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: C3 PLANT ISOTOPIC VARIABILITY IN A BOREAL MIXED WOODLAND: IMPLICATIONS FOR BISON AND OTHER HERBIVORES
Review round: 1
Reviewer: 2

Output:
Basic reporting: fine
Experimental design: fine
Validity of the findings: fine
Additional comments: This manuscript provides novel data on the carbon and nitrogen isotope compositions of plants in Elk Island National Park, Canada. There are not many studies that provide raw CN isotopic data for plants in such ecozones, so the manuscript is quite welcome from that standpoint. The main insights are not really novel, but I don’t see that as a problem. This is simple baseline work that many are too lazy to do before archaeological application, so score one for the author there. The sample sizes are small for a study of this nature, especially for the November sampling period. This is not something that can be remedied, but I don’t think it is fatal. It might be wise to acknowledge this, though, especially as it can make comparisons between various data subsets a bit problematic, and might have relevance for the discussion of skewness.

I would say the one area where this could really use some revision would be the discussion. For instance, in discussion of the skew in nitrogen isotope data, one can readily look to literature from other parts of the world. And one could dig a bit deeper into the habitat-related nitrogen isotope results. In short, the results are not terribly surprising, especially as the aridity effect is quite subtle--something like 2.6 permil for an order of magnitude change in precipitation. Thus, one might only expect miniscule changes here, and these should be largely undetectable given the variation and sample sizes. Much more likely to be driving things would be things like changes in nitrogen deposition by herbivores etc. and wetland related issues that abound in the literature (and yes, this complex and sometimes contradictory).

Anyway, my suggestion would be to take a good day to flesh out the discussion, and that would include using literature from other parts of the world when it comes to things like habitat or plant part based differences. C4 biomes are perfectly acceptable comparators in the discussion, and this would make the contribution more broadly useful.


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

Input:
Title: C3 PLANT ISOTOPIC VARIABILITY IN A BOREAL MIXED WOODLAND: IMPLICATIONS FOR BISON AND OTHER HERBIVORES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Metcalfe’s manuscript is well-written and provides good background information, but there are a few areas where some additional background and context are necessary.

Lines 48-59: I know this is just a summary of factors that may lead to variation in the carbon values of C3 plants, but the canopy-effect should be described here since it is offered as a potential explanation of results in the discussion later on.

Lines 74-75: This is a rather minor comment, but it is mentioned in a number of places that having plant isotopic baselines would be helpful to archaeologists/paleontologists. I do not disagree with this point, but a sentence or two specifically demonstrating the applicability would be helpful. A counter to the claim that these studies are important for researchers who study past ecosystems/animal diets would be that the plants occupying these regions of the world in the past were different and/or that the climatic and environmental regimes were different, so how do modern baselines or databases of plant values help archaeologists/paleontologists? Is it general principles – such as archaeologists/paleontologists should be aware that different parts of C3 plants might have different values? Is there value to archaeologists/paleontologists in knowing the specific baseline values?

Table 1: This may be a journal formatting issue, but the inclusion of full citations as a long footnote to the table seems a bit odd. Perhaps include a quick in-text like reference – for example, Zhou et al. 2016 in the ‘Ref.’ column – and integrate the full citations into the overall reference list.
Experimental design: Research question is clearly defined and both analytical and statistical methods are described in sufficient detail. The author provides information on research permits and the permit number is provided in the text.

There are two areas where components of the research design/methods could be clarified.

Lines 129-136: It would be helpful to have some of the numbers of whole-plant samples versus flower/seed samples provided here. Alternatively, an additional table showing the breakdown of the 165 total samples into how many from each habitat in each season, as well as whole samples versus part samples may be helpful to the reader. While this can be reconstructed from Table 2, it takes the reader quite a bit of time to figure out all of the sample numbers.

Lines 144-145: Where the parameters for excluding samples/values is described, the cut-off value for nitrogen gas peaks seems appropriate, but it is unclear whether this means that only nitrogen values were excluded or if both nitrogen and carbon values were excluded for those samples. From Table 2, there are 24 samples without nitrogen values. Are these the values that are excluded, or were there additional samples that did not make it into the final study? Also – what constituted ‘poor reproducibility’?
Validity of the findings: Some minor improvements can be made to the reporting of results and the conclusions.

In the Results section, a summary statistics table may be helpful with means, standard deviations, and medians reported for the various analytical groups (total samples, habitats, seasons, etc.). Some of these are provided in separate tables (Tables 3 and 4), but not all summary statistics are readily available. For instance, on Line 183 and Line 196 reference is made to median values, but the values themselves are not provided. Instead, reference to box-plot figures are provided from which medians can be deciphered/estimated, but it would be much easier on the reader to have these values listed in a table.

Line 182-183: With only 3 samples of horsetail I’m not sure that the statistically significant difference found with forbs is reliable. I am also not sure how such a small sample group would affect the overall ANOVA (for carbon) and Kruskal-Wallis (for nitrogen) tests. Perhaps re-run these without horsetail to see if there is a difference.

Throughout the results, p-values are sometimes given as negative exponents. This is only appropriate for those results with Bonferroni correction; otherwise, p-values reported as negative exponents do not provide useful information. Instead, p-values less than .001 should be reported as p<.001.

Lines 235-246: The finding that nitrogen values are skewed is certainly interesting, but is it possible that this is an artifact of the un-balanced collection of samples by habitat? In Lines 287-291 it is mentioned that nitrogen values are expected to be lower in wet versus dry environments (although, the finding here is in the opposite direction of that expected). Since only 10 nitrogen samples come from wet habitats, is the identification of the skew valid? I do not have an answer to this, but it may be worthwhile to provide other potential reasons for the skew or further justify that the skew is real.

Lines 310-312: It is clear that there are seasonal differences in plant carbon and nitrogen values, and that there are differences in values between plant parts, but at the level of 1-2‰ it is difficult to see how researchers, either archaeologists/paleontologists studying the past or even modern ecologists, could use this difference to infer seasonal patterns from values of animal tissues. When diet-tissue fractionation and individual physiological variation is figured in – will researchers be able to make any claims about seasonal differences or selective feeding on different plant parts (i.e. Lines 280-283)? Wouldn’t these differences just be swamped out unless one had incredibly large sample sizes (say in the hundreds per taxa)?
Additional comments: Metcalfe’s manuscript is an important contribution filling in a major gap in carbon and nitrogen isotopic baselines for C3 plants. In addition to the formal review sections, I have a few minor in-text comments that I will include here.

Abstract – Lines 21-22: I suggest here and elsewhere using ‘bulk’ to distinguish mixed-part samples from specific part (flower/seed) samples.

Lines 34-35: While it is clear what this sentence is getting at, it is awkwardly worded. In particular, the phrase ‘is the foundations’ as the ‘foundation’ being referred to seem to be the ‘significant difference.’ Perhaps just a need to clean up singular versus plural forms.

Line 41: This sentence reads as if herbivores and carnivores occupied different isotopic niches when I think the point is that different isotopic niches can be discerned between both groups of herbivores and groups of carnivores. The former would be expected – herbivores and carnivores should have different isotopic niches. Additionally, as this is a study about plant values, the inclusion of carnivores to the discussion requires additional explanation of how trophic-level plays into carbon and nitrogen values. Since this is not crucial to the current study, I may suggest just removing reference to carnivores.

Line 87-88: Is the motivation to understand bison dietary-selectivity in the present or the past? Or both? This is where the application to archaeology/paleontology versus ecology of the present study is a bit unclear.

Line 107: I think the climatic/environmental information for the Elk Island National Park should have a citation associated with it.

Lines 109-111: The distribution of wood versus plains bison is a bit unclear. In Lines 85-87 it is stated that, historically, wood bison territory was in the north and plains bison territory was in the south, but here plains bison are in the northern part of the park and wood bison in the southern part. Why the switch? When did this happen?

Line 229: Which other studies have found carbon values to be normally distributed? Citations would be helpful here.


