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: STEROIDAL GLYCOALKALOIDS FROM SOLANUM NIGRUM TARGET CYTOSKELETAL PROTEINS: AN IN SILICO ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: - In several lines, for example, 12 and 51, has been used an ambiguous expression, which needs to change and explain more details about another reported works. At the same time, some lines need commas (16-18).
- The cited references in this work are sufficient, inclusive I consider that which are exhaustive and can be selected fewer references.
- Figure 2 presents shorter labels of the reside; Figure 4 present structures with a different format, which is necessary homologate in the same format and depict a figure with a better resolution.
- This work is self-contained with relevant results to hypotheses, although the work needs most conscious results and diminish the excessive background.
Experimental design: - The work is an original primary research within aims and scope of the journal.
- The author defines, implicitly, the research questions, which have been well defined, based on the kind of interactions presented between the cytoskeletal proteins and steroidal glycoalkaloids and can be relevant to design some compounds directed to these targets.
- About the rigorous research, the homology model depicted in Figure 1, which show the modeled structure of TYB4, is very simple, and cannot homologate a complete protein, therefore it is not a model obtained with a rigorous research.
- The methodology about the optimization of the ligand structures is incomplete (lines 177-178), due is only reported the employed software, but not the level of theory. At the same time, in lines 180-186, the refinement of the selected proteins is mentioned, but the software is not cited.
Validity of the findings: - The obtained results of this work present a novelty, but one of the homology models, TYB4,  it is very simple and cannot represent the basic resides in a whole protein, for this reason, is necessary to obtain a better homology model and replicate the assays on this new model.
- The data is robust and controlled, the performed assays are sufficient to justify the results.
- The work conclusion needs to highlight more aspects of this work, due do not link completely the original research question with the results.
Additional comments: The author performs an in silico analysis of the interactions and pharmacological effects of them, about some steroidal glycoalkaloids from solanum nigrum with two cytoskeletal proteins. In general, the background is a lot of large and need some actual references, but justify the work done.
About the homology models, is clear the used methodology, but the percent of similarity and another measured parameters in a homology model, is not the only way to select the better model, at the same time is necessary to visualize the large and conformation of the protein, due to the possible allosteric interaction in the target surface, which is not the aim of this work, but is part of the obtaining models.


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: STEROIDAL GLYCOALKALOIDS FROM SOLANUM NIGRUM TARGET CYTOSKELETAL PROTEINS: AN IN SILICO ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Author presents an interesting molecular docking study of phytoconstituents against cytoskeletal proteins. Generally, the paper is well written, the Introduction is well laid out as to provide the readers with a general overview of the research question presented herein. However, there were some sentences that had grammatical errors and thus, it is suggested that the authors re-check and re-proof the manuscript for such language problems.

There are specific points for improvements as follows:
- Lines 137-138: The paragraph about Villin is too short. More details should be added here so that it would be of comparable depth and length as the other ABPs.
- Lines 164-168: It is suggested to delete this section as it does not any new information and is redundant with what is to come in the subsequent sub-sections.
- Line 176: Please correct "chEMBL" to "ChEMBL"
- Line 176: Please correct "Pubchem" to "PubChem"
- Line 178: Please correct "Auto Dock Tools" to "AutoDockTools"
- Line 181, 189-190: Please correct "protein data bank" to "Protein Data Bank"
- Line 208: Please correct "I-tasser" to "I-TASSER"
- Line 227: This section heading seems a bit confusing since the contents of this section primarily discusses mostly about Lipinski's Ro5. It is recommended to rename "PASS Analysis: Lipinski’s parameters" to "Lipinski’s rule-of-five". Please also integrate contents from Lines 243-247 to this section.
Then, please add a new section called "PASS analysis" and also provide more extensive details.
- Line 325: Please rename "SAR" to "Structure-activity relationship"
- Line 367: Correct "there have also been shown" to "they have also been shown"
- Figure 4:  All chemical structures shown in this figure seemed to each be obtained from different sources since it is heterogeneous. It is recommended that the authors redraw all structures using the same software using the same ACS style (refer to https://chemtips.wordpress.com/2012/11/19/drawing-chemicals/).
Experimental design: - Generally, the experimental design was well laid out. However, it would be nice if the author could provide a workflow figure that summarizes all the components of this study visually.
- In order to facilitate reproducible research, it is advised that the author also provide the predicted protein structures (as a PDB file) as a Supplementary File. All Binding pose of chemical structures (in MOL file format) should also be provided as Supplementary Files.
- It is advised that the author should discuss about the validity and reliability of the predicted protein structures and also show relevant Ramachandran plots as well as other relevant metrics.
Validity of the findings: This study reports interesting insights gained on the potential bioactivity of the investigated phytoconstituents as well as the binding modes against their target proteins. Results seemed to be experimentally sound.
Additional comments: Authors presented a well laid out study and the insights provided herein are expected to serve as useful guidelines for further investigations. There are specific comments on how to improve the manuscript as provided above. The author is highly encouraged to share all PDB files of the predicted protein structures as well as share the binding poses as MOL files for all investigated ligands as Supplements.


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: STEROIDAL GLYCOALKALOIDS FROM SOLANUM NIGRUM TARGET CYTOSKELETAL PROTEINS: AN IN SILICO ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The paper of Rumana Ahmad, deals with the study of Molecular docking analysis of Solanumnigrum alkaloids to cytoskeletal proteins Steroidal glycoalkaloids from Solanumnigrum target cytoskeletal proteins: An in silico analysis
The cytoskeletal target(s) for SN phyto constituents are identified by an in silico analysis. The results of molecular docking, SAR analysis, physicochemical properties and Toxicity prediction methods are appropriate. In my opinion the paper is suitable for publication in Journal of Peer j. Author has to mention the admissible ratio of physicochemical properties. In figure -2 the atom of substrate which binds to the  enzyme is not visible.
 I recommende this article may be suitable for publication in your esteemed journal.


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

Input:
Title: STEROIDAL GLYCOALKALOIDS FROM SOLANUM NIGRUM TARGET CYTOSKELETAL PROTEINS: AN IN SILICO ANALYSIS
Review round: 4
Reviewer: 1

Output:
Basic reporting: The article has been written better than the first version, presenting a clear and unambiguous, English.
The references are sufficient, and the article structure, figures, tables are more clear; the article presents self-contained results.
Experimental design: The article presents an original research within the aims and the scope of the journal, with a rigorous investigation.

