the front pane by the worker bees. On the other hand, movies 1, 2 and 4 and also the thermographic videos of the entire hive show many of the exposed cells with their cell walls reduced to half their normal length/depth, or less. E.g., in movie 1 the bee in the centre of the image has fully entered a cell but its entire abdomen is outside the cell due to the reduced cell length. This implies that many "construction workers" must have been visiting these cells.  Also in movie 1, the cell visitor in the bottom part of the right comb is clearly chewing pieces of wax off the cell wall and relocating them (e.g., during the first 25 s of this record, again on 8:32:57 PM, 8:33:04 PM, 8:33:31PM - 8:33:48PM of the original time in the image). Also the bee on the left side (third cell from bottom, 8:33:07 - 8:33:26 PM) is clearly chewing wax, not simply cleaning the cell.

Minor corrections (sorted by order of appearance in the text; no priorities):

Line 97: The citation of Flanigan 1973 seems to be missing "et al."

Lines 113-114: Kleinhenz et al., 2003, are cited correctly for observations of ventilation of bees inside cells. However, the mention of ventilatory pumping of bees outside cells should rather cite Bujok et al., 2002 (Bujok B, Kleinhenz M, Fuchs S, Tautz J: "Hot spots in the beehive", Naturwissenschaften 89: 299-301).

Line 279: the citation "Kleinhenz, 2003" seems to be missing "et al."

Lines 329-331: The authors claim that the publication of Kleinhenz et al. (2003) "did not report timing or durations of in-cell rest (or sleep)" although the observation time is stated and Table 7 in that publication provides durations of rest inside cells ranging from 4.8 to 14.5 min (5 resting bees).
Figure 4 (caption): According to the caption, the (correct) answers on the partially obscured behaviours (Movies 5-8) are included. However, I am unable to find these answers in the videos despite watching them until they end.

General comments/suggestions/clarifications (sorted by order of appearance in the text; no priorities):

Lines 115-117: The authors suggest that ventilatory activity may be a more suitable indicator of (honeybee) sleep under natural or close to natural conditions because the other indicator - antennal movement - is difficult or impossible to gauge in a nest. In this context, and to further support the authors' proposal, it may be worth mentioning that antennal immobility as indicator of sleeping bees may even be misleading: This is because brood-incubating bees on the comb surface, which may be mistaken as sleeping bees due to the absence of large body movements, also keep their antennae nearly immobile on the comb surface and only slow antenna movements may occur (Bujok et al. 2002, details see previous comment).

Lines 136-137, 350-352: the removal of cell contents to make the exposed cells accessible to cell visitors may present an additional bias  (in this case: on cell cleaning behaviour) that is not discussed/mentioned in lines 350-352.

Lines 137-138: if available, it would be interesting to know the approximate numbers of these different cell  types/contents on the comb pieces, and also the contents of those cells in the 2nd row (behind/adjacent to the exposed cells that were used to study cell visiting bees).

Lines 140-141: if available, an estimation of the error of thermographic temperature readings due to the colour marking, as determined in the preliminary tests, should be given.

Line 148: if available, the room temperature during the observation periods should be indicated. If standard temperature readings from inside the hive exist (e.g., air temperature measured via thermocouples or similar), those would also be interesting to know.

Line 149: Please add if there any thermal insulation covering the hive outside the recording periods

Line 167-168: Assuming that the color mark on the bees cannot be read from the greyscale infrared video nor from the thermographic video: how was the color mark identified (vocal record by the author when present during the recording?) Was the behaviour of unmarked bees also evaluated?

Line 178: The "surrounding" temperature was calculated as a  mean from "the surface temperature of anything" "within a circle (in the thermographic image) with radius of one bee's body length from middle point of thorax", "including a portion of the focal bee herself". This sounds as if the focal bee was (nearly) completely within the measurement circle... An image should be provided to illustrate where exactly the "surrounding" temperature was measured and how much the focal bee itself (or even other bees in neighbouring cells, if those were visited at the same time?) contributed to this measurement. Also, a statement may be necessary to explain that the camera settings were adapted for the bee thorax, not for, e.g., wax.

Line 179-180: The measurement circle for the "surrounding" temperature includes "a portion of the focal bee"(see previous comment) but is used to calculate temperature differences between the focal bee and the "surrounding" temperatue. This implies a systemic mistake that may lead to smaller temperature differences between the bees and the surrounding. The size of this error depends on the proportion of the bee within the designated circle area (see comment on line 178) and on the bees' temperature. Assuming that bees at rest/sleep have a temperature at or near ambient, this mistake would be small. In case of bees with elevated temperature (in particular, if the thorax temperature is actively raised above ambient temperature and if the thorax lies within the circle for measurement of "surrounding temperature") the calculated temperature difference would be smaller than it actually is. This should be considered by the authors.

Line 203: please specify whether the headlamp was a cold light source or one using heat producing bulbs, and if any interference of the lamp with the thermographic measurements may be expected (e.g., reflection of thermal radiation from the lamp on the plastic film covering the hive).
Line 205: please provide the source for the given accuracy, e.g. general specification by the manufacturer (camera manual), calibration certificate before start of the work, or own comparative measurements on dead bees.

Line 210: please specify how the spoken information in the audio record (Digital Voice Recorder) was synchronised with the video records (e.g., by speaking the video time index before identifying a bee and its behaviour)?

Line 255 and 260: should it read as "for event separations < 10 s", or why is there another limit value ("< 1 s") to filter certain ventilation events? This limit has not been introduced before.
Line 311: it is not clear what "the average heating bee" is.

Figure 9a: there seems to be a lot of noise or wrong calibrated pixels in the close-up thermal image (and in movie 9), making it unlikely to meet the given accuracy of 1°C or 1% (line 205). Any explanation?

Supplementary material (movies): Data collection took place on 23/24 Augut but movie 3 dates 22 August and movie 10 and 12 date 20 August. Auxiliary still images and videos for demonstration purposes only (not used to collect data) should be marked as such.


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: SLUMBER IN A CELL: HONEYCOMB USED BY HONEY BEES FOR FOOD, BROOD, HEATING… AND SLEEPING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The language and graphics are clear, correct, and thorough.
Experimental design: The research question is compelling and the study's design well-suited to answer it.
Validity of the findings: The conclusions are well-grounded in the data, and the authors are candid about limitations
Additional comments: The revised version of this manuscript is greatly improved and makes for a very interesting contribution about sleep in bees and about how it can be effectively studied. The experimental logic and methodology are now quite clear. I have only a few minor comments and suggestions, by line number.

177-179: Was the glass of the hive replaced with giftwrap only for the thermal recordings, or was it in place for the whole experiment? I would think that going back and forth would damage the comb attached to the glass.

351: What (roughly) were the magnitudes of the giftwrap correction factors?

427-439: The authors give a good measure for the sensitivity of their behavioral classifications: the proportion of true positives: e.g., cases of sleeping that were correctly identified as sleeping. For completeness, it would also be good to give a measure of specificity: i.e., the number of true negatives. For example, the authors could report 1 – p, where p is the proportion of all non-sleeping behaviors that were incorrectly identified as sleeping. They could do this for all four behaviors, or just for sleeping, since that is the main behavior of interest. If the measure I suggest seems a bit convoluted, they could also just report p.


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: SLUMBER IN A CELL: HONEYCOMB USED BY HONEY BEES FOR FOOD, BROOD, HEATING… AND SLEEPING
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors did a great job addressing all comments.
Experimental design: No further comments
Validity of the findings: No further comments
Additional comments: No further comments


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

Input:
Title: THE BRAIN-DERIVED NEUROTROPHIC FACTOR VAL66MET POLYMORPHISM INCREASES SEGREGATION OF STRUCTURAL CORRELATION NETWORKS IN HEALTHY ADULT BRAINS
Review round: 1
Reviewer: 1

Output:
Basic reporting: N/A
Experimental design: N/A
Validity of the findings: N/A
Additional comments: In this study, the authors created the brain-structural correlation network on each of two groups of samples with different BDNF val66met genotype followed by comparing their network-level patterns. As a result, they identified the average local clustering coefficient of the BDNF Met carrier's network was significantly higher than the Val homo group, and some other network measurements didn't show a significant difference.

Overall this is an interesting work. The writing is clear and organized. However, some major considerations need to be taken into account.

Major:
1. I am very confused about what's the biological meaning underlying such a structure co-variance network. Moreover, if there are two brain regions, and the correlation coefficient of their structural features is different between the two groups, then what's the represented potential biological meaning? It would be more straightforward and interesting to directly compare the structure difference in the individual region between the two genotype groups.

2. The authors treated the positive and negative correlations equally using absolute value. Back to my first question, if we are not sure the biological meaning of such a correlation between two regions, it's hard to figure out what's a better way to treat the sign of the correlation.

3. A little curious that is there any difference between Met/Val and Met/Met groups? It seems that currently, the authors treated them as the same group, by which the variance between them was ignored.

4. The sample sizes of the two groups are way more different (16 vs. 33). Some of the comparisons seemed not really fair, such as "a grid pattern was more clearly perceived in the Met carrier heat map than in the Val homozygote heat map."

5. A static power analysis or discussion was necessary, otherwise, the correlations, as well as the comparison on that, were not convinced me to the conclusions.

6. The authors failed to provide clear biological interpretations of their computational results. For example, they identified " Val homozygote networks consisted of weak connections whereas the Met carrier networks had more connections with middle strength.", then, what can we learn from this finding to explore the Met mutation influenced brain function?

7. The most important issue of the paper is that all the findings were generated from a small data set without any validations using other independent resources. Meanwhile, the data has a high variance on gender and age. There is limited information on other factors that could potentially influence the comparison. Consequently, from which the conclusions raised were not fully convinced me.

Minor:
1. The font of some figures is too small.

2. For figure.2, it's really confused to include positive and negative correlations in the same plot. It's challenging to understand what the heatmap wanted to introduce.

3.  For figure 3, I didn't find any obvious difference between those two plots.


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 BRAIN-DERIVED NEUROTROPHIC FACTOR VAL66MET POLYMORPHISM INCREASES SEGREGATION OF STRUCTURAL CORRELATION NETWORKS IN HEALTHY ADULT BRAINS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors here present structural covariance networks derived from inter-regional cortical thickness correlations, specifically focusing on differences between BDNF
valine (Val) homozygotes and methionine (Met) carriers. The authors found Met carriers’ structural covariance networks have denser connections with neighbors and are more distant from random networks than Val homozygotes’ networks.

The author has made an interesting research, but the significance of conducting this research should more clear.

1. The English language should be improved to ensure that an international audience can clearly understand your text. Some sentences are hard to understand. A careful proofread would be beneficial.

2. Abstract:
Line 46: Should the fourth part of the abstract be a ‘Conclusion’ or a ‘Discussion’? Please check the abstract again.
Line 50: ‘This could be the result of deficits in the BDNF Met carriers in axonal maintenance or synaptic revision’. This sentence is inappropriate here, the article did not conduct further research on this. Delete it would be better.
3. Introduction:
In the third paragraph of this part, the author lists many examples, which should be summarized to show the relationship with the current study would be better.
Line 94-95: Please provide relevant references to support ‘…. brain network will be important for understanding the neural basis of human brain function and disease…..’
Line 105-107: ‘If structure……brain structural networks’, the expression of this sentence is too absolute.
The introduction should be more concise.
4. The title ‘Methods’ should be ‘Materials & Methods’ according to the Standard Sections of ‘Peer J’.
Line 146: A single sentence into a paragraph is not good.
In the section of ‘MRI Acquisition’: Even all the subjects are the doctors with no health issues. Only one sequence, 3D-FSPGR, is collected, and it is not sensitive to the find of brain structure abnormal. I want to know whether there are any other sequences were acquired, such as T2WI and FLAIR, it can sensitively find brain lesions, which is more helpful for the subjects’ inclusion. You know, structure changes in the brain can have a potential impact on the results of the study. Furthermore, the detail of exclusion criteria do not mention in the study.
Line 183: what’s the mean for ‘….0.47kakeru×0.47×…..’
Line 222: double ‘(Váša et al., 2018)’ ?
Line 228: ‘using 31 subjects’ brains included in the Met carrier group and the 16 subjects’ brains included in the Val homozygote group respectively’, so, what the other 2 subjects ? (49 participants).
Line 310: the number of nonparametric permutation test repetitions was 2000, why not 5000 or 1000?  Please give an explanation or give the supporting literature.
5. Results: Line 355: ‘p-value ≥ 0.05’ , but in the abstract was ‘p values ≧ 0.05’, The expression of symbols should be unified, please check the whole text.

It would be better for the more concise section of post-processing methods and results

6. Discussion: More in-depth discussion regarding to current findings would be better.

7. Conclusion:
It would be better to delete the sentence ‘This might be the result of deficits in axonal maintenance or synaptic revision.’ There is no relevant research in the current study. Rewrite the sentence ‘Understanding the effect of the BDNF Val66Met SNP on brain structure will help us to understand human intelligence and disease.’ It is too absolute.

Overall, the authors present interesting results with Met carriers’ structural covariance networks have denser connections with neighbors and are more distant from random networks than Val homozygotes’ networks. However, the authors need to better explain the significance of conducting this research.