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

Input:
Title: COMPARATIVE ANALYSIS OF CODON USAGE PATTERNS IN CHLOROPLAST GENOMES OF FIVE MISCANTHUS SPECIES AND RELATED SPECIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study is original and the manuscript presents relevant data of scientific value. The English language should be improved, mistakes such as those found in lines 27, 29, 171, 186, and 187 make the text less clear and make the reading more difficult.  I would recommend you have a colleague who is proficient in English to revise the text or hire a professional editing service. It would improve greatly the quality of the manuscript.
You should describe better the taxonomic context of the chosen studied species. In line 80 it is said that species related to the genera Miscanthus would be included in the study. It would be clearer for the readers to explain the relationship between the species, all of them belonging to the family Poaceae.
When citing a taxon for the first time, please, include information of family and botanical authority:  Miscanthus Andersson (Poaceae). Once the species full scientific binomial name was written, you can use abbreviate the genera name, for example, Miscanthus sinensis Andersson for the first time, and from now on in the manuscript, you can refer to this species as M. sinensis. It is worthy to note that species scientific name is always in italic, while species family name and taxonomic authority are never in italic. In line 76 add the scientific name for “Strawberry” following the botanical taxonomic rules. Instead of using “higher plants” (like in line 75), a remnant of scala naturae, use the appropriate nomenclature, Tracheophyta, or vascular plants. The use of dicotyledonous should be avoided as well, instead, you should use eudicotyledons or eudicots (APG et al. 2016).
Please, standardize abbreviation. In line 53 you introduced an abbreviation for Chloroplast as “cp”, but you do not use it consistently throughout the rest of the text. Then, I would recommend you to use it throughout the manuscript or do not to mention it at all. In line 116 you start using ENc (lowercase “c”) to refer to “effective number of codons” but later in the manuscript (lines 145 and 148) you use ENC (uppercase “c”).
The figures, tables, and supplemental data available are informative and high quality. Still, I suggest increasing space between graphs in Figure 2 and in the supplemental tables, instead of using different tabs for each species, perhaps would be easier to visualize if all of them are gathered in the same tab.
Experimental design: The analysis you performed could be better explained. I suggest you justify the choices you made during your research. For example, it would be interesting to explain why you chose those three model species specifically or why you excluded specific codons from your analysis. It could help fellow scientists to learn from your paper and perform similar analyses.
Please, explain that the GC1, GC2, and GC3 refer to the GC content of the first, second, and third nucleotide of each codon, respectively.
The concepts of RSCU and RFSC, as well as its computation, were not clear, try to use the formula to explain it better, as done by Wang et al. 2020 (PeerJ 8:e8251 https://doi.org/10.7717/peerj.8251).
In line 124 you refer to an internet address for a tool you used, however, the page is not available anymore.
It would be very helpful for other scientists if you could add to the supplemental material the python scripts used for this study. It could likely improve the number of accessions and citations of this work.
Validity of the findings: The present manuscript sets clearly what are its aims and shows that your research was able to determine the codon usage patterns of the 7 Poaceae species used in this study as well as provide scientific evidence to suggest the use of the three model species as receptor systems for heterologous gene expressions of Miscanthus.
In the results section, I suggest you revise the species and other taxa nomenclature (as suggested before) and table number in line 300, it seems you mean table 3 instead of table 2.
Additional comments: The manuscript entitled “Comparative analysis of codon usage patterns in chloroplast genomes of five Miscanthus species and related species” wrote by Sheng and collaborators is original and presents relevant data of scientific value that will be useful to broaden our understanding of the evolution of codon usage in the plant lineage. However, the text needs some revision and correction by the authors in order to be published.


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 ANALYSIS OF CODON USAGE PATTERNS IN CHLOROPLAST GENOMES OF FIVE MISCANTHUS SPECIES AND RELATED SPECIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Jiajing Sheng and colleagues obtained the chloroplast genomes of five different Miscanthus plants and other two related species from the Genbank database. The values of RSCU and RFSC from seven represent species chloroplast (cp) genome were calculated by Codon W. Then multivariate statistical analysis combined by ENc, ENc-plot, PR2-plot, Neutrality plot analysis was conducted to explore the factors affecting the usage of synonymous codons. Their results showed that the chloroplast genomes of the seven represent species were preference to A/T bases and A/T-ending codons. The codon usage pattern of the seven chloroplast genomes is influenced by multiple factors, which the main influencing factor was nature selection. This study will provide important reference information for evolutionary analysis. However, I have several suggestions as listed below for helping the authors to improve their manuscript.
Experimental design: Rigorous investigation performed to a high technical & ethical standard. Methods described with sufficient detail & information to replicate.
Validity of the findings: Conclusions are well stated, linked to original research question & limited to supporting results.
Additional comments: 1. Line 91-94: The complete chloroplast genomes of Miscanthus floridulus (NC_035750.1), Miscanthus sacchariflorus (NC_028720.1), Miscanthus sinensis (NC_028721.1), Miscanthus x giganteus (NC_035753.1), Miscanthus transmorrisonensis (NC_035752.1), Sorghum bicolor (NC_008602), Saccharum spontaneum (NC_034802.1) with gene annotation were downloaded from the NCBI GeneBank database.

The chloroplast (cp) genomes of five different Miscanthus plants were selected as the research objects in this study. However, it is not clear as to why Sorghum bicolor and Saccharum spontaneum were also chosen as the research objects.

2. Line 95-99: To avoid sampling bias, the CDS sequences were screened from genome-wide data by python script according to the following principles: (1) CDS contains initiation codon (ATG), termination codons (TAA, TAG or TGA) and without intermediate stop codon in the sequences; (2) the number of bases in each CDS must be the fold of three (3) the length of sequence of CDS should be≥300 bp.

I believe that the filtering principle of sequence is reasonable. However, I did not find the reliable references for the filtering principle of sequence. I have to doubt whether this principle is rigorous.

3. Line 122-124: The RSCU of each codon was obtained from the sequence files of the high and low groups according to the cusp function of emboss (http://bioweb.pasteur.fr/seqana/interfaces/codonw.html).

The following occurs when the web address (http://bioweb.pasteur.fr/seqana/interfaces/codonw.html) is opened, Cannot GET /seqana/interfaces/codonw.html.

4. Line 131-135: To further explore the codon usage patterns in the seven species of Miscanthus and their relatives, codon usage bias data of four model species including Escherichia coli; Saccharomyces cerevisiae; Populus trichocarpa and Arabidopsis thaliana were downloaded from the Codon Usage Database (http://www.kazusa.or.jp/codon/cgi-bin/showcodon).

The detail of codon usage data of the four model species in this paper is not clear.

5. Line 151-154: PR2-plot is a graphical analysis takes G3/(G3+C3) as the abscisic and A3/(A3+T3) as the ordinate, which is performed to explore the composition of the four bases at the third site of amino acids (Sueoka 1999). The pattern of splashes around the central spot (A=T, C=G) indicate the extent and orientation of the base offset.

The most original literatures about PR2 calculation method are Sueoka 1995 and Sueoka 1999 (Sueoka N. 1995. Intrastrand parity rules of DNA base composition and usage biases of synonymous codons. Mol Evol 40:318-325. Sueoka N. 1999. Translation-coupled violation of Parity Rule 2 in human genes is not the cause of heterogeneity of the DNA G+C content of third codon position. Gene 238:53-58.). The four-codon amino acids, composing alanine, glycine, proline, threonine, valine, arginine (CGA, CGU, CGG, and CGC), leucine (CUA, CUU, CUG, and CUC) and serine (UCA, UCU, UCG, and UCC) (Sueoka 1995), should be considered in the calculation of PR2.

The whole paper is lack of innovation. There is no in-depth expression analysis of codons. In spite of this, I would like to thank the authors and encourage them to improve and complete their work.


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

Input:
Title: COMPARATIVE ANALYSIS OF CODON USAGE PATTERNS IN CHLOROPLAST GENOMES OF FIVE MISCANTHUS SPECIES AND RELATED SPECIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is a simple and interesting study that investigates codon usage patterns of cp of five Miscanthus and two related species. However, I suggest that a major revision needs to be done before the article is suitable for publication. Listed bellow are some major points that need to be fixed:

   1- The English language should be improved to ensure a clear understanding of the text. Some sentences are ambiguous or built using incorrect sentence structure. Typos were also observed. Some examples where the language could be improved are:
Line 54 – please remove “the” before small sizes
Line 72-74 – unclear sentence. Please re-write. Do you mean that the synonymous codon bias have been proven to exist in those cited species? Or the synonymous codon bias is applicable to something else? If so, for what?
109-110 – Sentence is not adequate. Please rephrase it as a possibility.
117 - please replace “reflecting” by “reflects
161 – The word “Meanwhile” doesn’t fit in the sentence
164-166 – Is this already a result or the possibilities? If the last, I would suggest adding an “if” or “when” at the beginning of the sentence.
171- please replace “compares” by “compared”.
206 – please replace “closely” by close

   2- Your introduction needs more detail. I recommend using the reference: Plotkin, J., Kudla, G. Synonymous but not the same: the causes and consequences of codon bias. Nat Rev Genet 12, 32–42 (2011). https://doi.org/10.1038/nrg2899  to improve the background information on lines 68-70. I also suggest that you improve the description at lines 79- 86 to provide more justification for your study. Please re-write the paragraph highlighting the aims, objectives or main questions that your work is going to address as well as possible hypothesis that you have.