The method described has been improved.
Validity of the findings: Data is robust and controlled. At the same time, conclusions are well stated, linked to the original research question and limited to supporting results.
Additional comments: The article can be published in the current form


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

Input:
Title: STEROIDAL GLYCOALKALOIDS FROM SOLANUM NIGRUM TARGET CYTOSKELETAL PROTEINS: AN IN SILICO ANALYSIS
Review round: 4
Reviewer: 2

Output:
Basic reporting: Authors have addressed all comments raised in the previous peer review comments.
Experimental design: Methods seem methodologically sound.
Validity of the findings: Reliability of findings have been addressed.
Additional comments: It is deemed that the manuscript is now ready 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: ISOLATION AND IDENTIFICATION OF MARINE STRAINS OF STENOTROPHOMONA MALTOPHILIA WITH HIGH CHITINOLYTIC ACTIVITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment'
Experimental design: no comment
Validity of the findings: Some improvement in the comparison of enzyme activity data is required. No new experiment suggestion but authors should compare their enzyme activity data more appropriately.
Additional comments: Manuscript by Roger Salas-Ovilla et al is an interesting study where authors have isolated microorganism with high chitinolytic activity from the marine source. Authors have characterized the isolated strains by analyzing morphological and molecular properties. Mostly isolated strains belong to Stenotrophomonas maltophilia. Authors have given sufficient literature background. Articles included appropriate figures, tables, and raw data. The article is easy to follow.

I have the following comments.
1)	Comparison of enzyme activity data:
(a)	The authors have defined the unit as “One enzymatic unit was defined as the amount of enzyme that produced 1 μmol of N-acetyl-D-97 glucosamine per minute under standard conditions.”
(b)	Liang et al definition: One unit of enzyme activity was defined as the amount of enzyme that produced 1 μmol of N-acetylglucosamine per min
(c)	Kim-Chi Hoang et al definition: One unit of chitinase activity was defined as capable of releasing reducing ends corresponding to 1 μg of N-acetyl-D-glucosamine from glycol chitosan in an hour.
(d)	Swiontek: The mentioned unit is not found in the referred paper.
As enzyme activity definitions are not uniform across the referenced papers, authors should reanalyze the data to make their points clear.

2)	As authors have mentioned that the maximum reported enzymatic unit of 16,598 U/mg of protein, so in what sense isolated strains are more important? This explanation is required as authors have proposed using isolated strains for the biotechnological purposes.  In what sense these isolated strains are more meaningful for the industry? Is the stain with the maximum reported unit not available commercially or is it not shared by that author? What will authors do to make their strains publicly available? Alternatively, authors should remove the proposal of its use in the industry if it fails on that ground.

3)	Excel data: Authors have reported the specific activity in values with five digits after the decimal. Such accuracy is not possible. Authors should round off the values to the nearest whole number. Word media is a typo error. Values are average of three readings and should be written as “average”. It is not even the median.

4)	The number of strains finally analyzed by authors should be mentioned in the abstract.

Minor comments
1)	1,000 ul of NaCl-EDTA: composition required for this
2)	at 10,000 rpm for 5 min.: Authors should report the value in RCF instead of RPM. Since different centrifuge machines may have different rotor sizes, so RCF is more reliable value.
3)	Swiontek BM, Donderski W. 2006. Chitinolytic bacteria in two lakes of different trophic status. Polish Journal of Ecology. 54(2):295-301. DOI: 381 https://doi.org/10.1016/j.soilbio.2017.09.019. Doi link is not leading to the right 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: ISOLATION AND IDENTIFICATION OF MARINE STRAINS OF STENOTROPHOMONA MALTOPHILIA WITH HIGH CHITINOLYTIC ACTIVITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the current manuscript authors have successfully isolated and identified chitinolytic bacteria
from shrimp residues and sea water. During first round of screening, more than 200 colony-forming units from chitinolytic microorganisms were isolated, after subsequent screening steps 10 colonies with high chitinase activities were selected for further characterization. Colonies on plate B4 demonstrated highest activities. 16S sequencing revealed that these bacterial colonies belong to belong to the species Stenotrophomonas maltophilia. Overall, manuscript is beautifully written, experiments are properly executed and results are well interpreted. I recommend this manuscript for acceptance in its current form.


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

Input:
Title: ISOLATION AND IDENTIFICATION OF MARINE STRAINS OF STENOTROPHOMONA MALTOPHILIA WITH HIGH CHITINOLYTIC ACTIVITY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is clear and literature references are sufficient for the background.
Experimental design: no comment
Validity of the findings: No comment
Additional comments: The aim of this paper was to isolate and identification of marine strains with chitinolytic activity. However, the scientific merit of this paper would be enhanced if the authors can address the following comments on the manuscript.

Introduction
1.	Line 55-57: Author mentioned isolation of chitinolytic microorganisms in marine environments has been rare in comparison to the terrestrial environment. Is there any specific reason? Explain it in a few sentences.

Materials and Methods
1.	Line 61-67: Author collected shrimp rubbish and seawater. Did they process the samples immediately or stored? If yes which condition and temperature before processing?
2.	Line 69-74: Author described the colloidal chitin preparation. Did they follow specific protocol? If yes mention reference/s.
3.	Line 76-81: Experiment used semisynthetic media to grow the microorganisms.  However, readers can understand more if the author includes detail information about media.
4.	Line 93-97: Chitinolytic activity was determined. Please explain this method in details.

Results
1.	Line 134-145: Chitinolytic activity was determined and separated into three groups; lowest, moderate and highest specific activity groups. How were three groups differentiated?

Discussion
1.	At the beginning of the discussion session, if the authors can start this section with their major findings, it would be more impactful.

Conclusion
Adding up the take-home message as well as the rationale of the findings or future directions would enhance the contribution of this study to the scientific community.


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

Input:
Title: THE ROLE OF CENTRAL AMERICAN BARRIERS IN SHAPING THE EVOLUTIONARY HISTORY OF THE NORTHERNMOST GLASSFROG, HYALINOBATRACHIUM FLEISCHMANNI (ANURA: CENTROLENIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: - Supplementary figures should be improved as follows: (i) Species names should be added. (ii) Terminals should not be overlapping.

- Table S1 needs improvement as follows: Add “species”, “elevation”, “Genbank code” and “source” as columns in this table. The table should include outgroups. “Source” should include the studies that generated the sequences.
Experimental design: - Most of the results and discussion are concentrated on a gene-by-gene analysis. However, all used genes are mitochondrial and, therefore, are linked. It is my opinion that the methods should focus on the joined analyzes of the three genes together.

- There are very few sequences from Venezuela and Ecuador. This might have an impact on the low genetic structure found by the authors.

- An additional potential dispersal barrier in South America is the Tachira Depression.
Validity of the findings: - ¿Could the low genetic structure found in South America be associated to the reduced number of terminals included, specially from Venezuela and Ecuador?

- Authors should mention the possibility of having cryptic diversity in Central America.

- The authors should discuss in more detail the taxonomic status of H. tatayoi.

- The authors should discuss a little bit more about the geographic origin of H. fleischmanni. Note that all species that are closely related to H. fleischmanni are endemic to South America. Thus, a South American origin of fleischmanni and dispersal to Central America makes a lot of biogeographic sense. The authors need to be explicit about this biogeographic patterns, and make their case for a Central American origin.
Additional comments: The ms is a very good contribution for our understanding of phylogeographic pattern and, potentially, diversification forces in both Central and South America. As mentioned before, however, I think that the study needs some reanalysis and a deeper discussion on some aspects.

I have included all my comments on a Word file (attached).


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

Input:
Title: THE ROLE OF CENTRAL AMERICAN BARRIERS IN SHAPING THE EVOLUTIONARY HISTORY OF THE NORTHERNMOST GLASSFROG, HYALINOBATRACHIUM FLEISCHMANNI (ANURA: CENTROLENIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is, for the most part, well-written and presents a mitochondrial dataset with very nice geographic coverage for a wide-ranging frog species. My two main concerns are described below in ‘Experimental design’.

Minor revisions throughout the manuscript are recommended to simplify overwritten sentences and would help improve clarity for readers. Some examples:

Line 21 – “has been useful for developing an understanding of the effects of different processes and barriers on the” could be simply “has greatly affected the distribution” or alternatively “has been useful for understanding processes that influence the distribution…”

Line 28 – suggested change: “three mitochondrial regions, two coding (COI and ND1) and one ribosomal (16S)”

Lines 164-165 – suggested change: “We performed spatial clustering analyses to identify patterns consistent with dispersal barriers.”

As written currently, the results are not clearly connected to the hypotheses. Lines 121-128 state several hypotheses, but it is not clear how these are tested. What results would indicate a South American origin and dispersal into Central America? What results would provide evidence that mountains are acting as barriers? What are the expectations for genetic structure if sea level change has had an impact? These predictions and outcomes should be integrated into the appropriate methods and results sections.

The authors have done a great and thorough job with data reporting, providing museum codes for vouchers, locality info, and GenBank accession numbers.
Experimental design: The two main areas for improvement relate to 1) a lack of explicit hypothesis testing, and 2) the reliance on effectively a single locus, mitochondrial DNA. The second concern is addressed in lines 152-161 indicating the authors are aware of potential shortcomings, but this section needs some improvement.

Specifically, the line “we could only perform our analyses…” should be rephrased to something like “we chose to perform our analyses with gene sequences that could be directly compared”. However, I am also unclear what exactly the authors mean by direct comparison. Do they refer to the GenBank/BOLD sequences they included, and thus did not have tissue for? Or are they referring to comparisons made about divergence timing or something else that might benefit from using common mitochondrial genes?

The main area of improvement would be to better connect the hypotheses with the analyses that were conducted. The last methods section suggests that hypothesis testing was done in some way, but it is very unclear. How were barriers “evaluated” or “tested”? Were these a priori regions tested using some sort of phylogenetic constraint analysis? I think this would greatly strengthen the paper if so.

Additional suggestions for improvement:

Line 180-182 – In my opinion, it would be more straightforward to combine the data (and just include missing data for some individual/locus combinations). I don’t think the differences in Fig. 2 are meaningful, since the mtDNA is effectively a single locus. The small differences are more likely related to the number of informative sites in each gene.

Line 183 – Clarify that by “landscape analyses” you mean the spatial clustering analyses conducted above, if true. Since there are some differences between methods and genes, how were the groups assigned?

The haplotype networks are somewhat redundant with clustering and phylogenetic inferences, all done with the same mitochondrial genes. Could be moved to the supplement.

Line 309-311 – Clarification needed - 95% credible intervals overlapped or did not overlap with what?
Validity of the findings: The conclusions are reasonable, but as described above, should be better presented to integrate the objectives, hypotheses, results, and interpretation.
Additional comments: No additional comments


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

Input:
Title: THE ROLE OF CENTRAL AMERICAN BARRIERS IN SHAPING THE EVOLUTIONARY HISTORY OF THE NORTHERNMOST GLASSFROG, HYALINOBATRACHIUM FLEISCHMANNI (ANURA: CENTROLENIDAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: I think that the authors have made a really good effort to improve the manuscript. This study shows very nicely the effect of biogeographic barriers on genetic structure and provides valuable information that may be related to diversification processes.


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

Input:
Title: THE ROLE OF CENTRAL AMERICAN BARRIERS IN SHAPING THE EVOLUTIONARY HISTORY OF THE NORTHERNMOST GLASSFROG, HYALINOBATRACHIUM FLEISCHMANNI (ANURA: CENTROLENIDAE)
Review round: 2
Reviewer: 2

Output:
Basic reporting: I noticed that the authors provided GenBank number accessions in Table S1. According to me, it would be better to report in the main text too that sequences generated in this study have been already submitted to GenBank.

Overall, the paper reads well and it is clear, fulfilling the four required criteria.
Experimental design: It is evident that the paper has been significantly improved after the first revision. The authors took into proper and serious considerations criticism raised by the reviewers.

However, in my opinion there are still at least two major issues that need to be fixed:
1. L326 and Table 2. Please, report the associated p-values to Fst. There's no point in discussing a differentiation index if it is unknown whether it is statistically significant.

2. I might be wrong, that no 'true' haplotype network is reported in the current version of the manuscript. In fact, what presented in Figures S1 and S2 are again phylogenetic trees. Haplotype network are meant to show the frequency of different haplotypes (with circle size proportional to frequency) and relationships among them in terms of number of mutations separating each haplotype in a kind of, loosely speaking, parsimony context.
I think that a phylogeographic investigation would greatly benefit from showing a proper haplotype network. User friendly software such as PopArt (http://popart.otago.ac.nz/index.shtml) are available for inferring haplotype network from sequence alignment.
Validity of the findings: No comment
Additional comments: The following minor points need to be considered:

1. L107. Can you, please, name some of the related species?

2. Please, provide adequate explanation for **


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: ANALYSIS OF THE COMPLETE GENOME SEQUENCE OF A MARINE-DERIVED STRAIN STREPTOMYCES SP. S063 CGMCC 14582 REVEALS ITS BIOSYNTHETIC POTENTIAL TO PRODUCE NOVEL ANTI-COMPLEMENT AGENTS AND PEPTIDES
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript by Chen et al. describes the genome analysis and biosynthetic potential of a marine Streptomyces strain. The genome of this new strain was sequenced and analysed with bioinformatic tools, and the strain’s phylogenetic relationships determined. Several specialized metabolite biosynthetic gene clusters were then identified and mutant strains were constructed to characterize NRPS cluster and their role in anti-complement agent’s production.

Although an extensive work has been carried out, for most part logically planned and carried out, several aspects are not thoroughly explained and prevent publication of this work in its present state

Questions and remarks:

-	The manuscript suffers from poor English and requires careful proofreading. It contains errors as well as repeated ideas. Examples: line 63, “screening of microbial strains producing anti-complement compounds in our group revealed the potential of various marine-derived streptomycetes to produce anti-complement agents”

-	The work is very much centred in anti-complement activities. As the authors say, it’s not very common to have streptomycetes producing these compounds so they should be better explained

-	Line 79, 83, etc – ds is not the official abbreviation for days, replace by “days.

-	Line 85 – the purpose of the E.coli culture explained here should be mentioned otherwise is no immediately clear why it was used (I’m guessing is the RecET protocol, gene deletion)

-	Line 141, reference to intergeneric conjugation should be added

-	The use of marine water fermentation should be further explored/explained apart from what is said in the abstract and conclusions

-	How do the results obtained with the 16S rRNA analysis compare to the whole genome analysis? Does the same S. badius strain appear in the OAT heat map? What is the ANI value with this strain? What can be said about the use of 16S rRNA and whole genome for phylogenetic relationship determination?

-	As BGC identification is part of the results and lead to cluster 21, this cluster should described in the results section. Then, if the authors think suitable, it can also be further developed in the discussion

-	Line 288 – authors state that the ACA from the studied strain should be a polar compound. This should be better explained, that this conclusion was reached because only the methanol extract showed activity. This is then explained in the discussion but the first time its mentioned is not immediately clear.

-	In figure 4 the negative controls of PCR with the wt and the same primers should be included.

-	Why RiPPs were not explored as possible BGCs responsible for the anti-complement activity observed?

-	There are two references Wang et al. 2016 that should be clearly distinguishable in text (for instance, 2016a and 2016b)

-	Line 344 – what is the advantage of using this strain as a production host, specifically as an alternative to the already described and available Streptomyces strains for heterologous expression? Is the strain’s tolerance to higher salt concentrations a factor?

-	Overall the discussion should be revised. It contains too many details and it should not be a repetition of the results.


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

Input:
Title: ANALYSIS OF THE COMPLETE GENOME SEQUENCE OF A MARINE-DERIVED STRAIN STREPTOMYCES SP. S063 CGMCC 14582 REVEALS ITS BIOSYNTHETIC POTENTIAL TO PRODUCE NOVEL ANTI-COMPLEMENT AGENTS AND PEPTIDES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written, but, few concerns has to be pointed by this reviewer.
At line 187 ,the authors state “...based on the 16S rRNA gene sequences revealed that Streptomyces sp. S063 may be a subspecies of S. badius NRRL B-2567T (100% similarity)”
Inference of kinship level using only one marker is very bold. Unfortunately, only 16s does not gives you such resolution. Besides The figure 2 does present any support for the branches, nor, the methology for this inference are present on material and methods. In this case, the authors should use internal transcribed spacer (ITS) a very currently used genetic marker for infering relationship (Check PNAS April 17, 2012 109 (16) 6241-6246; https://doi.org/10.1073/pnas.1117018109).

On General genome information section, line 198 it is state that “...most abundant categories are transcription (683), 199 carbohydrate transport and metabolism (456) and amino acid transport and metabolism (462)”. This is indeed expected, since those are on lower levels (generic) ontologies. An gene ontology enrichment  analyses, such as topGO (https://bioconductor.org/packages/release/bioc/html/topGO.html) might shed some lights on other high level (more specific) ontologies.

On lines 337 – 341 the authors selected four NRPS related BGCs but no result arise from this, so, why choose those? It was an empirical choice or there are some information available throughout the available literature to sustain this negative result? The last sentence starting on line 341 does not make any sense! The authors indicate that “Despite the negative results, we have demonstrated in this study that Streptomyces sp. S063 is easy to be genetically manipulated”. Compared to what organism? Wich data presented on the current manuscript lead to this finding?

On lines 349 – 351 the authors affirm “The rapid growth and abundant spores suggest Streptomyces sp. S063 is an optimal host for heterologous expression of BGCs”. As in previous comment this phrase is made in comparison to what? Other Streptomyces? Please, review your comparisons stated throughout the text, you always need to indicate to what are you comparing to.

On line  352 there is no clear cause/consequence on the following statement, regarding the growth on starch: “In addition, three cellulose-binding proteins and eight putative cellulases were also identified”. Is there some correlation present on the literature involving the presence of CBP and starch tolerance? This will enhance your discussion.

At lines 356 - 358 the phrases are also not much supported by the results presented on the current form of the manuscript. In this reviewer point of view this part might be suppressed from the text, or a revision must be done in a way to better conceptualize this affirmation.
Experimental design: The manuscript fits on the journal Scope. I felt that the discussion is quite superficial in some points, the authors indicate, more than once throughout the text, that some particular points need "further analyses". It leads the reader to a not a rigorous investigation feeling.
There is no methodology for the evolutionary 16s assessment present on figure 2.
Validity of the findings: The authors speculate more than their results are open to. Some comparisons seems more an affirmation than a  comparison. And some of them are not supported by their results.
Additional comments: The current reviewed manuscript entitled “Analysis of the complete genome sequence of a marine streptomycete Streptomyces sp. S063 CGMCC 14582 reveals its biosynthetic potential to produce novel anti-complement agents and peptides” present the Streptomyces sp. S063 CGMCC 14582 genome sequencing and some annotation, also, present a hypothesis of ancestry between the under research specimen with Streptomyces badius NRRL B-2567. In this reviewer point of view, the authors should revisit their results and improve phylogenetics proposition adding some branch support and explain how the inference was 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: ANALYSIS OF THE COMPLETE GENOME SEQUENCE OF A MARINE-DERIVED STRAIN STREPTOMYCES SP. S063 CGMCC 14582 REVEALS ITS BIOSYNTHETIC POTENTIAL TO PRODUCE NOVEL ANTI-COMPLEMENT AGENTS AND PEPTIDES
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This reviewer’s questions have been satisfactorily answered. The authors have made significant changes in the manuscript improving its overall quality. I have no further comments and support this work for publication in its present state.


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: ANALYSIS OF THE COMPLETE GENOME SEQUENCE OF A MARINE-DERIVED STRAIN STREPTOMYCES SP. S063 CGMCC 14582 REVEALS ITS BIOSYNTHETIC POTENTIAL TO PRODUCE NOVEL ANTI-COMPLEMENT AGENTS AND PEPTIDES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The english has been improved. My criticisms regarding superficial discussion is still on. But all the major flaws has been covered after first round of revision. This manuscript is more suitable for a descriptive manuscript than able to present novel findings to the area, and, might by eligible for publication under this descriptive bias
Experimental design: The manuscript fits on the journal Scope. I still felt that the discussion is, somewhat,  superficial in some points, even after previous criticisms the authors maintains that some particular points need "further analyses", even on the rebuttal letter. It leads the reader to a not a rigorous investigation feeling.
Validity of the findings: The overall presentation has been enhanced on this version, but the authors speculate more than their results are open to. It is OK for an descriptive paper.
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: TOXICITY OF TIO2, SIO2, ZNO, CUO, AU AND AG ENGINEERED NANOPARTICLES ON HATCHING AND EARLY NAUPLII OF ARTEMIA SP.
Review round: 1
Reviewer: 1

Output:
Basic reporting: The quality of the English needs to be improved. More relevant literature review should be added in the Introduction and Discussion to give a clear rationale of this study and explain the results better. All the figures need polishing. Remove the grid lines in the bar charts and increase the resolution.

Line 35 The abstract should summarize and refine the most significant findings in the study, rather than list the experiments and assays that have been done.

Line 50 The author should explain the rationale that why they chose these six metal and metal oxides nanoparticles in this study. The relevant literatures should be reviewed in the introduction.
Experimental design: No information about the DHE assay displayed in Figure 5 was mentioned in the Method section.

Line 86 “4 metal oxide nanoparticles (TiO2, SiO2 and CuO)”, should include “ZnO”.

Line 129 “Five types of nanoparticles (Ag, Au, SiO2, TiO2, CuO and ZnO) are tested”, should be “Six”.
Validity of the findings: The data is reasonable, but do not provide much of insights.

Line 172 & 174 The description of the size as “higher” is very ambiguous, the author should report the exact size of each particle in the text.

Line 203 “hatching rate was better…” crappy English, hatching rate was higher…

Line 240 More explanation should be added in the Discussion to reveal more insight about the data.

Line 258-259, Line 266 What is the mechanism of the hatching interference observed in the study? The author claimed that the metal ion release could be a reason for silver and copper oxide nanoparticles, so they centrifuged the nanoparticles, resuspended in the solution and considered them as “nanoparticles” after 72 h. The authors should provide evidence for this statement first, if this statement is correct, what could be the reason for the hatching interference in these non-dissolvable nanoparticles? The authors also reported that “Silicon nanoparticles at low concentration were very conducive in fact showed better hatching as compared to control.” More clear explanation should be added in this part.
Additional comments: This paper describes the toxicity of six different metal and metal oxides nanoparticles TiO2, SiO2, ZnO, CuO, Au, and Ag on the hatching and early nauplii of Artemia sp.. However, the paper is very descriptive and superficial and it fails to explain the data in terms of the physiology of the organism in any detail. The quality of the English, although reasonable, was quite clumsy in places. The figures need a lot of polishing for a publication. Overall, the manuscript won’t be suggested to be published in the journal in current form.


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

Input:
Title: TOXICITY OF TIO2, SIO2, ZNO, CUO, AU AND AG ENGINEERED NANOPARTICLES ON HATCHING AND EARLY NAUPLII OF ARTEMIA SP.
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript addresses an important subject. The authors examined the effect of 4 metal oxide and 2 metal nanoparticles on hatching and early nauplii of Artemia sp. in order to demonstrate the reability of the hatching assay.
The interest and justification of the paper is not very accurate and I have some comments in order to improve the manuscript
Experimental design: I have some concerns about the experimental design. This section needs a lot of refinement. The experimental set-up is poorly written in that it is very unclear.
Validity of the findings: There is not an adequate statistical analysis.
There is a weakness in the experimental design, it results confuse and not detailed
Additional comments: Introduction
The Introduction could use a bit more of a background on work that has been done in particular evidencing the recent work on this focus.
These papers can be an example:

-Libralato, G., Prato, E., Migliore, L., Cicero, A.M., Manfra, L., 2016. A review of toxicity testing protocols and endpoints with Artemia spp. Ecol. Indic. 69, 35–49.
-Libralato, G., 2014. The case of Artemia spp. In nanoecotoxicology. Mar. Environ. Res. 102, 38–43.
-A. Rotini, A. Gallo, I., Parlapiano, M.T. Berducci, R. Boni, E., Tosti, E. Prato, C. Maggi, A.M. Cicero,
L. Migliore, L. Manfra, 2018.  Insights into the CuO nanoparticle ecotoxicity with suitable marine model species Ecotoxicology and Environmental Safety 147: 852–860.

-A. Rotini, L. Manfra, S. Canepa, A. Tornambè, L. Migliore, 2015. Can Artemia Hatching Assay Be a (Sensitive) Alternative Tool to Acute Toxicity Test? Bulletin of Environmental Contamination and Toxicology. 5(6) 745-751.

 Materials and Methods
I have some concerns about the experimental design. This section needs a lot of refinement. The experimental set-up is poorly written in that it is very unclear.

Line 101 – Please use chemical symbols adequate.

line 103 –Precise which means PVP, since it is not referred previously.

Line 104-“Working dilutions of the nanoparticles are made in salt water (NaCl) for various studies” re-write this sentence is not clear.

-The authors measured the sizes nanoparticle suspension at 25μg/ml in milliQ water and salt water, but for toxicity test they used different concentrations of each nanomaterial (from 100mg/L to 0.01mg/L Why?

-I think it is needed at least more time points of for the NP dissolution (if possible a graph with the time evolution of the dissolution during the 3 days) and DLS of the NPs at different time points in the working media (with characteristics similar to the one used. Having a better characterization of the evolution of the NPs in the working media can be valuable for the scientific community.

-Please specify the preparation of stock suspension. In M&M is not reported about of three soluble salts : CuSO4, ZnSO4 and AgNO3.
-Line 145 LD50 change with LC50

-There is not an adequate statistical analysis.

Results
This section is not very clear the data are not well explained.

-The analysis of nanoparticles need a statistical analysis (one way ANOVA) to determine statistical significance among the treatments (distilled and sea water).

-Figure 1 are not ordered according to the text.

-Line 179 “The nanoparticles suspended both in Milli Q water and 25%NaCl solution (salt water) were measured up to 48 hours to see change in their dispersion pattern”. But the nanoparticle suspensions (in milliQ water and salt water) were measured thrice for three day???
-Line 187 “Brine shrimp normally showed  a hatching rate of 74%+5.5, the viability of these hatched nauplii was about 97%.” Pleas give a reference
-Line 191-193 “The hatching rate dropped to about 29% in the two nanoparticles; of these only 50% were motile and viable at a concentration of 100mg/L (Table 1)”.
Please give a graph or table with the viability of hatched nauplii.
-Why the authors reported also values of hatching rate for silver nitrate, copper sulfate, zinc sulfate. Maybe they use them reference toxicants? Give more information about this.

-Line 199-200 I don’t agree with this sentence, moreover the fig 4 referred on mortality data. Please, to compare the data use statistical analysis.
-Line 208 “The pH of salt water did not change upon …..” Please in M&M section reports how the authors measure pH , t°, salinity, oxygen.
-Line 211- Where is the Figures S2.1-2.6???

-Line 213 – Where is the Fig. S3. In the presentation of tables and figures I belive that there is a lot confusion.
-Line 221- “The most toxic was the silver nanoparticles and silver in ionic form”
Table 2 reported the lethal concentration only of nanoparticles for larval toxicity test. I do not see the silver in ionic form!!! The relased ions is crucial in determining the toxicity of NP suspension and for this purpose the ion concentration must be determined and the metal release was examined at 0, 24 h, 48 h, 72 h, and 96 h , after the stock suspensions were prepared.
-Line 225-“LC50 of gold was 2.53E-226  03mg/Lat 48h”. There is some prolem.

-Line 226. Order the text according to the increase value or according to the table.
Discussion
-Line 243. 25% is a high salinity condition??? I do no belive
-Line 243. This aspect is not reported in the results.
-Line 257. Together at silver and copper oxide also ZnONP showed high toxicity.
- Line 258- 259 As reported above, this aspect must be added to the M&M
-	Table 1 is not acceptable in this form:  lack the units, please indicate the  gold with  capital letter (as Au), moreover added a column where to put the used concentrations.
 -Table 2. In the column change with 24h LC50 and 48h LC50 because it is not a percent, the LC50 is a concentration of a chemical that determines the death of 50 percent of the  sample population.

-Figure 1 Make more readable

-Figure 2 Please, use the chemical symbols (also in fig 3)

-Figure 3 It is very bad, to lack of units

-Figure 4 Report the same graduation for all graphs

-resolution of Figure 5 is too low.
The English language should be improved


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

Input:
Title: TOXICITY OF TIO2, SIO2, ZNO, CUO, AU AND AG ENGINEERED NANOPARTICLES ON HATCHING AND EARLY NAUPLII OF ARTEMIA SP.
Review round: 2
Reviewer: 1

Output:
Basic reporting: The English still needs more polishing.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: All the comments in the first round have been addressed. However, there are still some problems need to be fixed in a scientific article.
Figures 2 and 3 need to be improved, they look fuzzy and ugly;
The citation format in the text needs to be consistent. Line 290, Line 300;
Line 197, the abbreviation of milliQ water needs to be consistent;
All the supplemental items need to put in a word file, with detailed figure legends.


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: TOXICITY OF TIO2, SIO2, ZNO, CUO, AU AND AG ENGINEERED NANOPARTICLES ON HATCHING AND EARLY NAUPLII OF ARTEMIA SP.
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I have read the review prepared by the authors.
The work has certainly been improved in some aspects.


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: FULLY AUTOMATED SEQUENCE ALIGNMENT METHODS ARE COMPARABLE TO, AND MUCH FASTER THAN, TRADITIONAL METHODS IN LARGE DATA SETS: AN EXAMPLE WITH HEPATITIS B VIRUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors of 'Fully automated sequence alignment methods ...' curate
and align large numbers of hepatitis B sequences (and fragments), and
use the alignments to estimate phylogenetic trees. They then compare
the accuracies of the tree prediction for a pipeline based on MUSCLE
against one based on UPP/PASTA. I found the paper very 'bitty'.  None
of the figures were very insightful, except for Figure 3.B, which is a
piece of art, and in any print journal would be a contender for the
title page. The introduction is very reference heavy for tangential
matter. The standard of written English is good.

[1] The title states that 'fully automated sequence alignment methods
outperform traditional methods'. However, the authors only compare
'fully automated sequence alignment methods' to *one* 'traditional
method' (MUSCLE) not 'methods'.

Action: either amend the title to summarize the work accurately or
        include other 'traditional methods'.

[2] How divergent are these sequences?  These are all isolates of one
virus type and should actually be relatively easy to align but the
divergence (average percent differences would do) would make that
clear. What would happen if one used GBLOCKS to remove regions of high
alignment instability?

Action: - Quantify similarity/divergence of data
        - Comment on using GBLOCKS

[3] On line 139 the authors say recombinants were removed. That makes
sense if one makes trees and compares them as recombinants (between
two or more strains) will have confusing phylogeny. On page 158, the
authors seem to say that recombination implies rearrangement along a
genome which is not the same?

Action: Clarify

[4] The authors pick MUSCLE as their method of choice to compare
against 'fully automated sequence alignment methods'. However, almost
immediately (line 175) they say that 'using MUSCLE to align all 55,353
sequences ... was not feasible'. Why choose MUSCLE, which allegedly
cannot align 55k sequences over CLUSTAL OMEGA, FAMSA, KALIGN or MAFFT,
which can?  Also, www.drive5.com/muscle/manual/compromise.html
suggests to use -maxiters 2 for large data-sets. This should make 55k
sequences (more) feasible for MUSCLE. What are and why use custom gap
parameters on line 155 for MUSCLE?

Action: - Explain why UPP/PASTA is compared to MUSCLE and not to any
        other aligner/s (or do compare to other aligners).
        - Explain why -maxiters 2 is not used for MUSCLE
        - Explain 'custom gap parameters' for MUSCLE

[5] In line 162 is the 'branch length' the distance from the root or
distance of a leaf from the nearest branch point?

Action: Clarify

[6] In line 214 five iterations of the NJ QC step may have somewhat
been 'previously described' (I could not recall) but 'NJ QC' was
definitely not formally defined.

Action: define 'NJ QC' before line 214

[7] The authors say that 'sequence alignment strategy did not have a
major effect on tree topology' (line 328). So the main advantage of
'fully automated sequence alignment methods' over 'traditional
methods' would be that they produce results 'in a more reasonable time
frame and repeatable manner' (line 342).

This is difficult to ascertain as (i) no times (or memory
requirements) are quoted, (ii) no 'traditional method', that can
actually align the required number of sequences without human
intervention, is used and (iii) the results are *not* repeatable.

Using PASTA and UPP I have repeatedly (UPP five times, PASTA twice)
aligned the same set of (small in number and short) sequences and
every time obtained a different alignment. In my example the alignment
lengths varied by 7%, the sum-of-pairs scores of one alignment against
another alignment varied from 96% to 97% and the total column scores
from 23% to 40%. For 'repeatable' results I would consistently expect
100%.

If I understand UPP/PASTA correctly, then there is a certain degree of
randomness involved in the assignation of the backbone
sequences. However, I did not see that there was (can be?) any random
seed specified on the command-line. If this is so, then the alignments
have to be repeated several times and the results have to be
somehow combined/averaged.

It is well known that MAFFT L-INS-i is not thread-safe. SATe/PASTA/UPP
use MAFFT L-INS-i (indeed it is my opinion, that much of the quality
of SATe/PASTA/UPP is due to this fact, see below) so a 'repeatable'
analysis should be run such that MAFFT L-INS-i is run single-threaded
(or by setting the MAFFT --threadit flag to 0).

Action: - Quote run-times and memory consumption.
        - Guarantee that alignments are repeatable.
        - If this cannot be done then repeat alignments and average.

[8] As mentioned above, SATe/PASTA/UPP use MAFFT L-INS-i, L-INS-i is a
high-quality consistency aligner and MUSCLE is not. It would be only
fair to compare like with like and either (i) use MUSCLE as the base
aligner in UPP (PASTA can do this, can UPP?) or (ii) use L-INS-i as
the 'traditional method'. L-INS-i would definitely have problems
aligning 55k sequences, but since in the current analysis sequences
are broken up into 1,269 FASTA files, this should be feasible.

Action: Swap MUSCLE for MAFFT L-INS-i (or vice verse)

[9] In line 77 MAFFT is mentioned but not cited

Action: Cite MAFFT

[10] Year of reference on line 506 (Nguyen et al., 2015) not same as
in text (line 83, Nguyen et al., 2016)

Action: make reference consistent

[11] Fix minor typos

l73-4
- the most important steps in a phylogenetic analyses
+ the most important steps in a phylogenetic analysis
+ the most important steps in   phylogenetic analyses

l90
- ... www.ncbi.nlm.nih.gov/genome/viruses/)as  a mix of
+ ... www.ncbi.nlm.nih.gov/genome/viruses/) as a mix of

l93/4
- Incorporation of these sequences in phylogenetic analyses are useful,
+ Incorporation of these sequences in phylogenetic analyses is  useful,

l98
- a hybrid of alignment methods—an  increasingly
+ a hybrid of alignment methods —an increasingly

l170
- PASTA v.1.6.3 (Mirarab 2015)
+ PASTA v.1.6.3 (Mirarab et al., 2015)

l248
- Robinson-Fould  (RF) distances (Robinson & Foulds, 1981)
+ Robinson-Foulds (RF) distances (Robinson & Foulds, 1981)

l321
- for most genome/s-region alignment types
+ for most genome/S-region alignment types

l574
- B) Robinson-Fould  distances
+ B) Robinson-Foulds distances

l575
- between two phylogenies  Darker cells indicate two trees
+ between two phylogenies. Darker cells indicate two trees

l579
- Bottom plots show Robinson-Fould  distances.
+ Bottom plots show Robinson-Foulds distances.

Figure 2.B
- Robinson−Fould  Distance
+ Robinson−Foulds Distance


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: FULLY AUTOMATED SEQUENCE ALIGNMENT METHODS ARE COMPARABLE TO, AND MUCH FASTER THAN, TRADITIONAL METHODS IN LARGE DATA SETS: AN EXAMPLE WITH HEPATITIS B VIRUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript describes a case study of sequence alignment and phylogenetic analysis of hepatitis B viruses.  The authors compared traditional and modern automated approaches for MSA.  They resulted in generally similar tree topologies.  I have several comments on Experimental design (points 1-4), but I think presentation of the data should be improved first (points 5-9; see the General comments section).
Experimental design: 1. It seems to be interesting to use the 10 genotypes for assessing the quality of tree.  However, it was difficult for me to follow the discussion on this issue (see also points 7, 9 below).  "The test for genotype monophyly showed consistent patterns among the different alignment types (genomes, S-region, total)" (lines 311-312), but in Fig.3A, genotype D, E, F and H show highly inconsistent pattern between "Genomes" and "Total" (and noted in lines 316-320).  Which is correct?

2. Related to the above point, it'll be possible to discuss more about why most genotypes are not monophyletic (line 55, line 414).  Possible factors would be: (1) convergent evolution, (2) difficulty in tree estimation, (3) gene transfers across different lineage, etc.  Factor (3) is consistent with the observation that the "genotype occupancy proportions" differ between different genotypes, in my understanding.  If this factor has to be assumed (ie, different regions on a genome have different evolutionary histories), then does it make sense to estimate a single tree from the whole genome?

3. The definition of "genotype occupancy proportion value" should be given as an equation.

4. MAFFT is referred to as a component of traditional analysis (line 77), but it has a function for the "modern" automated approach of this size of data (Katoh & Frith 2012):
% mafft --6merpair --addfragments fragmentary_sequences Aligned_Linearized_Genomes > output
https://mafft.cbrc.jp/alignment/software/addsequences.html#fragments
It'll be better to refer to this method.
Validity of the findings: no comment
Additional comments: I suggest improving/fixing the following issues:

5. The alignment by MUSCLE was seemingly provided as Genome_alignments/Genomes_MuscleAlign.fasta in the raw data, but actually the sequences in this file are unaligned.  Another file, Genome_alignments/Genomes_Pasta-Linearized.fasta is aligned, but the length (3423) differs from that (3387) explained in line 273.

6. In line 278, the sequence data set is explained using "Fig.1", but Fig.1 in the main part seems to be a different figure (comparison of trees).

7. In figure 3, different bars/squares have to be distinguished only based on color.  Moreover, in panel A, the order of colors differ between the bars (orange, yellow, light green, green, blue, purple, magenta?) and the legend (light green, orange, green, yellow, blue, purple, magenta?).  For readers with color blindness, it'll be impossible to interpret this figure.  Figure 2 has also a similar problem.

8. I was confused because tables S1 (mentioned in line 306) and S2 (line 313) are not in the Supplemental data in the journal's website, but they are in an external repository (Dryad).  Dryad is mentioned only one time in the main text (line 144).  It should be stated clearly every time the main text refers to the data in Dryad.

9. It would be helpful for readers if a figure is added to the main part to show a graphical representation of the HBV genome to illustrate the locations of S-region, other genes and the regions associated with the 10 genotypes.

10. A terminological issue: Is it correct to say that most genotypes are paraphyletic (line 361)? From fig.3B, at least genotypes A and B seem to be polyphyletic.


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: FULLY AUTOMATED SEQUENCE ALIGNMENT METHODS ARE COMPARABLE TO, AND MUCH FASTER THAN, TRADITIONAL METHODS IN LARGE DATA SETS: AN EXAMPLE WITH HEPATITIS B VIRUS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors of 'Fully automated sequence alignment methods outperform
traditional methods for phylogenetic analysis of hepatitis B viruses'
have responded satisfactorily to almost all the referees'
criticisms. However, their answers to point [1.4] are highly
questionable.
Experimental design: [A] Each of the alignment software that I had suggested in my original
review is able to align the full genome set, most of them on a modest
PC (8 cores, 8Gb RAM), and most of them in under 2 hours, using less
than 2Gb. I had to run MUSCLE on a machine with more memory. The
alignment lengths were comparable to the ones quoted by the authors.

CLUSTAL-OMEGA(v1.2.3)   T=101min        RAM=1Gb         L=3846
FAMSA(v1.2.2)           T=14min         RAM=1.4Gb       L=4631
KALIGN(v2.04)           T=108min        RAM=200Mb       L=4958
MAFFT(def, v7.245)      T=23min         RAM=600Mb       L=4578
MUSCLE(v3.8.31)         T=38hours       RAM=10Gb        L=4909

I was not able to run the full genome data set (or the larger
fragmentary one), using PASTA (v1.6.3 or v1.8.2), neither on a 8Gb nor
a 256Gb nor a 512Gb machine. The Java heap space was exhausted for
Opal.

CLUSTAL-OMEGA, FAMSA, KALIGN and MAFFT-PARTTREE were also able to
align the larger data set containing fragments, however, the alignment
lengths were much longer (8541, 10029, 29503, 83689 respectively).

So there are plenty of aligners that the authors could have used to
add weight to their assertion that 'Fully automated sequence alignment
methods outperform traditional methods for phylogenetic analysis of
hepatitis B viruses' which suggests that 'Fully automated sequence
alignment methods outperform *all* traditional methods for
phylogenetic analysis of hepatitis B viruses'.

It is laudable that the authors did run MAFFT and quote the alignment
length, but it is disappointing that none of the MAFFT alignments are
included in the rest of the analysis.

I do agree that it would not be feasible to use ClustalW, as a quick
scalability analysis (2,5,10,20,50,100 sequences) suggests that the
full genome data set might take more than a week. That is, why I did
not suggest this in my original report. If you do include it
nevertheless, then you should still cite it (Larkin et al, 2007; as
well as CLUSTAL-OMEGA, Sievers et al, 2011; Geneious, Kearse et al,
2012; Sequencher, Gene Codes Corporation, Ann Arbor).

        Action: Do include more aligners in your analysis
        or clearly state (in the text and in the title) that
        you are comparing Pasta/Upp with one method (MUSCLE) only
Validity of the findings: [B] I believe that the inclusion and referencing of software is very
defective.

I could not verify any of the statements concerning Sequencher or
Geneious, as both programs are commercial. It would be better to use
only programs that are free for everyone to use.

The fact that MUSCLE was afforded a dedicated 24-core machine is
pointless, as it is a sequential code. It would be much more useful to
quote the available memory.

The statement about MUSCLE running so much faster under Geneious than
as a stand-alone is intriguing. Since I cannot verify this myself (for
the above mentioned reason) I would need a very good explanation to
let this stand.

For the same reason it is totally unacceptable to run CLUSTAL-OMEGA
through SeaView. Citing the version of SeaView is pointless as (under
Linux) it picks up CLUSTAL-OMEGA from /usr/local/bin/, which is
whatever (unknown) version of CLUSTAL-OMEGA the user has
installed. Also, we cannot know if the (alleged) memory error occurred
in CLUSTAL-OMEGA or SeaView. I did align the genome data set and the
fragment data set with CLUSTAL-OMEGA through SeaView on a PC, and it
did not crash (SeaView v4.5.4, CLUSTAL-OMEGA v1.2.3).

        Action: remove all analyses of alignment software
        called by secondary programs or give satisfactory
        explanations which versions of the aligner is run
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: FULLY AUTOMATED SEQUENCE ALIGNMENT METHODS ARE COMPARABLE TO, AND MUCH FASTER THAN, TRADITIONAL METHODS IN LARGE DATA SETS: AN EXAMPLE WITH HEPATITIS B VIRUS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors have addressed all issues raised in a satisfactorily manner.
Experimental design: Experimental design appears to be sound.
Validity of the findings: I believe that the findings, based on the experimental design, are valid.
Additional comments: The manuscript is very much 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: FULLY AUTOMATED SEQUENCE ALIGNMENT METHODS ARE COMPARABLE TO, AND MUCH FASTER THAN, TRADITIONAL METHODS IN LARGE DATA SETS: AN EXAMPLE WITH HEPATITIS B VIRUS
Review round: 3
Reviewer: 2

Output:
Basic reporting: I suggest revising the title to reflect the observations more simply.  If a title says that method A outperforms method B, then readers expect:
* There is a reference that can be regarded to be correct based on a solid basis and
* Estimation by method A is significantly more similar to the reference than estimation by method B.
However, the observations here are:
* The results by methods A and B are not essentially different.
* The results are not fully consistent with an external reference (genotypes classification in this case).
It seems to be difficult to discuss which method is better based on these observations.  A possible conclusion would be that automated methods resulted in an apparently reasonable estimation for this specific data.  This might be informative since automated methods require less labour.

2. It seems to be a bit misleading to refer to MAFFT in the second sentence in Introduction: 'Traditionally, MSA methods for phylogenetic reconstruction are performed using alignment programs such as MUSCLE or MAFFT followed by manual, “by eye” corrections'.  Actually MAFFT has an option designed for adding fragmentary sequences to a backbone, as stated in the last sentence in Introduction.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: OTOLITH MICROCHEMISTRY AND DIADROMY IN PATAGONIAN RIVER FISHES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Very good in general. Some suggestions below in order of importance:

Line 22 – ‘…a number of diadromous fishes’. This is ambiguous as it could refer to a number of individuals or of species. Please clarify. Note that in other areas of the manuscript this ambiguity is not an issue.

Line 315-317 – In fact, we are increasingly realising that laboratory experimental approaches to characterising swimming performance are fraught with insurmountable issues regarding fish maintenance, behaviour and scale. Indeed, the species used in Laborde et al. (2016) were not selected on the basis of the need to design fish passage structures, but instead on the basis that they were the only species that would cooperate by surviving and swimming in the laboratory! Unfortunately, the best paper to substantiate this is currently with the publisher after acceptance, so I am unable to refer to it here. But, essentially, the most robust basis we currently have for designing fish passage structures in Chile and elsewhere in the temperate Southern Hemisphere is experience – see the models presented in Wilkes et al. (2018). Wilkes, M., Baumgartner, L., Boys, C., Silva, L.G., O'Connor, J., Jones, M., Stuart, I., Habit, E., Link, O. and Webb, J.A., Fish‐Net: Probabilistic models for fishway planning, design and monitoring to support environmentally sustainable hydropower. Fish and Fisheries.

Line 76 – More specifically, the galaxliids are distributed across the temperate Southern Hemisphere (McDowall, 2002). McDowall, R.M., 2002. Accumulating evidence for a dispersal biogeography of southern cool temperate freshwater fishes. Journal of Biogeography, 29(2), pp.207-219.

Figure 2 – This is a very useful illustration. However, there is no x axis label on the plot. This may be an issue with pdf conversion.

Figures 3, 4 & 5 – The scales of axes are at a much smaller font than axis labels in these plots. Please make them the same size.

Line 98 – ‘…from 6 locations…’ – Unless otherwise specified by the journal, it is convention to spell out numbers <10, i.e. ‘…from six locations…’.

Life 177 – Should read ‘affected the confidence of our interpretations’.

Line 184-186 – I find the way of listing these regimes a little awkward. Could the authors please provide them as bullet points?

Line 297 – I suspect few readers will understand what the ‘Baker corridor’ is. