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: COMPARATIVE PROTEOMIC ANALYSIS OF PEPPER (CAPSICUM ANNUUM L.) SEEDLINGS UNDER SELENIUM STRESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled "Comparative Proteomic Analysis of Pepper (Capsicum
annuum L.) Seedlings under Selenium Stress" by Chenghao et al. studies the regulated protein expression in Selenium stress in pepper plant. Overall the manuscript is satisfactory and well referenced & relevant to the concept.
However, I had some minor comments which I  mentioned in the next section, after that the manuscript is acceptable for publication.
Experimental design: Satisfactory.
Validity of the findings: Satisfactory.
Additional comments: In table 1, what is the cut-off ratio set up by author to include the peptide candidate in the table?

Did author got any percent coverage (% coverage) of peptide and can highlight the peptide sequences in full length protein sequence that got in Mass spec for the DEP that are highly regulated say 2 times the control.

Did author see any change in band pattern of total protein run on SDS-PAGE and probed by silver staining?

Background and borders of figures are unusual. Please keep it in canonical white background.


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: COMPARATIVE PROTEOMIC ANALYSIS OF PEPPER (CAPSICUM ANNUUM L.) SEEDLINGS UNDER SELENIUM STRESS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well written. There are no grammatical errors. However, the authors should carefully peruse the text to ensure that all Latin/scientific names are italicized.

Please check all the figure legends. the ones for Figure 2 and 3 are incomplete.

Also  the text in the figures is unreadable unless zoomed in considerably. If possible please increase the resolution of the figures.
Experimental design: Experimental design is well documented.
Validity of the findings: The authors discuss their findings in great detail. However, the discussion is limited as each type of DEP is discussed separately. The manuscript will have more impact if the authors could include a more global view of the processes in the plant at the end of their discussion.
Additional comments: This manuscript is very well written and shows showcases a well organized study into stress response in plants. I will be very interested to see further studies based on your current endeavor in the pepper plant and comparisons with other plants undergoing similar stress.


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: COMPARATIVE PROTEOMIC ANALYSIS OF PEPPER (CAPSICUM ANNUUM L.) SEEDLINGS UNDER SELENIUM STRESS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Clear and unambiguous
Experimental design: Original primary research within Aims and Scope of the journal.
Validity of the findings: Conclusions are well stated, linked to original research question & limited to supporting results
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: COMPARATIVE PROTEOMIC ANALYSIS OF PEPPER (CAPSICUM ANNUUM L.) SEEDLINGS UNDER SELENIUM STRESS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Manuscript has improved after revision. All questions have been answered satisfactorily by the authors.
Experimental design: Satisfactory
Validity of the findings: Satisfactory
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: COMPARATIVE PROTEOMIC ANALYSIS OF PEPPER (CAPSICUM ANNUUM L.) SEEDLINGS UNDER SELENIUM STRESS
Review round: 2
Reviewer: 3

Output:
Basic reporting: N/A
Experimental design: N/A
Validity of the findings: N/A
Additional comments: The authors have satisfactorily answered the issues raised by me in the last review.


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: COMPARATIVE PROTEOMIC ANALYSIS OF PEPPER (CAPSICUM ANNUUM L.) SEEDLINGS UNDER SELENIUM STRESS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The manuscript has significantly improved after revision. The authors had answered all the questions.
Experimental design: Valid
Validity of the findings: No Comments
Additional comments: The manuscript has more speculative/predictive datasets of MS identified proteins which is not of much information rather than jargon. However, it may be accepted for publication.


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

Input:
Title: COMPARATIVE PROTEOMIC ANALYSIS OF PEPPER (CAPSICUM ANNUUM L.) SEEDLINGS UNDER SELENIUM STRESS
Review round: 3
Reviewer: 2

Output:
Basic reporting: The authors have addressed my concerns from the previous reviews
Experimental design: The authors have addressed my concerns from the previous reviews
Validity of the findings: The authors have addressed my concerns from the previous reviews
Additional comments: The authors have addressed my concerns from the previous reviews


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: INTRA-INDIVIDUAL HETEROPLASMY IN THE GENTIANA TONGOLENSIS PLASTID GENOME (GENTIANACEAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: Professional English was used in the manuscript. sufficient background of the sytudy was also well desceibed. Professional article structure, figures and  tables were aslo well provided. All data were submited to  GenBank. And results have been discussed and analyzed.
Experimental design: The experiment of manuscript is well designed   based on a interst research question.  Methods described with sufficient detail & information to replicate.
Validity of the findings: The manuscript reported intra-individual plastid genome heterozygosity in Gentiana tongolensis, which will offer a new view for botanists to reconsider the heredity and evolution of chloroplast and be cautious with using chloroplast as genetic markers, especially in Gentiana.
Additional comments: The manuscript reported intra-individual plastid genome heterozygosity in Gentiana tongolensis, which will offer a new view for botanists to reconsider the heredity and evolution of chloroplast and be cautious with using chloroplast as genetic markers, especially in Gentiana.  As the first report about palstome heterozygosity in plants, it meet the aims and Scope of the journal, which should be published.  Nonetheless, authors need to make some revisions to inprove the manuscript. I marked  in the PDF 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: INTRA-INDIVIDUAL HETEROPLASMY IN THE GENTIANA TONGOLENSIS PLASTID GENOME (GENTIANACEAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Sun and colleagues examine the chloroplast of the angiosperm Gentiana tongolensis, they find evidence that individuals in the species exhibit chloroplast heteroplasmy. The authors should be commended for the work and thought they put in to this study.

Overall comments:

I had difficulty following a number of different aspects of the study due to grammatical issues and potentially when certain terms were used. Having a thorough read through for grammar would greatly help improve the manuscript.

The paper seems to focus on this being the first report of plastome intra-individual heterozygosity, but throughout the paper it is mentioned that this has been reported before. If it’s the first report, then that’s great it would be a new phenomenon found in plants. If this is not the first report and confirmatory findings, that’s great too. This study builds off of previous work on the subject and even if it is not the first to break new ground it does not mean the finding is not worth publishing.

Abstract
I believe the first sentence is a bit strong on this “Chloroplast is inherited strictly from the female parent, exhibiting haploid nature in most angiosperms”, there are limited examples of paternal inheritance. Saying something like “often” or “typically” rather than “strictly” would be better.
Example of non maternal:
McCauley, D. E., A. K. Sundby, M. F. Bailey MF, M E. Welch. 2007. Inheritance of chloroplast DNA is not strictly maternal in Silene vulgaris (Caryophyllaceae): evidence from experimental crosses and natural populations. American Journal of Botany 94: 1333–1337.
I believe intra-individual heterozygosity, or heteroplasmy has been documented a number of times so this is not the first. “This is the first report about intra- individual plastid genome heterozygosity in plants, and the cause is unclear from present data,  although biparental plastid inheritance seems to be most likely.”
Example of within individual heteroplasmy:

Hensen, A.K., L. K. Escobar, L. E. Gilbert, and R. K. Jansen. 2007. Paternal, maternal, and bipaternal inheritance of the chloroplast genome in Passiflora (Passifloraceae): implications for phylogenetic studies. American Journal of Botany 94: 42–46.

Introduction

I don’t think a citation is necessary in the first sentence and if a citation is used it would preferably be the first to describe the value of the chloroplast.

At line 48 you get into the crux of the study and make a less definitive statement than in the abstract, this would be a good place to put some citations regarding the rare instances of intra-individual heterozygosity.

At line 73 the claim of this being the first report is made again but I don’t think that’s true. The authors are likely better read on the subject but it seems as though other studies have reported this. The authors mention structural heteroplasmy at line 47 and I’m not sure how you could get structural heteroplasmy without having an ancestral population that exhibited intra-individual heterozygosity.

Results

Line 143-144: I’m not really following why it would not be hard? Or why it was hard?

At line 178 is there a reason these could not be supplementary material?

Discussion

I’m a bit confused as to lines 181-182. These state that “intra-individual heterozygosity has been reported” but previously the authors have stated this is the first study to report it.
Line 193: It is stated that biparental inheritance is rare (that seems to contradict previous statements).
Line 200: It is stated that this is a relatively widespread trait (This also seems to contradict previous statements)
Experimental design: Line 96: I could be missing something but I do not see the results of OGdraw or a plastome map?

Line 126: What does it mean by aligned and edited? Which aligner from Genious was used? How were these edited?

Line 128: Which model of evolution was selected as the best?
Validity of the findings: no comment
Additional comments: Hopefully these comments are helpful and can be of assistance with your manuscript. Good luck as it continues forward!


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: INTRA-INDIVIDUAL HETEROPLASMY IN THE GENTIANA TONGOLENSIS PLASTID GENOME (GENTIANACEAE)
Review round: 1
Reviewer: 3

Output:
Basic reporting: This article does not meet several of the criteria established by PeerJ for basic reporting. Below, I identify the areas that need attention, along with suggested improvements.

1. The language used throughout the manuscript is not clear, unambiguous and professional English. There are also many clear typos. Here are a few examples: “duo” instead of “due” on line 58; “palstomes” instead of “plastomes” on line 68; “palstome” instead of “plastome” again on line 73. Furthermore, there should be more care given to the usage of proper scientific terms. For example “heteroplasmy” is often incorrectly used to describe genetic variation among species or populations (lines 47, 180). Its definition is genetic variation among organellar genomes within a cell or within an individual. The authors apply this term correctly when characterizing their intra-individual genetic variation in plastomes, but not when describing variation within populations or species. In lines 154 – 155, it would be better to use the terms biallelic, triallelic, and quadriallelic sites. In lines 160-161, they describe these as the 1st, 2nd and 3rd codon positions. To solve these problems, I recommend that the authors have their manuscript edited by a scientist who is a native English speaker or a professional scientific editing service.

2. The authors have cited the appropriate literature in most cases, but are still missing a few relevant citations. For example, the sentence in lines 46-48 is missing a citation. The authors cite the literature concerning the transfer of individual chloroplast genes to the nucleus in various plant species in lines 186 - 188, but ignore the vast body of work showing that integrations of the plastid genome into the nucleus can be quite large and contain multiple genes and intergenic sequences (Michalovova et al. (2013) Heredity; Huang et al. (2005) Plant Physiology, and Noutsos (2005) Genome Research). This work is especially relevant to the major finding in this manuscript (which I will discuss in a later section of this review). Finally, in line 135, they say that their plastid genome assembly “constituted a closed circular molecule”. A circular-mapping assembly does not indicate anything about the linearity or circularity of the molecules themselves, as circularly permuted linear molecules or head-to-tail linear concatemers can also produce a circular map and genome-sized circular molecules are rarely, if ever, observed (see Bendich, 2004, Plant Cell). This is actually a pretty common misconception in the field of organelle DNA research and I encourage the authors to note this as a way of correcting this misconception.

3. The structure of the article is professional and appropriate. The authors share the Genbank references for their plastome assembly and various PCR clones, but they do not indicate that they have deposited the raw Illumina read data in the short read archive or any public repository.

4. The article is self-contained, but there are some inconsistencies in the reporting. In the Abstract and Introduction (Lines 32-33 and 73), the authors claim that their study is the first report of heteroplasmy (intra-individual plastome variation). However, they later say in the Discussion that “rare intra-individual heterozygosity has been reported (Medicago sativa, Fitter et al., 1996; Cynomorium coccineum, Garcia, Nicholson & Nickrent, 2004) (lines 181-183). Those statements are directly opposed.
Experimental design: 1. This article is an original research article and it is within the Aims and Scope of the journal.

2. The research question is reasonably well-identified and the authors clearly identify their research objective for this manuscript as part of an ongoing larger research project aimed at characterizing the plastomes of the genus Gentiana.

3. This investigation suffers from some technical flaws that may affect the major conclusions they draw from their results. I will detail these insufficiencies in the “Validity of the findings” section.

4. There are a few places in the Methods where more information is need to allow for replication of their study. In lines 89-90, the authors need to include additional BLASTN parameters, such as the word size, gaps allowed, reward for a match and penalty for a mismatch. The assembly parameters for NOVOPlasty should be included in lines 90-91. The methodology for calling SNPs in lines 103 to 105 needs to be more detailed. What criteria were used to determine a SNP? How did they distinguish the quality of the SNP calling? In line 146, how did the authors decide whether a base was low quality or not? In lines 147-148, I am not sure what is meant by “among the 145,757 nucleotides of G. tongolensis plastome, SNPs were counted in 100,260 nucleotides (68.78%).” I think it would be better to just mention how many of the total 145,757 nucleotide positions had a SNP.
Validity of the findings: 1. The authors conclude that the existence of hundreds of SNPs at >1% frequency in the plastome of Gentiana tongolensis is an indication of heteroplasmy. In my opinion, the authors have not effectively ruled out alternative possibilities that explain their data. Thus, the data presented in the manuscript are not robust, statistically sound and controlled. Here, I will outline several alternative explanations and make suggestions for how the authors can rule these out.

Alternative explanation #1: Sequencing errors / errors in SNP calling
While the error rate of Illumina sequencing is often low, it is highly variable and can be affected by the Illumina platform used, whether a base is in read 1 or read 2, and whether the base is near the end of the read. In some situations, the error rate can approach 1-2% (see Schirmer et al. 2016 Bioinformatics). From Table S2, many of the SNPs reported as >1% are indeed very close to the error rates described for the ends of reads and could be sequencing errors. Also, false-positive SNPs can occur frequently due to misalignments of the reads or having a position only covered by the ends of reads. The authors attempt to confirm these SNPs through Sanger sequencing of four “loci”. It is not clear how they are defining a locus in these cases. It sounds like each of these “loci” may span several SNPs, but in other places in the manuscript, they use the term “loci” to refer to individual SNP positions. From context, I gather that these four sequenced “loci” span several SNPs. They give the primer sequences used to amplify these “loci”, but it is hard as a reader to match their “loci” with the SNP positions in their plastome assembly (i.e. those listed in Table 2 and Table S2). The authors state that “four loci with a high frequency of SNPs were randomly chosen” on line 162. It's not clear if they mean that each locus spans several SNPs and they are all high frequency (i.e. more than 10%) or if only some of them are. It would be better to include a table giving the starting and ending coordinates of each of the four “loci” and list the positions and frequencies of SNPs that fall within the region amplified by their primers. They later describe genes that are included in the GTcpSNP3 and GTcpSNP4 loci, but they do not provide any information about what SNPs were found in the Illumina data and which ones they were able to confirm by Sanger sequencing. The authors then clone the PCR products amplified for each of these four “loci” and do Sanger sequencing of variable numbers of clones for each.  For two of them (GTcpSNP-1 and GTcpSNP-2), they did not find any SNPs among the clones that they sequenced. The numbers of clones sequenced in both cases were quite low, so it is possible that the SNPs (even if >10% per individual) would not be sampled among the clones. Or it could be that these are SNPs that failed to be verified by cloning and sequencing. It's not clear. They were able to recover SNPs in the third locus, GTcpSNP3, from 25 sequenced clones among 2 individuals. Was one of these individuals the same one that was sequenced to obtain the Illumina data? They report finding 24 SNPs and 7 indels that make up a total of 19 haplotypes. It isn't clear here whether the SNPs they found are the same SNPs that were identified in the Illumina sequencing data or if one can even compare these data to the Illumina data because they might come from different individuals. The authors state in lines 169-170 that the region includes “parts of rps19, whole trnH and parts of psbA”, but there are no SNPs indicated in the Illumina data for these genes in table S3. They also did not report finding any indels in the Illumina sequencing data. Table S3 shows the 19 haplotypes, but they are not aligned to the consensus plastome assembly, making it additionally difficult to interpret whether these data confirm or contradict the Illumina data. For the fourth locus, GTcpSNP4, the authors obtained 35 clone sequences from 5 individuals. In this case, they found 17 SNPs and 2 indels that made up a total of 13 haplotypes for a region that spanned part of the rpoB gene. There were many SNPs at a frequency of >10% that were identified in rpoB in the Illumina data. In this case, they had sequences for 5 individuals, so one of them must be the same individual that gave rise to the Illumina data. However, it is also not indicated anywhere which SNPs were present in the Illumina data and confirmed in their clone sequencing. Further complicating all of this is the fact that cloning also introduces errors. From my own personal experience cloning genes to make transgenic constructs, I find errors in my clones at frequencies that are similar to the frequencies of SNPs and indels reported in the cloning data that are presented by these authors. To really confirm heteroplasmy, the authors should make a clear comparison between their Illumina data and the clones (if indeed the same individual is represented in the Illumina and cloning data). SNPs that are identified in both the Illumina data and in the clones are likely to be real, as getting the same error in both cases is pretty unlikely. From there, the authors can compare different individuals. I suggest not presenting the data in a pie chart, as there are probably only 5 or 6 sequences in total. I suggest presenting an alignment, like those shown in Tables S3 and S4, where the sequences are aligned to the consensus plastome. Indicate the positions that were found to be variable in the Illumina data with a bold letter and then color code the aligned sequences by individual (and clearly indicate which individual was the one that was sequenced).

Alternative explanation #2: Plastid sequences in the nucleus.
The authors do attempt to rule out this possibility in the Discussion, but the explanations they give do not eliminate nuclear-located plastid sequences as the source of the SNPs in this study. The authors acknowledge that transfer of plastid sequences to the nucleus does occur, but suggest that it only occurs at the level of individual genes (lines 186-188). The authors seem to be unaware that many plant genomes contain segments of plastid DNA that are tens of kb in length. They say that they don't think that their SNP data can be explained by plastid sequences in the nucleus because there are SNPs in intergenic regions. However, many plastid-derived insertions in the nuclear genome come from genic and intergenic regions. I direct the authors to read Michalovova et al. (2013) Heredity; Huang et al. (2005) Plant Physiology, and Noutsos (2005) Genome Research to start with. Nuclear plastid DNA sequences (NUPTs) often show a bias toward C-to-T and G-to-A transitions (due to deamination of methylated C nucleotides in the nuclear genome). When I was just casually browsing the SNPs reported in this manuscript, it seemed to me like there were a lot of C-to-T and G-to-A transitions. The best way to rule out NUPTs as a source of the SNPs they observe in their data would be to isolate chloroplasts first and then obtain DNA from the isolated chloroplasts for Sanger sequencing of SNPs to compare with the Illumina data. However, I don't believe that this option is available to these authors because these plants were sampled in the field. What the authors could do is calculate the frequency of C-to-T and G-to-A transitions and determine whether they are indeed the predominant substitutions. This would be a sign that the mutations that occurred due to 5-methyl-cytosine deamination, which would only happen if the sequence was in the nucleus. They could compare this to the pattern of substitutions among the plastid genomes of the other species of Gentiana, which should show a lower frequency of C-to-T and G-to-A transitions. Another experiment would be to do a restriction digest of the total genomic DNA from G. tongolensis using a methylation-sensitive enzyme. They could then do a southern blot and probe the blot with a plastid-derived sequence. If there is a lot of plastid DNA in the nucleus, this would be evidenced by a strong hybridization signal in the uncut fraction of the DNA. It is possible that there are just far more NUPTs in G. tongolensis than in the other Gentiana species studied by these authors, which is why they have not encountered this situation before.

Alternative explanation #3: DNA damage
Is it possible that the DNA is damaged and that this is the reason why so many SNPs appear in the Illumina read data. This could be for technical reasons, such as the silica drying process used to preserve the samples collected in the field or for biological reasons, such as the age of the tissue collected or that perhaps this particular species of Gentiana is compromised in plastid DNA repair. Silica gel has been used reliably with other species, but I have also heard reports of silica gel leading to DNA damage. I would be curious to know whether the authors examined their DNA on a gel before sequencing to verify that it did not show any signs of degradation before sequencing. DNA damage that occurred due to biological processes would still lead to heteroplasmy, but not of the type that the authors propose. In this case, there would be random haplotypes present in an individual, but they are not maintained as distinct haplotypes. It is hard to distinguish between these possibilities given the data they present.
Additional comments: This study suggests the possibility of heteroplasmy that is maintained within individuals of a species, which is really interesting. However, there are several alternative explanations that cannot be ruled out given the current state of the data and the analysis. I would really like to see this paper be eventually published and I encourage the authors to follow through on the suggestions I have outlined in my review.


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: INTRA-INDIVIDUAL HETEROPLASMY IN THE GENTIANA TONGOLENSIS PLASTID GENOME (GENTIANACEAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have greatly improved the quality of the language and have adopted the correct usage of scientific terms in the revised article. However, some typos still remain and there are some areas where more clarification is needed. These are:
Line 59: I am not sure what is meant by "(title = chloroplast AND complete genome). Are these search terms used to identify how many complete chloroplast genome sequences there are in Genbank?
Line 111: "111 Reads were BLASTED with the assembled plastome using BWA-MEM (Li, 2013) ...". Usually when people use "BLAST" as a verb, they are referring to using BLAST to align sequences. From the second part of the sentence, it looks like the authors used BWA to align the sequences. If BWA was used, then "BLASTED with" should be changed to "aligned to".
Line 192: "one individuals" should be changed to "one individual".

The cited references and context provided are sufficient. The structure of the article is professional. The authors indicate that the raw data have been shared. However, I cannot retrieve the assembly from Genbank using the accession number MK251985. The authors deposited the raw read data in the National Genomics Data Center, BIG Data Center under accession number CRA001588 in response to my last request and I was able to easily locate the files.

The article is self-contained, but the relationship between the results and the hypotheses is still unclear. Please see more details about this in the "Validity of the findings" section.
Experimental design: No comment.
Validity of the findings: I appreciate the authors' efforts to respond to my earlier comments. This revision has improved a lot, but I have still identified one major issue that needs to be resolved before publication in PeerJ.

Regarding the statements in lines 192-196:
"Focusing on the individual for Illumina sequencing, only two sites (a SNP, position 23411; a indel, position 68770) in the sequences obtained from verification were matched with the sites in the plastome on which our primers were designed. On the other hand, lots of new variable sites were detected through verification."

This statement is a little unclear, but I have been through the data and I think I know what they mean. The authors selected three regions, each of which covered more than one SNP or indel that had been detected in the Illumina data. Only two (of many) sites (the SNP and the indel they mentioned in the statement) were also identified in the Sanger data. In addition, they found many more sequence variations in the Sanger clones that were not observed in the Illumina data.

Since the same individual was used both for the Illumina sequence and the Sanger sequencing, I would expect a complete overlap between the two datasets. However, there is variation present in the Illumina data that is not present in the Sanger data and there is variation in the Sanger data that is not present in the Illumina data. If the disagreements between the two datasets was only a minor fraction of the variable sites, then I think that this would indicate that there is true biological variation with some variation due to technical artifacts. Because there is so little overlap between the two datasets, it is really hard to tell what this means.

One explanation for the failure to detect polymorphisms that had been observed in the Illumina data in the Sanger sequences is low number of clones per individual. For example, in GTcpSNP-4, there should have been two SNPs that had been detected in the Illumina data present in the Sanger data. I am inferring this by cross-referencing the coordinates given in the Materials and Methods with Table S2. The authors should make it easier to find this information, perhaps by highlighting the SNPs in Table S2 with color-coding that indicates which SNPs are covered by which GTcpSNP regions that were Sanger sequenced. Those two SNPs should have an allele frequency of around 15%, meaning that each should be present in 3 of 20 clones (15%) for a single individual. The authors only sequenced two clones for the individual that was used for the Illumina sequencing, so it is not surprising that only one of these two SNPs was detected. They were lucky to find one of the SNPs among only two clones, given that the probability of this happening is low.

Similarly, I inferred that GTcpSNP-5 should have contained 5 SNPs and 2 indels. According to Table S2, the allele frequencies for these polymorphisms ranged from 8.9% to almost 26%. Only 6 clones were sequenced for this individual, meaning that at best only the polymorphism with the highest frequency (an indel at position 68516) would be found in one or two of the six clones. However, it was the indel at position 68770 (listed at 68769 in TableS2) that had a lower allele frequency that was detected in the Sanger data.

I was unable to make a similar comparison for GTcpSNP-3 because the coordinates were not given in the Materials and Methods (although the coordinates were given for all of the other regions). Cross-referencing the positions in Table 3 with Table S2 did not help because none of the positions in Table 3 were also found in Table S2. Also, the genes in the GTcpSNP-3 region (rps19, trnH, and psbA) are not listed in Table S2. This needs to be clarified.

It is difficult to know how to interpret the polymorphisms that were present in the Sanger data that were not found in the Illumina data. I think that the most likely explanation is that these are mutations that were introduced during the cloning process. If that is the case, then why are there so many? I would expect maybe a few here or there, but I would not expect nearly all clones to have an independent artifactual mutation, which is what I see in Tables 3-5. Another possibility is that the Sanger sequencing data is of low quality. In either case, they would be technical artifacts. The only other interpretation is that they represent true biological variation that is somehow missed in the Illumina data, but this seems unlikely given the massively high coverage of the Illumina data.

To summarize, there is very little overlap in the polymorphisms detected in the Illumina data and those detected in the Sanger data. Based on the data presented by the authors, it is not clear which polymorphisms represent true biological polymorphisms and which are technical artifacts.

I would recommend sequencing more clones (at least 20 per individual per region). If the frequency of the mutation (based on Illumina sequencing) is 15%, then there is a probability of 0.85 of not finding it if you sequence only one clone. If you sequence two clones, then the probability of not finding the mutation is (0.85)^2, or 0.72. If you sequence 20 clones, then the probability of not finding the mutation is (0.85)^20, or 0.039. Thus, if the authors sequence 20 clones per individual for each region, then they have a good probability of finding all of the mutations present in the Illumina sequencing data.

If the authors do this and there are still polymorphisms that appear in the Sanger sequencing data from the clones that do not appear in the Illumina data, then I think that the polymorphisms present only in the Sanger data should be dismissed as possible technical artifacts and thus they should not be scored as new haplotypes.
Additional comments: I have a few more suggestions for the authors to improve clarity and the presentation of their data.

1. Please consider presenting Figures S1 and S2 as main figures instead of supplemental figures. There are no figures in the main document and adding these visual representations of the data would be a welcome addition.

2. For the plastome map in Figure S1, it would be great if the authors could also indicate the regions that were cloned and subjected to Sanger sequencing.

3. For the plastome map in Figure S1, the authors should consider plotting the locations of the SNPs.

4. Please consider making a table of the coordinates for each of the 5 regions used in the cloning and Sanger sequencing analysis.

5. Line 164: I don't think it is necessary to have two decimal places for the depth of coverage, as this level of precision is not meaningful. I would change "2698.72" to 2699x (with "x" indicating that it is 2699-fold coverage).

6. Lines 171-172: Thanks for including this analysis as a response to my earlier critique. I think that there needs to be more clarification here. I think it is necessary to mention why an excess of C -to-T and G-to-A transitions in comparison to T-to-C and A-to-G transitions would be an indication that these are polymorphisms that occur due to nuclear integration.

7. Line 173: More explanation is needed here. It could be something along the lines of "To verify the polymorphisms detected in the Illumina data, we selected three regions that spanned multiple polymorphisms and two control regions without polymorphisms for cloning and Sanger sequencing. You could then refer to a table of the coordinates if you do make this table, as I suggested earlier.

8. Lines 176-178: It is not clear what this means. If you detected 7 SNPs among 12 clones, that could mean that there was one clone out of 12 that had 7 SNPs in it. Or it could mean that 7 of the 12 clones each had one SNP. Or it could mean anything in between. The language for all of the results described in these lines needs to be clarified so there is only one possible meaning.

9. Lines 208-212: Some rephrasing is needed here. Thanks for including this discussion in response to my earlier critique. However, I think that it needs to be stated at the beginning that you are evaluating the possibility that the heteroplasmy is due to DNA damage - either DNA damage due to technical reasons (sample storage) or to biological reasons. Then present the reasons why you are ruling out these possibilities.

10. Table S2: Please indicate which are located within the regions used for cloning and Sanger sequencing. Also, please revise the language in the header.  "Allelic depths of Reference Base" can simply be changed to "Depth of coverage". Finally, I don't see any entries in the "location" column that say "intergenic" even though you mentioned in the text that there were 45 polymorphisms in intergenic regions.

11. Table 1: the term "Rate (%)" is unclear. Consider changing it to "Percentage of polymorphic sites" or "% polymorphic". Add (bp) to the "Length" in the header.

12. Table 2: Change header to match Table S2.


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: FIELD EVALUATION OF MOSQ-OVITRAP, OVITRAP AND A CO2-LIGHT TRAP FOR AEDES ALBOPICTUS SAMPLING IN SHANGHAI, CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors compared Ae. albopictus sampling with MOTs, OTs and CLTs in Shanghai, China. They present that more eggs were collected in MOTs compared to OTs and that the minimum time MOTs should be deployed is 7 days.
The manuscript is of very good quality  – clear and prof. English – and presents relevant Figures and Tables.
Experimental design: The research question is well defined and methods described.
It is clearly mentioned and discussed that OTs were slightly modified.
Validity of the findings: The authors clearly describe the advantages of MOTs compared to OTs for albopictus egg sampling. Moreover adult sampling is compared of MOTs and CLTs.
Conclusions are well stated.
Additional comments: However some modifications are recommended
1)	Sometimes the authors only mention Aedes. Do you mean albopictus or also other Aedes species. Please change to albopictus if you only mean Ae. albopictus
2)	entire manuscript – change to CO2 (2 smaller; e.g. line 58)
3)	Mosquito processing: Which keys were used for specification?
4)	Line 243: change to MOTs (Li et al… - Space – also at Line 255
5)	Figure 9: Legend: When mentioning Aedes - only albopictus included?


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: FIELD EVALUATION OF MOSQ-OVITRAP, OVITRAP AND A CO2-LIGHT TRAP FOR AEDES ALBOPICTUS SAMPLING IN SHANGHAI, CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: # please use more scientific phrasing  e.g. do not use dangerous (line 52), but most important

# improvement English need significant improvement for the complete manuscript  “egg hatch” (line 164), “use generalized linear mixed model for analysis“ (lines 175-175), “percent positive was“ (line 182), etc.
Experimental design: # lines 75-89: Please highlight the lack of knowledge and the aim of this study to tackle this lack of knowledge. In the moment this is a little bit lost in the presentation of the literature.

# line 95: What do you mean by “one group”? Did you just put all traps in one place? Why did you not use a latin-square approach to account for small-scaled differences between trapping sites and to avoid direct competition between the traps?

# lines 107-: How did you observed that gravid mosquitoes typically do not escape? Please present data and/or publication.

# lines 110-: “Traps were also…”  at the end of the “Traps Tested”-section

# lines 125-131: How were the sampling with the CLT conducted? How often? Time? Etc.

# lines 133-140: I thought you reared the mosquitoes to adults (see line 150)?

# line 149: Which taxonomic keys?

# line 150: How many eggs were selected?

# lines 154-160: The statistics are unclear. Which kind of GLMM did you use (poisson, etc.). What is the random effect? What did you mean by “preliminary”? Why do you use different tests?

# lines 174-176: This belongs to the methods section
Validity of the findings: # lines 181-188: You should not use the phrase “increasing” as you do not observe this process, but comparing different groups (3d, 7d, 10d)

# lines 181-188: You need a p-value correction (e.g. Bonferroni) for multiple tests

# lines 189-191: Please analyse the correlation between the MOT and OT samples over time

# lines 191-192: Discussion belongs to the discussion section

# lines 196-197: I do not understand how and why can compare eggs and adults?

# lines 198: test statistics of the Pearson correlation?

# tables/figures: 9 Figures and 6 Tables are to many and make it difficult the capture the important results. Please think about to move some of the tables/figures to the supplement (e.g. Table 1 and Table 4)
Additional comments: # lines 52-63: please explain more precise why ovitraps are much easier to implement (e.g. no electricity, no CO2, etc.)

# lines 61-70: please give a more comprehensive review on studies showing the correlation or the opposite

# 240-242: I do not understand this sentence. What do you mean by “secondary index?”

# lines 269-280: What are the consequences from this?

# lines 292-296: The paragraph appears a little bit hasty. Please give a comprehensive discussion of the potential problems of the here presented study (e.g. why did you not use a latin-square approach)

# lines 299: “more productive”  do you mean efficient?


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: FIELD EVALUATION OF MOSQ-OVITRAP, OVITRAP AND A CO2-LIGHT TRAP FOR AEDES ALBOPICTUS SAMPLING IN SHANGHAI, CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors signiciantly improved their manuscript and responded to all of my comments,
Experimental design: The authors signiciantly improved their manuscript and responded to all of my comments,
Validity of the findings: The authors signiciantly improved their manuscript and responded to all of my comments,
Additional comments: The authors signiciantly improved their manuscript and responded to all of my comments,


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

Input:
Title: EFFECTS OF ELEVATED PCO2 AND NUTRIENT ENRICHMENT ON THE GROWTH, PHOTOSYNTHESIS, AND BIOCHEMICAL COMPOSITIONS OF THE BROWN ALGA SACCHARINA JAPONICA (LAMINARIACEAE, PHAEOPHYTA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: Good
Experimental design: Good
Validity of the findings: Good
Additional comments: This manuscript investigated the combined effects of two critical environmental factors, ocean acidification (OA) and eutrophication on physiological performances in the economy important macroalga Saccharina japonica. The authors measured relative growth rate (RGR), net photosynthetic rate, and biochemical compositions (including chlorophyll (Chl) a, Chl c, soluble carbohydrates, and soluble protein) and found the negative effect of OA and interaction of OA and eutrophication on some key parameters. This study provides useful understanding on how production and chemical composition of S. japonica would be affected in the future oceans.  The experiments were well designed and performed. The data analyses were satisfactory. I thus think this manuscript can be published in Peer J after revision.

Specific comments
Line 22 For pCO2, the unit of uatm rather than ppmv is usually used. Please correct it throughout the text.
Line 25 400 ppmv of what?
Line 29 Aggravate is not a proper word here.
Line 34 The section of Introduction is well written.
Line 92 How quickly?
Line 102 Culture period should be pointed out in this section
Line 104 the concentration of nitrate and phosphate in the two nutrient levels should be presented.
Line 113 Please provide the mode, brand and product place for the two incubators

Discussion section
I am wondering whether the combination of OA and eutrophication led to reproduction events, which caused the decrease of growth, particularly after fragmentation (1.4 cm in diameter). This could happen in Ulva species (Gao et al., 2017ab, 2018). Please discuss it in the text.
Gao G, Clare AS, Rose C, Caldwell GS. Eutrophication and warming-driven green tides (Ulva rigida) are predicted to increase under future climate change scenarios. Marine Pollution Bulletin, 2017a, 114:439-447.
Gao G, Clare A S, Rose C, Caldwell GS. Intrinsic and extrinsic control of reproduction in the green tide-forming alga, Ulva rigida. Environmental and Experimental Botany, 2017b, 139: 14-22.
Gao G, Clare AS, Rose C, Caldwell GS. Ulva rigida in the future ocean: potential for carbon capture, bioremediation, and biomethane production. Global Change Biology Bioenergy, 2018, 10: 39-51.

Line 279 Positive interaction sounds weird.
Line 296 I cannot see “eutrophication exacerbated the negative effect of OA on growth” based on Figure 1. Please describe the interaction in the Results section.
Line 514 Correct 2012 to 2017.
Figure 1 Add space between number and letter. Correct it throughout the figures.
Figure 2 The unit of net photosynthetic rate is not correct.
Table 2 Italicize a and c for chlorophyll


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

Input:
Title: EFFECTS OF ELEVATED PCO2 AND NUTRIENT ENRICHMENT ON THE GROWTH, PHOTOSYNTHESIS, AND BIOCHEMICAL COMPOSITIONS OF THE BROWN ALGA SACCHARINA JAPONICA (LAMINARIACEAE, PHAEOPHYTA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: The experiment was well designed.
Validity of the findings: no comment
Additional comments: This study focuses on the effects of pCO2 and nutrient enrichment on growth, photosynthesis, and biochemical compositions of the brown alga Saccharina japonica (Laminariaceae, Phaeophyta). The paper is well written and the results are clearly presented. but a number of points need to be addressed. The language needs considerable help by a native speaker. Sentence constructions are awkward throughout the whole manuscript leading to major misunderstanding or non-understanding of the text passages.

Introduction: Hypothesis missing.

The whole growth experiment last for only 6 days, why did you do the experiment for longer time because the growth rate of this brown alga was less than 2%?

Please provide the information about nutrient condition at nature seawater and enriched seawater.

Figure 2, μmol.

The unit of PCO2 in seawater is μatm, not ppmv

Table 1, please provide the unit of the parameters of seawater carbonate system.

References: Xu Z, Gao G, Xu J. 2017. Physiological response of a golden tide alga (Sargassum muticum) to 515 the interaction of ocean acidification and phosphorus enrichment. Biogeosciences 14: 671-681.


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

Input:
Title: EFFECTS OF ELEVATED PCO2 AND NUTRIENT ENRICHMENT ON THE GROWTH, PHOTOSYNTHESIS, AND BIOCHEMICAL COMPOSITIONS OF THE BROWN ALGA SACCHARINA JAPONICA (LAMINARIACEAE, PHAEOPHYTA)
Review round: 1
Reviewer: 3

Output:
Basic reporting: This study showed the changes in the growth, photosynthesis, and biochemical compositions of the brown alga Saccharina japonica under elevated pCO2 and nutrient enrichment.

1.	It’s not clear why the authors didn’t determine the concentration of carotenoid, which is important photosynthetic pigment as important as Chl a and Chl c for the Saccharina japonica.

2.	One would strongly disagree with claim that “Despite the important effects of OA and eutrophication on kelp species, very few studies have considered the combined effects of these factors on the physiological characteristics of kelp species”.

I suggest the authors to well search the literatures. At least, there are several cited in this manuscript already:
Gao G, Gao Q, Bao M, Xu J, Li X. 2019. Nitrogen availability modulates the effects of ocean acidification on biomass yield and food quality of a marine crop, Pyropia yezoensis. Food Chemistry 271:623-629 DOI 10.1016/j.foodchem.2018.07.090.

Gao G, Beardall J, Bao M, Wang C, Ren W, Xu J. 2018. Ocean acidification and nutrient limitation synergistically reduce growth and photosynthetic performances of a green tide alga Ulva linza. Biogeosciences 15(11):3409-3420 DOI 10.5194/bg-15-3409-2018.
Experimental design: 1.	What are the nutrients and their concentrations in the 50% PESI enriched seawater? The authors need to explain why this level was set up. This is key information for the whole study.

2.	Along with my concern on the nutrient level used in this study, a further question is what about the nutrient scenarios in the previous studies on the responses of seaweed to OA and eutrophication? This may also help explain the different results observed in these studies. Now the authors simply conclude that “These results indicate that the interactions of elevated pCO2 and nutrient on seaweeds are algal species-specific.” A better approach could be setting up a series of nutrients, considering that the CO2 is only 1 treatment and 1 control group (air).

3.     The concentration of carotenoid is recommended to be detected in such a study.
Validity of the findings: The authors need to expand the statements for their novelty.
Additional comments: English writing need to be improved; and the authors need to check throughly for the minor issues.

For instance, L65-66: “The canopy-forming kelp Saccharina japonica (Areschoug) Lane, Mays, Druehl and Saunders inhabits subtidal zones”, what’s the Lane, Mays, Druehl and Saunders?


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

Input:
Title: EFFECTS OF ELEVATED PCO2 AND NUTRIENT ENRICHMENT ON THE GROWTH, PHOTOSYNTHESIS, AND BIOCHEMICAL COMPOSITIONS OF THE BROWN ALGA SACCHARINA JAPONICA (LAMINARIACEAE, PHAEOPHYTA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors have revised the manuscript based on my comments. I thus suggest accepting this manuscrip.


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

Input:
Title: EFFECTS OF ELEVATED PCO2 AND NUTRIENT ENRICHMENT ON THE GROWTH, PHOTOSYNTHESIS, AND BIOCHEMICAL COMPOSITIONS OF THE BROWN ALGA SACCHARINA JAPONICA (LAMINARIACEAE, PHAEOPHYTA)
Review round: 2
Reviewer: 2

Output:
Basic reporting: The basic written English has been improved throughout the manuscript.
Experimental design: To keep the readers aware of the major flaw in their experiment design. The authors need to add explanations in the methods and discussions that, they didn't determine the concentration of carotenoid, which is important photosynthetic pigment as important as Chl a and Chl c for the Saccharina japonica.

The authors need also consider my previous comment: "A better approach could be setting up a series of nutrients, considering that the CO2 is only 1 treatment and 1 control group (air)."
Validity of the findings: no further comment
Additional comments: No additional comments


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

Input:
Title: EFFECTS OF ELEVATED PCO2 AND NUTRIENT ENRICHMENT ON THE GROWTH, PHOTOSYNTHESIS, AND BIOCHEMICAL COMPOSITIONS OF THE BROWN ALGA SACCHARINA JAPONICA (LAMINARIACEAE, PHAEOPHYTA)
Review round: 3
Reviewer: 1

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


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: SEASONAL VARIATION IN APPARENT CONDUCTIVITY AND SOIL SALINITY AT TWO NARRAGANSETT BAY, RI SALT MARSHES
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: The methods should be improved or revised prior to publication to improve the strength of the manuscript. First, I had questions about the spatial interpolation method, that should be addressed in the context of the manuscript revision.

First, quantification of uncertainty in the salinity calculations needs to be done explicitly.

Second, how was it decided to use this method for interpolation mapping? Where any validation techniques used to arrive at this method, such as leaving out a sub-set of data points, and estimating prediction error? Did you walk specific transects when you did the surveys? If not, do you think that you produced different maps because different paths were taken during different seasons? Did you do any analysis of how maps might vary based on different paths taken? How closely spaced where your measurements and what role do you think that spatial autocorrelation plays in your analysis?

Third, why were RTK-GPS measures made of elevations if LiDAR, with many thousands of points, and much more elevation detail, is available? How do your generated DEMs compare with the bare earth LiDAR?

Fourth, are the cumulative rainfall values correlated? Does this influence your data analysis? I would recommend trying out different synthetic lag-terms as well to see if some fit your data better than others.

I have added specific line comments below, as well.

Line 143 – At how many points were sipper readings taken per survey?

Line 163 – Again, is there any evidence that this interpolation method is the most appropriate and accurate for mapping elevation? And why not use LiDAR?

Line 168 – How did you adjust NAVD88 to sea level ?

Line 196- Explain how spatially averaged salinity was calculated.

Line 196-7 – Also was there consistent variability by season? That could be examined for using something like GLM or linear mixed effects models?
Validity of the findings: The discussion of findings could be improved by more reference and discussion of primary literature on spatial structure of salinity gradients in coastal wetlands.
Additional comments: This manuscript focuses on the use of electro-magnetic conduction surveys to identify spatial and temporal patterns in coastal marsh salinity. This can make a valuable contribution to the literature. Below I have identified locations where the manuscript prose can be improved to increase clarity.

Line 72 – The phrasing in this sentence is somewhat awkward and should be revised. Tidal wetlands have a continuously saturated zone, and so it is not that porewater is “absent;” it can just be difficult to sample if the saturated zone is deep, or if there are clay or fine sediment layers with low hydraulic conductivity.

Line 90- I know that EMI is defined in the abstract, but it would be helpful to define it at first use in the manuscript as well.

Line 105 – I suggest replacing the second “and” with a comma.

Line 122/123 replace spp with spp.

Line 122- Likely your “Scirpus” species have been renamed to genera such as Schoenoplectus and Bolboschenus.

Line 183 – What models?  Models haven’t been discussed yet.

Line 191-194- This is unclear and needs to be better explained. What is 1.0 to the proportions?

Line 212 – Producing biplots is not a quantitative assessment of association I would recommend removing completely the “visual comparison of maps.”

Line 220-1 – This sentence could be more clearly written.

Line 222-4  - Can this be written more simply? Such as, “salinity was high at the seaward edge and low at the terrestrial border across the spring to fall growing season”

Results section should present results of calibration of porewater salinity vs. conductivity and prediction errors (ie, not just R2, but actual prediction errors). Bulk density was measured – how does that play into the calibration?

Line 230 – “sampling” not “sample” ? Also, these values seem overly precise given unspecified prediction errors that are likely quite large (r2~0.5), e.g., salinity +/- 10‰.

Line 239 – Are you referring to mean salinity for the whole marsh?

Line 266-267 – This sentence could be more specific. What were the results, beyond salinity varied spatially and temporally?

Line 268 – Why was this salinity pattern expected?

Line 269-272 – This section is results.

Line 274- I’m not sure that a lower water table in the marsh would be caused by reduced precipitation. Isn’t the source of most of the shallow groundwater the tides? Do you have evidence for lower water tables in marshes during droughts? Maybe revise “lower water table” to “reduced groundwater recharge from uplands.”

Discussion – Different coastal marsh salinity patterns are found in different places based on seasonality in precipitation and/or tidal flooding (e.g., some marshes are flooded more by wind events than tides). The discussion should be revised to identify these differences. As it stands now, statements such as “Seawater influence has been shown to diminish as distance from tidal creeks increases (Hemmond and Fifield 1982)” are overly centric on northeastern marshes. While these marshes are well studied, the discussion should be drawn on more from the international literature to show how these surveys both confirm and refute past studies of northeastern marshes, and how this approach could be valuable in e.g., Mediterranean marshes that are often hypersaline at the marsh upland border.

Do these marshes have similar hydraulic conductivity values? Are the tides the same? Wouldn’t these things help explain differences in spatial patterns of salinity?

Line 284 – It doesn’t follow to me that high freshwater discharge to the marsh would translate into homogenous salinity patterns.

Line 288-291 – The prose could be simplified here.

Line 299 – Why would freshwater have a salinity of 5ppt? Isn’t that by definition not freshwater?

Line 319 – Can you be more specific than “relatively slow” ? Days? Months? Years?

Line 362 – Sentence is unclear.

Line 368 – The implications here seem not specific enough. Refer more specifically to spatial patterns in salinity and plant zonation.

Line 372 – Vulnerable to what? Erosion? Salt water intrusion?

Table 1 – How many points were sampled for porewater conductivity? Normally, dates should be in international format, e.g., 30 Oct 2015, as most countries use the day-month-year formate.

Figure 1 – “Salt marsh study sites” not “Salt marshes study sites”

Figure 2 & 3 – Sampling points should be demarcated. Also, it would be easier to interpret this maps if they had the same legend (e.g., yellow represented the same thing on the different plots). If they are  - I apologize – it is hard to tell. Perhaps just have one legend that shows all the possible salinity levels?

Figure 4 – x axis should be “day of year-” Also I suggest making the figure more professional, rather than using excel default style. I suggest the authors look at figures published in journals, especially PeerJ for examples of publication quality figures.


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: SEASONAL VARIATION IN APPARENT CONDUCTIVITY AND SOIL SALINITY AT TWO NARRAGANSETT BAY, RI SALT MARSHES
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: The authors have presented an interesting study that follows up prior work using electromagnetic induction (EMI) and apparent conductivity ECa measures as a valid/dependable proxy for pore water salinity measures in marshes, clearly demonstrating EMI’s value in tracking interannual changes in soil salinity patterns in a new and highly efficient manner.

I think they have done a fine job in designing, implementing and interpreting their study for the most part. That said, I have some concerns that I’d want the authors to consider and explain, as well as some minor comments and suggestions they could address to help make the study more valuable to readers and/or users of EMI technology in the future.

One such concern is why they did not interpret results by major habitat type (high vs low marsh for example) and rather treated the marsh as a whole/single unit. My sense is that some interesting patterns were lost by not considering how the results shake out for low vs high marsh areas, esp since different plants, different rates of water uptake, different root depth and biomass/bulk density, and often different soil types (greater percentages of silt/clay, for example that can affect ECa). This is a fact the Authors note in Lines 337-338, but more could be done to account for potential influence of clay minerals/iron sulfate soils. While they did list the dominant plant species (Lines 119-129), there may be more analysis to be done here. Keep in mind most researchers in New England are documenting shifts in high/low marsh plant communities, where low marshes are drowning and high marshes are converting to short form S. alterniflora or worse… EMI used as detailed in this manuscript may help show the salinity patterns that will drive these shifts BEFORE the plants are forced to react and migrate. THAT is very interesting and underplayed in the ms as presently written…

Lines 138-139: Were pore water samples (via sipper) gathered at each monthly sampling event or were representative calibration samples taken at some other time of year and used to correct/convert ECa to salinity at each monthly sampling event? Obtaining pore water in Dec-Mar must have been a real challenge!! Must be warmer than I thought in Narragansetts Bay in mid-winter! Table 1 suggests actual pore water sample collected each month, correct? Please clarify.

Lines 143-145: The Authors note their pore water sampling depth of 0.25 m, which is rather shallow. Depending on the plant community, that mean depth of live roots may be closer to 0.40 m and often there is a marked difference in pore water salinity near the surface (< 0.10m) and deeper depth (~ 0.06m). My concern is that using the 0.25m as the calibration value against ECa via EMI is mismatched. EMI integrates the entire profile, with the sipper is a fixed point. During seasonal extremes (min/max salinity) this could significantly influence interpretation or misinterpret actual conditions in the rhizosphere that affect plant zonation patterns. And Typha (present at the upper edge of the marsh can produce significant root/rhizomes well below 0.25m…, for example). Sampling at two depths might have resolved this potential confounding factor. Since they can’t go back and take that second depth, they should do more to acknowledge the potential flaw (in addition to text they already provided in Lines 341-345).

Lines 267-269: I have often found a bimodal pattern of salinity along a gradient of creek edge to upland, with the highest salinity often midway along that gradient of well-drained creek edges and freshwater collecting upland edges. High marsh can often collect and retain salty water as freshwater evaporates out… However, in smaller marshes, the pattern may be more as the Authors describe. I guess I just caution them against saying “expected pattern” without qualifying why its “expected”.

Lines 366-368: This line underscores my concern that vegetation data is lacking in this manuscript. At the very least, consider results by high vs low marsh habitat.

Lines 365-375: In my opinion, the Authors miss a good opportunity to highlight how efficient EMI can be for this type of sampling. If we are to believe that ECa is a dependable proxy for direct pore water sampling once calibrated with actual field data, then we should be equally convinced that mapping large or repeated spatial areas with EMI is far more efficient than traditional approaches – its wouldn't be impossible to do this with sippers, but it would have taken 10-100x more person hours/effort! EMI makes this type of study practical/possible.

Table 1: So, the Authors were able to extract a field pore water sample in Dec, Jan, Feb, etc.? Impressive!

Table 4: Since the Authors indicate bulk density samples were obtained from low vs high marsh habitats, I’m hoping they can sort out which ECa and pore water samples came from which habitat and will consider analysis of results through this additional lens. This is PARTICULARTLY important as New England is watching high marsh convert to Low marsh due to SLR and prolonged flooding. EMI data will track nicely (if not be predictive of) vegetation pattern shifts… I STRONGELY ENCOURAGE THE AUTHORS TO RECAST RESULTS/DISCUSSION IN THIS TIMELY PERSPECTIVE.

Figures 2-3: Add a high/low marsh line to maps to help direct readers to potential patterns in each key habitat.


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: SEASONAL VARIATION IN APPARENT CONDUCTIVITY AND SOIL SALINITY AT TWO NARRAGANSETT BAY, RI SALT MARSHES
Review round: 2
Reviewer: 1

Output:
Basic reporting: See recommendation below.
Experimental design: See recommendation below.
Validity of the findings: See recommendation below.
Additional comments: After careful review of the revised manuscript, I believe  the authors have made all necessary revisions (or provided acceptable explanations) to warrant publication as is.


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

Input:
Title: DIATOMS AS INDICATORS OF THE EFFECTS OF RIVER IMPOUNDMENT AT MULTIPLE SPATIAL SCALES
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Comments on "Benthic diatoms as indicators of river impoundment at multiple spatial scales"
I have no real comment on the study design, data collection or analyses as the two former are part of a national standard and the multivariate and univariate analyses are common-place for ecological data. There are a few statements in the text that need explanation and I have a few additional comments, as follows.
1.	The title needs rethinking as planktonic diatoms are included in the data analysis, results and discussion
2.	Line 101 page 3 – "Only monitoring sites with sufficiently good water quality…" More details are required for this. How did the authors define good water quality? What parameters were used? The authors find that water quality is an important driver of the diatoms even with "sufficiently good water quality", so the data set is already confounded, so why not include those with less quality?
3.	Page 4 Community structure – Is it possible for the authors to get access to the Primer 7 software package in order to do a bootstrapped ordination. Only bootstrapped PCAs are currently available in R and probably would not be appropriate for the diatom data. Unless the authors have sufficient programming skills to write a bootstrapped mds? A mds bootstrapped ordination would be extremely useful to look at the entire data set in one diagram, rather than separating all the samples into the two seasons and having multiple ordinations for the different regions.
4.	Page 4 Diatom indices – The authors need to provide hypotheses as to why the different ecological guilds would react differently to river regulation, i.e. provide an explanation why they were grouped in such a way and analysed separately.
5.	Lines 231 and 232 page 6 – "on average higher at downstream sites than control sites" is repeated in the following line "Ntaxa was typically higher at downstream sites". This paragraph need rewording.
6.	Tables 3 and 4 – Can the authors provide a percentage variance explained as in Tables 1 and 2?
7.	Lines 403 and 404 page 10 – The term "may guide water managers" is very vague. The authors need to be much clearer about what that means and then the paper would be more valuable to the scientific and management communities. For example, some managers (and maybe conservationists) would see it as a good thing that there are more taxa at the downstream sites as there is more biodiversity.
8.	Line 412 page 11 – "suggested the influence of other factors including flow regime". With no hydrological data this statement cannot be made.


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: DIATOMS AS INDICATORS OF THE EFFECTS OF RIVER IMPOUNDMENT AT MULTIPLE SPATIAL SCALES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study presents a big dataset of diatom data to reveal the differences exist between diatom communities at sampling sites downstream of water supply reservoirs and unregulated control sites at the regional and national scale. I have noticed that the authors have published the paper Krajenbrink et al. (2019, STOTEN) which also is based on the same dataset from the SHEBAM monitoring network. This manuscript answers questions similar as those in the 2019 STOTEN paper from the angle of diatom communities. I think the conclusions raised here are of high scientific validity and I am happy to see some discussion on the connections between the two research (i.e. ecological difference between diatom and macroinvertebrate communities). The current ms is written in clear and professional English and all the statistic analysis are sound and appropriately used.
Experimental design: Well designed and rigorous performed
Validity of the findings: Conclusions are well stated and scientificly justified.
Additional comments: I have only one comment. Is it possible to give more description on the diatom ecology and species (i.e. the dominant species from downstream and control sites?


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: DIATOMS AS INDICATORS OF THE EFFECTS OF RIVER IMPOUNDMENT AT MULTIPLE SPATIAL SCALES
Review round: 1
Reviewer: 3

Output:
Basic reporting: All good.  One final read through in order to prune back surplus wordage and check for clarity would be useful.
Experimental design: No substantial issues here.  Meets the journal's requirements
Validity of the findings: Some suggestions to the authors are included below.  These relate primarily to data interpretation, not the execution of the work per se.   Some speculation in the Discussion is not evidence-based and could be pruned back
Additional comments: I am happy to recommend this paper for publication, subject to a few reservations discussed below.  It is a generally well-written contribution to the literature on this topic, and is based around thorough statistical analysis of a large dataset.   My primary reservation about the paper is summed up by the complete absence of reference to individual diatom species which is also reflected in the lack of profound insight in the Discussion.  It is, nonetheless, a workmanlike paper that does the job it sets out to do.

My own experience is that the phytobenthos in rivers immediately below impoundments often take on the characteristics of the phytobenthos in the impoundment itself.  This is true, to some extent, for both natural lakes and for artificial water bodies and probably is due to inocula from the standing water body but also to the nature of the regulation itself.  Inclusion of samples from upstream and downstream of natural lakes would have made the paper more interesting, as would inclusion of samples from within the reservoirs.

I disagree with some of the data interpretation presented here.  First, I am not convinced that biogeographical factors are responsible for differences at the national scale.  Second, the reasons for the observed changes in the downstream assemblages need a rethink.

The experience in the UK is that, in the absence of anthropogenic pressure, it is alkalinity (reflecting underlying geology) that is the primary factor controlling diatom assemblages.  I suspect that the differences you see between northern and southern England are probably a consequence of underlying geology and I would not regard this as “biogeography” per se, simply as a shuffling of environmental drivers that happens to have a spatial correlation.   I’m happier when you refer to this as “regional influence”

Lines 344-347 say “The results involving TDI and the motile guild point towards the influence of water quality gradients on benthic diatom communities, even in largely oligotrophic regions, and provide evidence for the influence of river impoundment on downstream water quality.”   This is an easy default interpretation but does your data actually support this?   Table S4 and associated figures suggest that N variables have a larger effect than OP whereas dogma would suggest that it is OP that should be driving these changes. First, are there reasonable grounds to argue that N is limiting, relative to P?   Second, are there unmeasured variables that may correlate with these that are actually driving the patterns you observe?  A further possibility is that the biological changes are only partially captured by the diatom assemblage – that there is greater accumulation of biomass in regulated rivers immediately downstream of impoundments (possibly due to filamentous greens accumulating due to the less scouring conditions) and that this favours certain diatom taxa that also happen to thrive when the thick biofilms are the result of eutrophication.  I don’t have an easy answer either, but I think that, in trying to explain your results, you need to acknowledge that there is not a direct cause-effect relationship between impoundments and diatoms and that much of the evidence on which you rely for this part of the discussion is also based on statistical associations within datasets rather than experiments.   In the absence of a well-thought through conceptual model to explain the effects observed, the easiest way forward may simply be to trim back some of the speculation in this part of the Discussion.  I certainly find lines 369-371 rather strong, in light of the evidence. Perhaps “most important drivers …” should be replaced by “strongest correlates”?

Minor comments
Please refer to the diatom “assemblage”: the diatoms are part of a wider phytobenthic/periphytic community

Line 128-129: what are “EA quality assurance protocols”.  I did not know that they had a standardised process for this.

Why did you use TDI3 when TDI4 is the EA’s current approach?

To what extent is Ntaxa affected by the counting policy described in lines 123-124?   If there is an overwhelmingly dominant taxon then the “tail” of rare species will be correspondingly longer due to the greater count size and Ntaxa for such samples may be misleading.

It would be useful to have a Table similar to S5 that describes the inter-relationships between the diatom indices that you calculate, and a comment on the extent to which each is contributing unique information.


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: DIATOMS AS INDICATORS OF THE EFFECTS OF RIVER IMPOUNDMENT AT MULTIPLE SPATIAL SCALES
Review round: 1
Reviewer: 4

Output:
Basic reporting: See comments below
Experimental design: See comments below
Validity of the findings: See comments below
Additional comments: General comments:
This study test if diatom community, taxonomic diversity, diatom guilds etc.  show consistent response to impoundment with data from multiple scales. Overall, the author did a good work. But I also have some comments and suggestions.
1) Line 86-91, the author clarified their research question, which is fine. But I would suggest to include some expectations/hypothesis, e.g., what does author expect diatom community, diversity etc. differ among control VS impacted sites? Are these show consistency in different region?
2) Line 274-278, for exploring the relationships between water quality and benthic algal responses, the author only employed a correlation analysis, which in my opinion is not sufficient. With such results, we are not sure if these correlations are consistent in different regions, i.e., for some indices, the main driver will be different. Therefore, I would recommend a linear mixed model for this part, that is, take regions and season as random effects, while water quality variables as fixed effects.
3) Line 323-325, author stated that higher TDI in Midlands indicated more eutrophic conditions. I am wondering if nutrient concentration support this?
4) Line 352-354, the author said that motile and high-profile taxa are typically prevalent in low disturbance conditions. This is not right, only high-profile taxa adapt to low disturbance, see
(Wu et al., 2019). This also means Line 354-355 is wrong, please check below references.
Wu, N., Thodsen, H., Andersen, H.E., Tornbjerg, H., Baattrup-Pedersen, A., Riis, T., 2019. Flow regimes filter species traits of benthic diatom communities and modify the functional features of lowland streams - a nationwide scale study. Sci. Total Environ. 651, 357–366. https://doi.org/10.1016/j.scitotenv.2018.09.210
5) Suggestions for visualization. The author showed their results mainly with tables, which is fine but not very intuitive. Especially for Table 3 and Table 4, I noticed that author do have plots for these two tables in supplementary, so please do the reverse way. In addition, for the plots, significance can be indicated with letters. As for tables, more details like standard deviation, chi-square value for Kruskal-Wallis analysis should be included.


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

Input:
Title: EVALUATION OF PERFORMANCE OF LEADING ALGORITHMS FOR VARIANT PATHOGENICITY PREDICTIONS AND DESIGNING A COMBINATORY PREDICTOR METHOD: APPLICATION TO RETT SYNDROME VARIANTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: a.	The overall manuscript is not difficult to follow, however, some sentences and terms used are still confusing. For example, they used meta predictors and empirical in-silico predictors interchangeably.
b.	Some mis-spelled words are present which is disturbing for the audience to read, such as in line 125 ‘tis’ and line 138 ‘photogenic’.
c.	Some references are not in the same style as the rest, for example, line 220 ‘Li et al.[24]’. Also it was not correctly indexed. Some material lacks reference. For example, line 69, ‘ACMG recommendations’.
Experimental design: a.	The design of the experiment is standard. The research lacks novelty and the idea of ensemble scores have long been adopted in the field.
b.	The methods described are not clear enough for the audience to understand the logic and steps behind the actions. For example, 1) how to decide which predictor outperforms another based on sensitivity, specificity, accuracy and MCC altogether? 2) How are the different cut-off points for pathogenicity decided and why there are NAs in them?
Validity of the findings: One major concern regarding the quality of the manuscript is their method. The rationale to select top four predictors in each category to form their ensemble predictor is not clear. It is possible that other combinations of predictors would yield better result, but the authors presented no such information for the audience to judge their method. Also, ClinVar and HGMD (used as training data by many predictors being compared) have overlapping SNVs, and the study did not take this into consideration which would lead to bias in their conclusion. In sum, the conclusion is not well-supported by the study design and results.
Additional comments: No additional comments


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

Input:
Title: EVALUATION OF PERFORMANCE OF LEADING ALGORITHMS FOR VARIANT PATHOGENICITY PREDICTIONS AND DESIGNING A COMBINATORY PREDICTOR METHOD: APPLICATION TO RETT SYNDROME VARIANTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: see General comments for the author
Experimental design: see General comments for the author
Validity of the findings: no comment
Additional comments: This paper describes the selection of a suitable subset of the clinvar database and the use of a machine-learning method to combine the results from eight predictive programs to predict the clinical consequence of mutations.  They started with twelve predictive programs and selected the best eight for their method.  The method appeared to perform better than using each program alone or to using only two methods as in a previous study.  They then used the method to predict the clinical outcome of the mutations that were previously labelled as possibly benign, possibly pathogenic, or variants with unknown consequences.  The findings should interest scientists in the field.

The authors might want to address the following issues before publication:
-	The best method used appear to be random forest but inconsistent statements occur earlier in the manuscript. For example, “Purpose: We implemented feature selection and support vector machine (SVM) classification methods”
-	How were the results in Fig. 1 obtained?  Did they run the twelve predictive programs themselves?  Or the results came come https://sites.google.com/site/jpopgen/dbNSFP?
-	Were the results in Fig 2 averages from the independent predictors and averages from the ensemble predictors?
-	Why were the results for random forest from Table 5 and Table 6 different?
-	In table 5: recall is the same as sensitivity: can remove one of them.
-	There are a number of grammatical mistakes in the manuscript.  The authors might want to proof-read the article.


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

Input:
Title: EVALUATION OF PERFORMANCE OF LEADING ALGORITHMS FOR VARIANT PATHOGENICITY PREDICTIONS AND DESIGNING A COMBINATORY PREDICTOR METHOD: APPLICATION TO RETT SYNDROME VARIANTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment, the article is very well written and background and aims are both well established with sufficient references
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors performed classifications of pathogenic and benign variants using multiple predictors by selecting the best combination. In general, the manuscript is well written and rational was very nicely explained. Since there are multiple variant scores both independent and ensemble, and they are all widely used to annotate variants, this manuscript would be a great resource to refer to when one would like to compare multiple scores and their performance. However I have some concerns in the decision of the feature selection and application of the model. My specific comments are below.

1.	I see that the authors evaluated each predictor to rank them. But I did not get why top 4 from each independent and ensemble are the best combination. It could be top 3 or only top 4 from ensemble predictors may perform better than including independent predictors. If the authors tested other combinations, those results should be mentioned in the manuscript. Otherwise, it needs to be clarified how and why they decided to take top 4.

2.	Authors used known pathogenic and benign variants from Rett syndrome to validate their model. Those are the only independent datasets used in the manuscript. Even though the authors showed the high performance of the model with the cross-validations, the number of variants used in the application for Rett syndrome was considerably small, especially there was only a single benign variant. It would greatly improve the validation if there were larger independent sets to test.

3.	In practice, if someone wants to apply the model proposed in this manuscript, they would have to re-do what authors did (i.e., train the model again). I believe it should be able to share the pre-trained model from Weka pipeline. The authors should consider depositing the pre-trained model into a free online repository.

Minor comments
1.	For fig. 1, it would be more readable if independent and ensemble predictors are labeled and also ordered by the rank (so that it’s easy to see which ones are used for the final model)
2.	For fig. 2, the legend in the figure is “Meta” predictor while it is “ensemble” in the legend text. Not limited to the fig. 2 but across the manuscript, it is better to harmonize to one of the other.
3.	Typo in line 125, “this reduced datasets tis” should be “this reduced dataset is”
4.	Typo in line 135, “as like benign/pathogenic” should be “as likely benign/pathogenic”


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

Input:
Title: EVALUATION OF PERFORMANCE OF LEADING ALGORITHMS FOR VARIANT PATHOGENICITY PREDICTIONS AND DESIGNING A COMBINATORY PREDICTOR METHOD: APPLICATION TO RETT SYNDROME VARIANTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No further comments.
Experimental design: I am not totally convinced by the authors' response but it's up to the editor to decide.
Validity of the findings: I don't think the authors addressed my question regarding the overlapping SNVs.
Additional comments: No additional comments


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

Input:
Title: EVALUATION OF PERFORMANCE OF LEADING ALGORITHMS FOR VARIANT PATHOGENICITY PREDICTIONS AND DESIGNING A COMBINATORY PREDICTOR METHOD: APPLICATION TO RETT SYNDROME VARIANTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: satisfactory.
Experimental design: satisfactory.
Validity of the findings: satisfactory
Additional comments: the manuscript has been 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: EVALUATION OF PERFORMANCE OF LEADING ALGORITHMS FOR VARIANT PATHOGENICITY PREDICTIONS AND DESIGNING A COMBINATORY PREDICTOR METHOD: APPLICATION TO RETT SYNDROME VARIANTS
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: My questions/concerns were sufficiently answered and I have no further 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: ARBOREAL TWIG-NESTING ANTS FORM DOMINANCE HIERARCHIES OVER NESTING RESOURCES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript presented by Yitbarek and Philpott  presents a very comprehensive and novel statistical analysis of the dominance hierarchies of arboreal twig-nesting ants  over nesting resources. The most prominent shortcoming that I find in this manuscript is the lack of focus towards emphasizing the significance of the findings of the study and their contribution for filling the identified research gap. The effort displayed by the authors in working out a comprehensive and novel statistical method for quantitatively analysing the dominance hierarchies of arboreal twig-nesting ants is highly commendable. However, I observe that the authors have not pitched the manuscript in a way that emphasizes the significance of these findings and their contribution for filling the identified research gap. Provided that these minor revisions will be addressed upon submission, I recommend this manuscript for publication.

As an immediate revision, I suggest the title to be changed as “Arboreal twig-nesting ants form dominance hierarchies over nesting resource”, since this study has focused only on twig-nesting ant species. The existing title, in my opinion, covers a wider range which includes all sorts of arboreal ants, not just twig-nesting ants.

The results that the authors have obtained for dominance hierarchy uncertainty/steepness are quite an interesting and significant finding. To enhance the weight of this finding I suggest including a more detailed description of steepness and how it contributes to community structure and species coexistence in the discussion section. For example, Sánchez-Tójar et al., 2017 provides a descriptive insight of steepness as a source of uncertainty of dominance hierarchy (Sánchez‐Tójar, A., Schroeder, J. and Farine, D.R., 2018. A practical guide for inferring reliable dominance hierarchies and estimating their uncertainty. journal of animal ecology, 87(3), pp.594-608).
Experimental design: While appreciating the effort of authors for identifying the significant research questions existing, I suggest supporting the existence of these research gaps with appropriate citations. For example, Stuble et al., 2017 provides a good insight of the existing knowledge and limitations involved with this research question (Stuble, K.L., Jurić, I., Cerda, X. and Sanders, N.J., 2017. Dominance hierarchies are a dominant paradigm in ant ecology (Hymenoptera: Formicidae), but should they be? And what is a dominance hierarchy anyways. Myrmecological News, 24, pp.71-81.).

I agree with the authors’ point presented in lines 285-290, that there can be factors other than dominance hierarchy, influencing distribution and coexistence of arboreal ant communities. I suggest  that this statement will carry more weight in the manuscript if a suitable regression analysis can be conducted to determine to what percentage does ant distribution and coexistence depend on dominance hierarchies. According to the information given in 116-118 I presume that the data collected from surveys on ant abundance/distribution and the dominance hierarchy rankings stated in Table 1 will serve this purpose.
Validity of the findings: Since the beginning of the manuscript the authors’ main objective had been to present  novel statistical analyses to explain dominance hierarchies of arboreal twig-nesting ants over nesting species. Hence, the paragraph coming under discussion section (lines 228-251), elaborating existing knowledge on dominance hierarchies over food resources in ant communities fails to make a direct connection with the results obtained by the authors and the ultimate research question addressed by the manuscript. I suggest further elaboration on resource utilization in nesting sites and connection of that information with the results obtained on competition shown by different species over nesting sites. For example, Adams et al., 2019 provides some insight into how resource availability in nesting sites contribute to community structure.  (Adams, B.J., Schnitzer, S.A. and Yanoviak, S.P., 2019. Connectivity explains local ant community structure in a Neotropical forest canopy: a large‐scale experimental approach. Ecology, 100(6), p.e02673.)  In fact, the information included in lines 290-303, on how nest site factors such as canopy structure, tree size and nest entrance size, gives a comprehensive explanation on how dominance hierarchies over nesting sites affect the species coexistence, based on existing knowledge and findings as well as existing limitations and knowledge gaps.  Hence, I suggest combining these two sections.  It will better establish the  relationship between dominance hierarchy and  variability observed in the results in resource utilization in nesting sites, shown by coexisting species arboreal twig-nesting ant species. This relationship will add more weight to the answers provided by this study when addressing the major research question of how dominance hierarchies contribute to species coexistence.

The authors have presented a very interesting and novel approach for quantifying dominance hierarchies in arboreal ant communities and have made a good attempt to explain species coexistence in ant communities through it. However, in the conclusion section, I fail to see the weight and significance of those findings and their contribution to answering the main research question stated at the beginning. Rather, I see background  information and suggestions for followup research included in the conclusion, which are content that could be moved to the discussion section. Hence, I suggest re-writing the conclusion in a way that the findings of this study are highlighted and emphasizes its contribution for filling the existing research gap.
Additional comments: I suggest moving the sentence, in lines 168-169, “The nodes in the network represent…” to the figure caption to improve the comprehensibility of the figure.

I commend the authors for their successful attempt of analyzing dominance hierarchies of arboreal twig-nesting ant species using a novel and comprehensive statistical approach, based on extensive field and lab work added to thorough literature review provided. Also I appreciate the manuscript written in unambiguous English. So I recommend this manuscript for publication, given that the above mentioned minor revisions are addressed and the manuscript is pitched in a manner that the significance and contribution of this study is emphasized to make a bigger impact.