  3- Please improve the caption of the figures so they are self sufficient. Also, Figure 1 needs a title on the X axis.
Experimental design: I liked the idea of performing a study only with data that is already available on a public database. It is an interesting work but as mentioned before I would like to see the research questions clearer defined. I would also recommend inserting a small explanation on why choosing the four model species. There was some ambiguous information that needs to be addressed, for example:
Line 125-127 – Have you calculated the RSCU as described on lines 107-110 or have you obtained them from the files?
Line 164-166 – Is this already a result or the possibilities? If the last, I would suggest adding an “if” or “when” at the beginning of the sentence.
Validity of the findings: The study found interesting results and suggests appropriate exogenous expression receptors for Miscanthus and related species. However, I think that the results and discussion sections need to be restructured. There was lots of information contained in the tables that were repeated along the text. I also noticed that some of the discussion is embedded in the result part. I suggest the authors to include further comparison with other studies in the discussion section as well as clarifying what are the implications (e.g evolutionary consequences) of your findings. For example, in lines 340-342 please cite which are those multiple factors influencing the codon usage bias. You also cite that: "no common optimal codon was defined in the seven represent species", are there any hypothesis on why that occurs? Was that anticipated?
Additional comments: The study has a lot of potential but it needs to be restructured according to the major concerns listed above. There are also minor revisions that require attention:
1. Please pay attention to species names along the text and the reference list - they all need to be in italics.
2. Line 140-148 – Is Enc or ENC the correct abbreviation?
3. Line 59-60 – is there any reference for this sentence? Might be out of context.


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 ANALYSIS OF CODON USAGE PATTERNS IN CHLOROPLAST GENOMES OF FIVE MISCANTHUS SPECIES AND RELATED SPECIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The writing quality of this paper has increased greatly since the first submitted version. However, I noticed some inconsistencies in the botanic taxonomic nomenclature, such as genus names not italicized (lines ) or the absence of taxonomic authority (lines ). I recommend checking on the botanical taxonomic nomenclature to correct these minor mistakes.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: COMPARATIVE ANALYSIS OF CODON USAGE PATTERNS IN CHLOROPLAST GENOMES OF FIVE MISCANTHUS SPECIES AND RELATED SPECIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article must be written in English and must use clear, unambiguous, technically correct text. The article must conform to professional standards of courtesy and expression.
Experimental design: Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.
Validity of the findings: Impact and novelty not assessed. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.
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: EFFECT OF VASCULAR RESECTION FOR PERIHILAR CHOLANGIOCARCINOMA: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: - Peri-hilar cholangiocarcinoma should be used instead of hilar cholangiocarcinoma (Cholangiocarcinoma Working Group, Dig Liv Dis 2020)

- I can't find any of the following ref: previous meta-analyses Wu 2013, Chen 2014, Abbas 2013, nor the last italian guidelines (see above). wu and chen's reviws were only on PV resection, whereas Abbas' was on vascular resection generally

- Manuscript would (really) benefit of a review by a native English speaker

- Fig 4B is cited after FIg 4C, Fig 4A is not cited in the results section

- Fig 4: legends are not clear, the authors should state in which direction vasc resection or no vascular resection favors (i.e. I have the impression that no vasc resection favors stage 3/4 UICC, but when I read your text it is quite the opposite) ; maybe the scale of fig 4C should be changed so we can actually see all the plots?

- for more clarity, an axis "favors XXXX" should be added to each funnel plot so the reader easily understand what vasc resec favors
Experimental design: - Introduction/research question/discussion : I think that portal vein resection and hepatic artery resection are totally different attitudes. However, the authors do not distinguish between both in the introduction. On the one hand, there is a intimacy in the hepatic pedicle between hilar confluence and PV, does routine PV resection improves oncological results without significant increase in morbidity? on the other hand, HA resection is a rare event and is performed mostly in highly selected patient, sometimes with anatomical variations.

- in table 1, it would be useful to distinguish PV resection/HA resection/PV+HA resection in the numbers of patients assessed

- methods and ethics are OK
Validity of the findings: - The rationale distinguishing PV from HA should be clearer both in introduction, results and discussion
Additional comments: There are not many meta-analyses on that specific topic, the last one being Abbas' in 2014. The present meta-analysis has almost 2,000 patients more and newer studies. However, from my surgical perspective, (i) it does not bring new data to the pre-existing literature, (ii) it would be very important to distinguish between PV and HA resection. As for now, I think that most surgeons agree that a PV resection should be made in specialized centers when deemed necessary because of suspicion of PV encasement, and that HA resection should be either not recommended or reserved to very selected cases. Introduction, aims, methods, results and discussion should be made according to PV or HA resection, but not "vascular resection".

Minor remarks
- line 50: resection surgery and transplantation
- line 178: capital letter is missing
- please review English


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: EFFECT OF VASCULAR RESECTION FOR PERIHILAR CHOLANGIOCARCINOMA: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Authors could provide more clarity on the primary and secondary outcomes of the study. I would suggest an additional paragraph in the Methods section of the manuscript detailing the primary and secondary outcomes that the authors intended to study in this meta-analysis, together with definitions for these outcomes. For instance, it is not precisely apparent what the authors mean by “total morbidity” or “hepatic insufficiency”.

There were clerical errors in the paper:
1.	Line 129: Figure 2B shows a total of 2189 participants in the study, but the authors report 11 studies with 1182 patients in total. Please clarify.

Certain phrases were cumbersome and could use some editing. Kindly make edits to the following:
1.	Line 59: “efficacy and security of vascular resection” – the authors might have meant safety instead of security. If I have misinterpreted what the authors meant to convey, please make edits correspondingly
2.	Line 62: “efficacy and security of vascular resection” – see above
3.	Line 148: “who did and did not treated with” – please paraphrase. This is grammatically incorrect. Something along the lines of “between patients who were treated with and not treated with” might be acceptable.
4.	Line 151: “who did and did not treated” – see above
5.	Line 159: “who did and did not treated” – see above
6.	Line 160: “who treated with hepatic” – please paraphrase.
7.	Line 181: “just simple bile duct section initially” – please paraphrase.
Experimental design: Line 112: Article selection process was not immediately clear. In figure 1, before the authors reviewed the abstracts to decide if they were suitable for inclusion, 642 records were deemed irrelevant and excluded. This represents more than half of the initial number of records that were identified after duplicates were removed. Kindly elaborate on why these records were excluded and how they were deemed to be irrelevant. Article selection is of paramount importance and when improperly performed, would render the conclusions derived naught. I would suggest to the authors that they use the PRISMA Flow diagram, accessible at http://prisma-statement.org/prismastatement/flowdiagram.aspx, to report their article selection process. I would suggest to the authors to elaborate further on their article selection process in the manuscript as well.

Line 94: Could the authors elaborate on the methods used to approximate the Hazard Ratios when they were not immediately available in the article?

Line 105: I did not note the use of any inverse-variance methods for meta-analysis of dichotomous variables in the manuscript. Could the authors clarify when this method was employed?

Line 106: Suggest to the authors include Mean Difference as an effect measure in their statement for completeness. Something along the lines of “… forest plots and presented as Odds Ratios and Mean Differences and 95%CIs” would be acceptable.

Lines 240 to 245: The authors highlighted important limitations in their discussion. Could they elaborate on some of the processes that they undertook to overcome these limitations when conducting their study?
Validity of the findings: Line 144: The meta-analysis of Intraoperative blood loss is quite heterogenous and has an I2 of 89%. Could the authors kindly investigate further and discuss why this might be the case.

Line 160 and 161: “hepatic artery resection and reconstruction showed lower survival rates”. This statement, at face value, is not supported by the findings laid out by the authors in Figure 3E, which shows statistically insignificant differences in the Odds of survival at 1- and 3-year intervals. I would suggest to the authors to qualify or retract this statement.

Line 231: The meta-analysis of R0 resection margin is quite heterogenous and has an I2 of 70%. Could the authors kindly investigate further and discuss why this might be the case.
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: EFFECT OF VASCULAR RESECTION FOR PERIHILAR CHOLANGIOCARCINOMA: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Appreciate the response
Clarity of text has significantly improved
Experimental design: Queries adequately addressed and changes reflected in the manuscript
Validity of the findings: 1. Analysis of high UICC stage, Vascular Invasion and Lymph Node metastasis seem to suggest that more advanced disease could be a confounder for poorer clinical outcomes. The authors' own analyses showed that these factors were more commonly present in patients undergoing vascular resection, yet these factors could also independently lead to poorer oncological outcomes, as evidenced by other authors in the past (eg Lurje et al. 10.1016/j.ejso.2019.04.019). Authors themselves also appear to be cognizant of this possibility - as evidenced by authors' arguments within lines 297 to 300 of the manuscript. I would suggest to the authors that it may be possible to implement further statistical methods to see if poorer clinical outcomes were indeed attributable to the procedural risks incurred by undergoing vascular resection, such was by performing subgroup analyses or meta-regression. However, I am also appreciative of the fact that there may not have been enough data for this to be possible, especially in the context of hepatic artery resection, where there are already limited number of studies for each meta-analysis. Suggest the authors to further look into this.

2. Authors suggested in line 304 that PVR may be acceptable for selected patients - suggest to the author to elaborate upon which patients may belong to this category.
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: LARGE-SCALE DIFFERENCES IN DIVERSITY AND FUNCTIONAL ADAPTATIONS OF PROKARYOTIC COMMUNITIES FROM CONSERVED AND ANTHROPOGENICALLY IMPACTED MANGROVE SEDIMENTS IN A TROPICAL ESTUARY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The major reported findings includes: 1) the environmental factors of organic matter, pH, salinity, dissolved oxygen and lead were significantly correlated with the observed shifts in the communities; 2) contributions of specific taxa to the functional potentials was negatively correlated with biodiversity, such that fewer number of taxa in the conserved area contributed to the majority of the metabolic potential; 3) the contamination by domestic sewage generated a eutrophic environment that may have led to the extinction of taxa observed at the conserved site.
Experimental design: Good. more detailed information reference the "General comments for the author".
Validity of the findings: Good. more detailed information reference the "General comments for the author".
Additional comments: In this MS, the authors focused on the 16S rRNA gene sequencing to investigate the difference communities between impacted and conserved in mangrove sediment. The major reported findings includes: 1) the environmental factors of organic matter, pH, salinity, dissolved oxygen and lead were significantly correlated with the observed shifts in the communities; 2) contributions of specific taxa to the functional potentials was negatively correlated with biodiversity, such that fewer number of taxa in the conserved area contributed to the majority of the metabolic potential; 3) the contamination by domestic sewage generated a eutrophic environment that may have led to the extinction of taxa observed at the conserved site. The weakness as described below, which should be improved upon before acceptance.

More specific notes below:

Line 49: The reference format in the text should be unified. Checking the reference format in the full text.

Line 101-102: Coordinate symbols should be unified

Line 112：What is the material of the cylindrical sampler mentioned, and does it have an effect on environmental factors of the mangrove sediment such as heavy metals?

Line 121: provide what instruments and method were used for analyzing the environmental factors of heavy metal.

Line 154: “(S02 Fig.,)”, check this sentence.

Line 154-159: “SILVA database (Silva SSU 132), (McDonald et al. 2012), (see Supplemental Methods).” check journal style of language in the full text.

Line 160: “… (Dixon 2003)in …”, check this sentence.

Line 163, check this sentence.

Line 218: It is mentioned that fig2B is a NMDS plot, and the annotations are a PCoA plot. If it is a NMDS plot, please provide the stress value.

Line 220: The statistically significant of p values required less than 0.05, checke the sentence “We found significant (p < 0.1) differences between the population
structures of the two communities, suggesting that the environmental effects of the urbanization on the mangrove has generated a significantly different microbiome.”

Line 236-237: “Many of the families that were abundant in the impacted mangrove site were absent in the sediments of the conserved area.” Rewrite this to provide some details.

Line 285: What is the typical range of salinity in mangrove sediment? Will the salinity variation be significant and expected to impact the bioavailability in the study?

Line 302-309: How do these environmental factors (dissolved oxygen, pH …) affect the microbial community. Rewrite this to provide some details.

Line 302-309: “This is in accordance with our results where the Desulfobacterales taxa was also an important contributor to carbon and nitrogen metabolisms in both mangrove areas.” refer this sentence to the figure/table.


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: LARGE-SCALE DIFFERENCES IN DIVERSITY AND FUNCTIONAL ADAPTATIONS OF PROKARYOTIC COMMUNITIES FROM CONSERVED AND ANTHROPOGENICALLY IMPACTED MANGROVE SEDIMENTS IN A TROPICAL ESTUARY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The standard of English is good and generally well structured. Raw data has been provided.

Some detail is missing from the introduction and discussion (see general comments for more information) and the references provided are not always the most suitable (or relevant) to the associated statement.
Experimental design: The study contains original primary research within the aims and scope of the journal.

The reasoning behind the need to fill the knowledge gap is sound.

A lot of important details are missing from the methods section.

The hypothesis or hypotheses need to be adjusted to match what was actually measured in this study.

The replication of samples is very low which does not give the reader strong confidence that the study questions have been adequately answered.
Validity of the findings: Conclusions are well stated but the authors need to give more consideration in the discussion section to the limitations of their study, particularly in relation to level of replication and the methods used to asses sediment function.
Additional comments: General comments
Please be consistent throughout in how the sites are named, sometimes it is “preserved” and other times it is “conserved”.

It needs to be made clearer in all sections that the prokaryotic functional profiles are estimated from the prokaryotic taxonomy and the limitations of this method need to be fully considered in the discussion section.

Introduction
Line 49: Mangroves do not account for more than 70% of the world’s coastline. Please recheck the literature. Of the papers that you reference here, none seem to be relevant/appropriate for this statement: Yao et al 2020 is a study on mangrove bacterial epiphytes, Singh et al 2005 is a review on mangrove nutrient cycling, and Imchen et al 2018 looks at metagenomes for effects of heavy metals an antibiotic resistance. I suggest you seek out some of the many papers that actually measure or review  mangrove area cover.

Also multiple references at the same point should be in the same brackets, not in separate adjacent brackets.

Line 50-51:Again, the references provided here are perhaps not the most appropriate. Find actual studies or reviews that provide mangrove areas for different countries.

Line 53: Again, Moitinho et al 2018, referenced here, is not relevant to the statement at hand. Just because they provide a summary of mangroves in their opening paragraphs, they are not the source of this information. Seek out papers that actually assess carbon sequestration capacity and coastal protection.

So far nearly all of the referenced papers are not particularly suitable as supporting evidence for the accompanying statements. I am not going to check every single reference in the paper to see if it is relevant to the statement. I highly recommend that during the revision the authors take the opportunity to go through and double check that the references they provide are actually relevant to the accompanying statements.

Line 86-88: this study doesn’t really provide understanding of the consequences of urbanisation in the larger ecosystem. It is evaluating local effects near the impact. Consider revising this statement.

Line 89-91: “We hypothesized that the constant input of nutrients present even in only light urban domestic runoff and sewage would lead to a more eutrophic environment in the mangrove sediments close to the urban area.” This is not the hypothesis of the study, nutrients were not even measured. Please adjust the wording of this paragraph to relate your hypothesis to the thing you are actually testing (that proximity to urban environments will increase/decrease/change prokaryotic diversity/composition/function/other)

Line 91-94: “The higher diversity and bioavailability of nutrients favors the colonization of exogenous opportunistic prokaryotes while leading to the extinction of more sensitive endogenous taxa, ultimately creating significantly distinct communities with distinct metabolic profiles.” The authors have not provided any information in the introduction to support this statement. Please provide more information in the introduction summarising what has already been found in studies that have assessed effects of sewage/increased nutrients on mangrove sediment prokaryote community composition/diversity/function. This should inform your hypotheses for what you expect to find.

Please provide justification for why only prokaryotes were assessed in this study and why they are important in the functioning of mangrove sediments.

Line 96: “microbial communities” should be changed to “prokaryotic communities” as the whole microbial community was not evaluated in this study.

Methods & Materials
Line 101-102: saying “located ..between the coordinates...” and then only listing one latitude coordinate and one longitude coordinate does not make sense. You either need to give two sets of coordinates, or reword to say the coordinates are for the centre of the area, or instead just provide two sets of coordinates – one for each of your sampling locations.

Line 112: I do not think “superficial” is the word the authors meant to use here, should this be surficial?
Please provide much more information relating to the sampling sites, sampling locations, and sampling methods. How far are the two sites from each other? How far apart were the replicate samples collected at each site? Where in the tidal zone were the samples collected – was it similar at each site? Were they collected at high or low tide? When were they collected – month/year/season? What mangrove tree species are present at each site? How far from trees were cores collected? Was the sediment type similar at each site – sand/silt/mud/mixed? Were the samples stored and transported on ice to the laboratory? It says 3 composite samples per site – how many cores were combined for each composite sample? What kind of area was covered by the sampling? What was the diameter of the sampling core? Were the samples collected along a transect? From plots? How were sampling locations determined? How far from habitation were the impacted samples located?
This section does not mention how the heavy metals were analysed.
Is says D.O., conductivity, pH and temperature were measured in the water column – how far were these measurements taken from the collected sediment cores? Directly above? Was the sediment underwater at the time of sampling?

Line 128: says amplification of the bacterial 16S rRNA gene but you also assess archaea from this data so “bacterial” should be removed here.

Were any negative or positive controls included in the PCRs and were they sequenced?

What was the range of DNA concentrations of the extracts? It says 2.5 uL of each sample was added to the PCR reactions but that gives no indication of how much DNA was added.

What cleanup method was used after PCR?

How were libraries quantified and pooled?

Line 153-155: was dataset normalised (eg. rarefied) before diversity and community structure comparisons? I note that sequencing depth was lower in the pristine samples (Fig S1) and so therefore without normalisation would already be expected to have lower diversity than impacted samples which is not necessarily wholly related to the sediment samples themselves. I see that the authors have rarefied data prior to differential abundance analyses (lines 175-177) even though it is arguably not considered the best normalisation to use for this type of analysis (eg. McMurdie & Holmes 2014).
Was there any trimming of the dataset based on numbers of reads? For example were ASVs kept in the dataset even if they only had a total of 1 read across all samples?

Line 165: It would be useful to start this sentence saying what PICRUSt was used to assess ie. to  predict the functional profiles of the prokaryotic communities based on their taxonomic composition

It might be helpful to split the Data analysis section up into the bioinformatics processing of the sequencing data, and then statsistical analyses performed. There appears to be information missing relating to statistical analyses. What statistical tests were used to compare diversity measures between sites? ANOVA? What test was used to assess community composition between the areas? Were the data checked for normality and other assumptions? The results section refers to a permanova but this is not mentioned in the methods. The results figures include an NMDS and a PCoA but these are not specifically mentioned in the methods.

Results
I think it is important to know what differences there are in environmental variables between the two sites you are comparing to understand why there are differences in prokaryotic communities. Instead of presenting this in the supplementary information, I suggest that it is included in the results and that differences between the sites are tested statistically.

Line 204: I would be cautious about stating that the communities are mainly composed of bacteria as sequences reads do not necessarily equal abundance. qPCRs with bacteria and archaea specific primers would be required for such a comparison. I would suggest rewording to say that the ASVs in the dataset were mainly composed of bacteria.

Line 204-206: In this sentences when it is referring to number of sequences it is not entirely clear whether the authors are referring to the number of unique sequences (ie. ASVs) or the number of sequence reads. I am assuming the latter but this should be clarified. It would also be useful if the authors could include how many ASVs were in the entire dataset, how many ASVs were detected at each site, and how many ASVs overlapped between the two sites. It should also be made clear whether the numbers provided are from normalised or non-normalised data.

Line 216-220: I would suggest rewording of this sentence to improve readability and accuracy. It makes it sound like the NMDS plot is calculated using a permanova test which is not correct. It would make more sense to present the permanova results when mentioning the permanova test. Also it is probably not necessary to include all the additional methodology wording here that is already presented in the Methods section and the figure caption. Suggested alternative: “Prokaryotic community composition differed between the two sites (insert permanova results) with the samples of each mangrove site forming separate clusters on the NMDS plot (Figure 2B).”

I would prefer to see more detailed stats outputs eg. the degrees of freedom, F, R2 and p values so that the reader can assess for themselves how strong the relationships are. Is the p value presented in line 220 (p<0.1) the results of the permanova? If so, this would not normally be considered significant and should be reworded to marginally significant.

Why is there only one impacted point on the NMDS plot? Is it because the two sites are too different to present on the same plot and the impacted ones are all overlapping because they are less variable? This needs some kind of explanation as this is not a normal NMDS plot. Nowhere in the results is it mentioned that the composition is more variable at the non-impacted site, this is an important point to note and consideration should be given to why. I see that the authors also present a PCoA in Figure 5 which effectively renders the NMDS plot redundant. I would suggest removing the NMDS plot and refer to the PCoA instead of the NMDS when talking about differences in community composition.

I can’t seem to see anywhere in the results where the authors refer to the result presented in Figure 2C. This should be included in the text or the figure removed.

In this same paragraph when presenting the differences in community composition, it would make more sense to present the environmental variables that relate to the community composition instead of coming back to that later on in the results section.

Line 221-222: “suggesting that the environmental effects of the urbanization on the mangrove has generated a significantly different microbiome.” This should be kept for the discussion section rather than the results. I also do not think that this is necessarily supported by the results presented, considering there is only one impacted and one non impacted site with low sample numbers in different locations of the estuary.

Line 223: “Alpha-diversity measured were performed...” diversity measured are not performed, they are not tested. The measures were calculated. Also please include more of the stats output than just the p value eg. “ Shannons diversity index was significantly higher in the impacted sediments compared to the non-impacted sediments (Kruskal-Wallis chi-squared =...., df = ....., p-value = ... )”. Also Figure 3 presents Simpsons diversity but this is not mentioned in the text. Either include in text or remove from Figure 3 to supplementary.

It would be good if the authors could also present the richness (ie. number of ASVs) results here too. In the text and in Figure 3.

Lines 228-240: Much of the text in this paragraph belongs in the methods or the discussion section rather than the results. For example: “Biotic and abiotic selective pressure at the impacted site can alter the relative fitness, and therefore abundance, of taxa. At its most extreme this can lead to native taxa becoming locally extinct and allow for the colonization of exogenous prokaryotes” this is discussion not results. “In order to identify possible opportunistic and extinct taxa in the two mangrove sediments, we identified families with significant (p < 0.05) differences in abundances between sites” this is methods not results. “These results suggest that some taxa present in the conserved site, here representing the most natural conditions, adapted well to the changes at the impacted site, while other groups struggled in the modified environment, being led to extinction” this is discussion not results.
Perhaps provide more info from this analysis like how many taxa were identified as being differentially abundant? How many of these taxa were only found in the impacted and how many were only found in the non-impacted area? Are any of these families known to have important functions in sediments?

Line 258-262: “Importantly, Formaldehyde is a common organic compound found in animal waste, building materials, pesticides, and fertilizers. While formaldehyde assimilation is a common intermediary in the oxidation of methane by methanotrophs, it is also known that the influx can alter microbiomes (Dixon 2003), suggesting that the prokaryotes at this site may have been selected to take advantage of this additional carbon source present in the urban runoff” this is discussion not results.

Discussion
Once the authors have adjusted their hypotheses in the introduction, they should then come back to this in the discussion in relation to how their findings do or do not support the hypotheses.

The authors attribute increased prokaryotic diversity to eutrophication, but nutrients were not measured in this study, and nitrogen addition has mostly been found to lead to decreases in microbial diversity in terrestrial soils (see Wang et al 2018), with similar results observed in nutrient addition experiments with coastal soils (eg. Aoyagi et al 2015; Wang et al 2016; Bulseco et al 2019; Craig et al 2021). The authors should either not attribute the findings to something that wasn’t measured, or consider their findings in the context of other studies and suggest reasons why their results differed.

In relation to the discussion on salinity, although salinity did differ between the two sites, in biological terms the difference between a salinity of 13.3 and 15.1 is unlikely to have much impact.

Please provide discussions around the limitations of the study (eg. small sample size, no replication of sites, functional profiles estimated from taxonomic data, nutrients not measured). Conclusions have been drawn from a very small number of samples from only one “conserved” and on “impacted” site. Due to the different locations in the estuary, two such sites would be expected to have differences in their environmental conditions anyway and this paper provides no clear evidence that the differences in are the direct result of anthropogenic activity.

Perhaps include suggestions for future work that could be done to further assess affects on functioning and the implications. eg. Actual measures of carbon and nutrient cycling in the field need to verify the functional profiling results

The authors continually relate the results to sewage but as far as I can tell from the information provided, the samples weren’t collected specifically near a sewage outlet. Therefore could there be other potential anthropogenic impacts at play? Chemical pollutants? Physical disturbance?

What are the potential implications of the “higher functional abundance of the methane and carbon fixation pathways at the impacted site? Could this lead to changes in greenhouse gas emissions?

Line 400: I think “organs” is supposed to be organisations. Or bodies

Figures
Figure 2A: Each of these panels (impacted and conserved) had three samples. Are the results presented in this figure a sum of all the read from each of the three samples? Or the mean? Median? Please make this clear in the caption. Also, there is a grey colour that is not on the legend. I assume that it means they were unassigned at the class level  but this should be specified.

Figure 2B: The figure caption says it is a PCoA plot but the figure axes and methods in the report say that it is an NMDS plot.

Figure 2C: Why does the x-axis say conserved n=9? should this not be n=3?

Figure 3: I would like to see richness (number of ASVs) added to this figure. Also, Simpson’s index is presented in the figure but not mentioned in the figure caption or in the text. And I don’t understand the difference between the plot on the left and the plot on the right. They both seem to be showing shannons index for impacted and conserved. But the numbers are different??? Is the plot on the left showing mean? and the box plot showing median with interquartile ranges? Even then the differences in numbers don’t make sense. More info needed in caption and I think one of these figures is incorrect. There is no need to present both types of figure so choose one and make sure that the numbers are correct.

Figure 4: This is a nice figure. The authors should extract some of the more important details to present in the results text too.

Supplementary files
Environmental variables: this file provides no units for the numbers in the table. Is it air or sediment temperature? Please specify what D.O. and O.M. abbreviations stand for. Which variables were measured in water and which were measured in sediment?

I can’t find titles or captions for any of the supplemental figures.

Figure S1: should the y-axis title be observed number of ASVs? It does not mention in the paper anywhere clustering of the ASVs into OTUs

Figure S3: y-axis text is too small to read. Also S3B x-axis has a typo “Conservedd”

Figure S4: I have no idea what this figure is showing. It either needs some explanation, or if it is of no use then remove it.

Figure S5: The y-axis title should read proportion not percent to match the numbering used on the scale. I am surprised that only 70-80% of taxa were unassigned at the species level, I typically get a much higher number.

References
McMurdie & Holmes 2014 Waste Not, Want Not: Why Rarefying Microbiome Data Is Inadmissible. https://doi.org/10.1371/journal.pcbi.1003531

Aoyagi et al 2015 Dynamic transition of chemolithotrophic sulfur-oxidizing bacteria in response to amendment with nitrate in deposited marine sediments. https://doi.org/10.3389/fmicb.2015.00426

Bulseco et al 2019 Nitrate addition stimulates microbial decomposition of organic matter in salt marsh sediments https://doi.org/10.1111/gcb.14726

Craig et al 2021 Nitrogen addition alters composition, diversity, and functioning of microbial communities in mangrove soils: an incubation experiment. https://doi.org/10.1016/j.soilbio.2020.108076

Wang et al 2016 Responses of bacterial and archaeal communities to nitrate stimulation after oil pollution in mangrove sediment revealed by Illumina sequencing. https://doi.org/10.1016/j.marpolbul.2016.05.068

Wang et al 2018 Decreasing soil microbial diversity is associated with decreasing microbial biomass under nitrogen addition https://doi.org/10.1016/j.soilbio.2018.02.003


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: LARGE-SCALE DIFFERENCES IN DIVERSITY AND FUNCTIONAL ADAPTATIONS OF PROKARYOTIC COMMUNITIES FROM CONSERVED AND ANTHROPOGENICALLY IMPACTED MANGROVE SEDIMENTS IN A TROPICAL ESTUARY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The standard of English is good and generally well structured. Raw data has been provided.
Experimental design: The study contains original primary research within the aims and scope of the journal.
More detailed information reference the "General comments for the author".
Validity of the findings: Conclusions are well stated, linked to original research question.
Additional comments: The font size and the language style in the full text need to check. The full text requires a strict checking.
For example:
Line 49: “Introductio”. Spelling mistake.
Line 50: “… 137,760 km2 of the world’s coastline …”. Checking this sentence.
Line 162: “Briefly, 0.5g of the …”, and line 175: “… from 0.25g of each sediment …”. A space is required between letter and number.
Line 420: “We calculated the functionalFunctional profiles …”. Spelling mistake.
...


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: LARGE-SCALE DIFFERENCES IN DIVERSITY AND FUNCTIONAL ADAPTATIONS OF PROKARYOTIC COMMUNITIES FROM CONSERVED AND ANTHROPOGENICALLY IMPACTED MANGROVE SEDIMENTS IN A TROPICAL ESTUARY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The language is good but some further clarification in parts of the sampling design is required (see additional comments). Background/context information and references is sufficient. Article is well structured, and raw data is available. The hypothesis is now better suited to the study.
Experimental design: Research question is well defined. The revised version now has much more methodological detail which is useful, there are just a couple of things that could be further clarified (see additional comments).
Validity of the findings: Although replicate numbers are low, and there are multiple additional factors that differed between how samples were collected and analysed between the two sites, the study does provide some evidence that mangrove sediment bacterial community composition is impacted by urbanisation/human factors.
Additional comments: •	Have I interpreted correctly that one site was sampled in July 2018 and the other in February 2020? And different tidal zones were sampled at each site? And the samples for each site were sequenced on different sequencing platforms MiSeq vs NovaSeq with different read lengths (300 cycles vs 2x250 cycles). And the data for each site were processed with different settings for the denoising step. Each of these factors are introducing so many additional differences between the samples that how can you confidently attribute and bacterial community differences to the conserved vs impacted factor? I do concur though with the comments in L472-474 that “…some of the taxa uniquely found in the impacted sediments are recognized as pathogenic and gut microbiome colonizers, we consider that these important differences are correlated with the observed human impacts”. I think this point which is based on presence/absence is sufficient to show that urbanisation does have some impact on the community composition even if some of the other findings, which are reliant on differential abundance of sequence read numbers are a bit more questionable.
•	With regards to the heavy metal analysis – was this conducted on the same sediment samples that were used for the DNA extractions? Or are you using existing data from a previous study on previous samples? If the latter, approximately how far away and when were the heavy metal samples located? It is not ideal to be using heavy metal data from different samples collected from different locations at different times for drawing conclusions of the effects on bacterial communities.
•	L40 “…originally accounting for more than 137,760 km2 of the world’s coastline…” The word originally does not make sense in this sentence.
•	L90 “…we calculated the general patterns of functional potentials in these microbiomes…” I would suggest rewording this to “we estimated” or “we calculated estimated functional potentials”
•	L98: there appears to be a typo here with the letters “remo” after the reference
•	L121-122 “The mangrove trees are composed of the species Rhizophora mangle, Avicennia schaueriana and Laguncularia racemosa” could you please clarify if these same three tree species were present in the vicinity of the sampling area at both sites?
•	L133-134: it says that DNA was extracted from 0.25g of each core. The results indicate that there are a total of three samples for each site, but more than three cores are mentioned for each site in the sampling methods section. Please provide some clarity as to whether replicate cores were pooled prior to extraction (if so then this line should say DNA was extracted from 0.25g of each composite sample), or if the DNAs for each core were pooled after extraction. Or from the NCBI database it looks like there is sequencing data for 9 samples at the pristine site, 3 from each tidal zone, was the data pooled instead for the site? It does not seem to be the same for the other site which appears to have sequencing data for just three samples.
•	L188: It is not understood what the authors mean by “Alpha-rarefaction was calculated…”. Are they referring to the production of rarefaction curves, which are presented in Article S1 Figure 2, or are they saying that they rarefied (normalised) the data prior to analysis of alpha- and beta-diversity? Rarefy and rarefaction are two different terms, one is a normalisation technique and one is a technique assessing species richness from the results of sampling
•	L287-288 “The analysis revealed significantly higher richness and diversity (Shannon’s diversity, Kruskal-Wallis H = 3.857, df = 1, p-value = 0.050)…” Why are statistical results provided in this sentence for Shannon’s diversity but not richness?
•	I note that the authors have added more information regarding the locations of the samples and the number of cores, but it is still a little unclear and requires rewording. For example (L108-111): “At the conserved mangrove site (13°44'34.6"S, 39°03'30.6"W), collection points were 15 meters distant from each other and 3 sediment samples of each tidal zone (supralittoral, intertidal and sublittoral), composed by 3 sediment cores randomly extracted, were collected in July 2018 (de Santana et al. 2021).” From what I understand there were three samples for this site – did each sample consist of one core from each zone mixed together? If so, were these collected along a transect perpendicular to the water? Or were the three samples each representative of one zone? And how do the other three randomly extracted cores fit in? Was each subsample core 15m apart? Or a transect for each sample were 15m apart from each other?
•	Also, with the impacted site (L114) it is unclear if the subsample cores for each sample were 5m from each other, or if the areas in which cores were collected for each sample were 5m apart.
•	Were the sublittoral samples within the mangrove tree area or were these in the water channel beyond the tree line?


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: RELIABILITY OF THE WEIGHT-BEARING ANKLE DORSIFLEXION RANGE OF MOTION MEASUREMENT USING A SMARTPHONE GONIOMETER APPLICATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The reporting is clear but insufficient in its use of existing evidence in some areas. I have covered this more completely in the last section.
Experimental design: The research is well within the Aims and Scope of the journal of submission. The research question is well defined. There are some concerns with how it rationales the 'gap' it fills by not referring to current evidence in the field. The method of investigation needs further explanation but maybe appropriate if described more fully.
Validity of the findings: The findings add to a body of knowledge on the use of smartphones. Further information is needed in the methods to determine the reach or interest it may have. Data has been provided. Analysis requires some attention but what has been completed appears robust. Conclusion is sound.
Additional comments: Thank you for the opportunity to review the manuscript “Reliability of the functional ankle dorsiflexion range of motion measurement in young adults”. The article is well written, however there are some concerns that are prohibiting the recommendation for publication at this point.
Major concern: the measure capabilities of the app are not being compared to standard practice or ‘gold standard’ measures (e.g. analogue or digital inclinometers). The reliability of the app is not in question but the authors need to be very clear that this does not guarantee the outcomes are valid. The app could consistently measure incorrectly. Determining validity of the app against a known standard is strongly recommended prior to continuing for publication. This can be done comparing outcomes of the app to known angles (e.g. compare the app to a digital inclinometer and 0 degrees, 15 degrees and 45 degrees using solid and immovable reference points, such as inclinations on ramps).
Second recommendation would be to reduce the use of abbreviations, particularly non-standardised abbreviations such as ADROM. It introduces a risk of confusion, breaks sentence flow for the reader and rarely has an adequate effect on word count to be of value. I would recommend the abbreviation be replaced with ankle dorsiflexion ROM throughout. Furthermore, it may be prudent to identify the measure as weight bearing (e.g. weight-bearing ankle dorsiflexion ROM).
Final issue is that much of the literature cited in the background and discussion is > 5 years old. Many newer publications review Android and IOS apps in adults and children. It would be good to update some references, particularly as the Tilt-meter app, which is mentioned several times throughout the manuscript, was decommissioned in 2018.

Abstract
including the number of measures made and more information on the participant cohort would benefit.

Background
Good rationale for the need to measure weight-bearing ankle dorsiflexion ROM is given. Further breadth of referencing could be done for the last section where it is currently limited to Williams et al., for using IOS based measures given many other studies have investigated ankle dorsiflexion – e.g:
IOS measures of weight bearing ankle dorsiflexion:
Banwell, H.A., Uden, H., Marshall, N. et al. The iPhone Measure app level function as a measuring device for the weight bearing lunge test in adults: a reliability study. J Foot Ankle Res 12, 37 (2019). https://doi.org/10.1186/s13047-019-0347-9
Carlos Balsalobre-Fernández, Natalia Romero-Franco & Pedro Jiménez-Reyes (2019) Concurrent validity and reliability of an iPhone app for the measurement of ankle dorsiflexion and inter-limb asymmetries, Journal of Sports Sciences, 37:3, 249-253, DOI: 10.1080/02640414.2018.1494908
This section would also benefit from further rationale on why targeting android apps is important. It erroneously states that there are no publications reporting reliability of Android smart phone apps, which is incorrect (see below) – however the argument could be make that the majority of papers published within the last 5 years are all using Apple IOS based goniometers, and those using Android apps are not measuring dorsiflexion, e.g:
Motaz Abdalla Alawna, Bayram H. Unver, Ertugrul O. Yuksel; The Reliability of a Smartphone Goniometer Application Compared With a Traditional Goniometer for Measuring Ankle Joint Range of Motion. J Am Podiatr Med Assoc 1 January 2019; 109 (1): 22–29. doi: https://doi.org/10.7547/16-128

Methods
Could the authors begin this section with a study design description.
Participants: It would be good to include the age, height and body weight range of participants and offer some indication of where they were recruited from (e.g. a convenience sample of university students) and how recruitment was conducted..
Please indicate if inflammatory joint conditions were also an exclusion criteria.
Examiners: Raters need to be explained further. Does the experienced physiotherapist use this measure frequently in practice and had the final year physiotherapy student being instructed on how it was conducted prior to the study? Did they pilot the study first and refine the protocol?
Measurement of the functional ADROM: How was the measurement demonstrated to all participants, as a group on one on one.
Given the physiotherapist measured first each time, does this introduce potential order effects (e.g. increasing available ROM due to stretch from the first measure)?
Can the authors describe the timing and protocol between raters, e.g. could the person rest between? And how were the measures collected (on paper?).
Did the experienced physiotherapist complete and record measures in the presence of the student (or vice versa)?
I also question if 32 people is a large enough sample to ensure that raters don’t have memory of previous results. Also, can the authors discuss the limitations of ‘double dipping’ when you use both legs on the one person (Menz, 2004)
Menz, H. (2004). Two feet, or one person? Problems associated with statistical analysis of paired data in foot and ankle medicine. Foot, 14, 2 - 5.
Further information on the App, e.g. reference to its developers and which operating systems are required etc.,  and is it free to download or involve a subscription etc., .
Statistical procedure
How was all the data managed? It would benefit to include the participant characteristic management here (e.g. Descriptive statistics were used to describe participants…).
Can the authors rationalise the use of ICC random rather than two-way mixed effect (i.e. Model 2, 1 rather than Model 3, 1)?
The authors could consider using Bland Altman Plots to ensure the limits of agreement of raters is within an acceptable level. This could also be used to explore validity if the app is compared with a digital goniometer as recommended.

Results
Please change the word smallest to minimum in line 134.
Discussion
You could incorporate this paper into your discussion given it included a final year physiotherapy student and a novice (parent). It would offer you a clearer comparison that the Williams study.
Gosse G, Ward E, McIntyre A, Banwell HA. 2021. The reliability and validity of the weight-bearing lunge test in a Congenital Talipes Equinovarus population (CTEV) PeerJ 9:e10253 https://doi.org/10.7717/peerj.10253
Sentence beginning line 206 requires a reference.
Please replace comma’s for full stops when reporting the Minimal Detectable change (Lines 220 to 228).

References:
Evans, Rome & Peet, 2012 is not in the reference list. Please review to ensure all other references are 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: RELIABILITY OF THE WEIGHT-BEARING ANKLE DORSIFLEXION RANGE OF MOTION MEASUREMENT USING A SMARTPHONE GONIOMETER APPLICATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript was very clear, easy to read and was conducted well. Considering the language, I just found few suggestions and typing errors. In lines 79-83 (at the end of introduction) I would suggest to make two different sentences instead of one, and in the heading of table 2 there is a small spelling mistake.
In the Table 1, the Range is somewhat confusing, I would suggest to leave that out or show the range more understandable, like 19-27.
Experimental design: Fine, no comments to add.
Validity of the findings: Interesting study, no comments to add.
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: RELIABILITY OF THE WEIGHT-BEARING ANKLE DORSIFLEXION RANGE OF MOTION MEASUREMENT USING A SMARTPHONE GONIOMETER APPLICATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: Some comments in general section
Validity of the findings: no comment
Additional comments: I have read the article ID 58796 under title “Reliability of the functional ankle dorsiflexion range of motion measurement in young adults”.

In this article, the authors reported their work using a mobile goniometer application Spirit Level Plus from Android smartphone and recruited 32 subjects in study. The overall, article writing is good and the results analyses are reasonably thorough. But although, the authors used new application to measure the ankle dorsiflexion, the study is still preferable that if the authors can take the following comments into consideration in preparing the final version.

I recommend that the authors make changes related to the comments below.

Reviewer comments:

-Figure 1, shown (weight bearing lunge test) but at the same time what the degree of knee flexion during test. Maybe the same subject when did re-test the degree of knee is different so I think that the best position is for the knee to be parallel to the foot, and the distance (to the nearest 0.lcm) from the end of the big toe to the wall using a tape measure on the floor and, for more information could see references:

- In line 297(Konor et al.);
- Bennell et al, 1998. Intra-rater and inter-rater reliability of a weight-bearing lunge measure of ankle dorsiflexion. Australian Journal of physiotherapy, 44(3), pp.175-180.
- O'Shea, S. and Grafton, K., 2013. The intra and inter-rater reliability of a modified weight-bearing lunge measure of ankle dorsiflexion. Manual therapy, 18(3), pp.264-268.
- Cejudo et al, 2014. A simplified version of the weight-bearing ankle lunge test: Description and test–retest reliability. Manual therapy, 19(4), pp.355-359.
Therefore, could the authors write how to adjust the angle of the knee during the test and retest please?

 Some comments:

1-	Line 2, I think the title is general, so it’s could be “Reliability of Android smartphone to measure the range of motion of ankle dorsiflexion” or smartphone application or…
2-	Line 25-29, the authors should mention the application or the software name in the Methods
3-	Line 66, “Several different measurement…” should put in new Paragraph
4-	Line 74, the cited “Williams et al. (2015) not mention in references
5-	Line 130, in results “Descriptive…...in Table 1” should be after line 93
6-	Line 221, cited Williams et al. (2013) is the same (Williams, Caserta &Haines, 2013)?
7-	Lines 220-227, these results for left leg or right leg or both?
8-	In conclusions and depending on the results would you recommended for adoption the goniometer application Spirit Level Plus to measure the ankle ROM?


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: RELIABILITY OF THE WEIGHT-BEARING ANKLE DORSIFLEXION RANGE OF MOTION MEASUREMENT USING A SMARTPHONE GONIOMETER APPLICATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The updated manuscript is clearer and more 'relevant' in its literature use.
Experimental design: This is a reliability study reviewing outcomes from a smart-phone App when used by different people over different days. The test-retest design answers the relaibility but does not determine if the measure is true (e.g. validity of the device).
Validity of the findings: Appear sound
Additional comments: Thank you for your attention to the previously raised issues with this manuscript. I hope you agree that it is a more robust article for the effort.

I am surprised at the author's decision to not test the App against known angles (or compare with a goniometre) and calculate validity. This would take less than an hour to conduct/analyse and would give the study credibility. As it stands, the authors have taken considerable effort to determine that a phone App can measure the same outcome (moderately at least) when used by different people but there is nothing to show that it measures correct angles.

The other concern (minor) is that there is still not enough information regarding the App used for readers to understand how they access it. Can the authors please indicate if the App comes pre-installed in Android phones or requires downloading separately (and if so, is there a cost involved) and credit the developers please. I note the third reviewer also commented on this.


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: RELIABILITY OF THE WEIGHT-BEARING ANKLE DORSIFLEXION RANGE OF MOTION MEASUREMENT USING A SMARTPHONE GONIOMETER APPLICATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: I have no further comments on this article. The authors have thoroughly revised the manuscript according to the reviewers comments and suggestions.


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: RELIABILITY OF THE WEIGHT-BEARING ANKLE DORSIFLEXION RANGE OF MOTION MEASUREMENT USING A SMARTPHONE GONIOMETER APPLICATION
Review round: 2
Reviewer: 3

Output:
Basic reporting: Non
Experimental design: Non
Validity of the findings: Non
Additional comments: Most of the comments have been corrected
Thank you


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: TRANSCRIPTOME AND STRUCTURE ANALYSIS IN ROOT OF CASUARINA EQUISETIFOLIA UNDER NACL TREATMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article of Wang et al. addresses an important topic under the context of saline agriculture that will cover more than 50% of the agricultural land within few decades. Thus, understanding the mechanisms underlying plant resilience to salt is of utmost importance to develop appropriate stress mitigation strategies. Casuarina equisetifolia belongs to the group of actinorhizal trees which are distributed worldwide and are able to thrive in a wide range of extreme environments. Together with legumes, these plants belong also to the restricted group of plants that are able to establish root-nodules with N-fixing bacteria, reinforcing their importance in plant research.
Experimental design: The work is original and the research strategy and the experimental design have been carefully drawn. The methods are well described.
Validity of the findings: All data are provided, robust and statistically sound. However, there are some aspects that should be improved (see comments below).
Additional comments: Although the research strategy and the experimental design has been carefully drawn and is clear and concise, overall the MS has major flaws that should be overcome before publication.

(1)	The Language should be fully edited, preferably by specialized services or by a fluent speaker

(2)	The Introduction should include a chapter with the state of the art of the research developed in actinorhizal plants, particulary in the casuarinaceae. Actually, this topic is not included, neither in the introduction nor in the discussion and from the ca. 45 cited publications only 6 concern casuarina research (4 of which are self-citations). I have included in point (6) a list of bibliography that, in my opinion, should be included to enrich the extent of the paper.
(3)	In the Materials and Methods sections, please explain:
-	the characteristics (in relation to salt stress) of the genotype used (selection criteria)
-	the choice of 200 mM NaCl (particularly taking into account that in the introduction 500 mM are referred, it is not clear why did the study was conducted with 200 mM)
-	why was hyrdroponic culture used instead of sandy soils, where C. equisetifolia occurs naturally?
-	why was the impact of the symbiosis with Frankia excluded?
(4)	The results are clear and concise.
(5)	The Discussion is focused on anti-transporters and PCD, rather than in the four gene clusters and their respective interactions to promote stress tolerance; also, it is not clear if the genotype used in this study is tolerant, moderately tolerant or susceptible; a deep comparison with other Casuarina spp., actinorrhiza as well as halophyte species, should be mandatory to increase the relevance of the research conducted under this study, clearly highlighting the novelty of this work; equally important is to discuss the putative advantages (or not) of the symbiosis with Frankia and mycorrhiza.
(6)	Relevant bibliography to be included:
10.3389/fsufs.2020.601004
10.1007/s11738-020-03088-y
10.3390/ijms21010078
10.1016/j.envexpbot.2019.103808
10.1007/s11306-017-1234-7
10.1007/s13199-016-0386-y
10.1007/s13199-016-0425-8
10.1007/s13199-016-0424-9
10.3389/fpls.2016.01331
10.1016/j.jplph.2016.03.012
10.1007/s11104-015-2666-3
10.1016/j.plaphy.2015.07.021
10.1111/j.1399-3054.2006.00633.x
10.1111/j.1747-0765.2006.00005.x
https://www.jstor.org/stable/23616578?seq=1


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

Input:
Title: TRANSCRIPTOME AND STRUCTURE ANALYSIS IN ROOT OF CASUARINA EQUISETIFOLIA UNDER NACL TREATMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: See below
Experimental design: See Below
Validity of the findings: See beow
Additional comments: The manuscript of Wang et al describes an investigation into the salinity resistance of Casuarina equisetifolia via a transcriptome approach. Building off the released C. equisetifolia genome sequence, the system is ripe for global expression studies. However, there are number of flaws or absence of information that affects this manuscript. Some of these problems are significant or others may be explained or rationalized but are not in the text. In general, reads well and the data presented clearly.

1.	The RNASeq data as all high-throughput sequence data to needs to be submitted a repository for public access via NCBI GEO. This requires metadata and actual data submission. You must write about this fact in a data deposition statement. It must be access via the public for validation of these results.  This information is not present.  However, the raw reads are available at NCBI, but the GEO information should also be deposited and described in text.  The statement on raw reads should also be moved to the Material and Methods section under a separate section.
2.	Causuarina equisetifolia and C. glauca that are in symbiosis with Frankia are more tolerant to salt effects than uninnocualted plants (Ngom et al 2018).  Why were these studies done with uninoculated plants? Explain?
3.	How do you know what is a salt effect and not an osmotic effect?  Were any controls run to eliminate that possibility?
4.	There is a lot of literature that is missing in the introduction or discussion on Casuarina and salinity effect For example Ng0m et al 2018; Craco et al 2020 and others.  A quick review of all of the literature would help this study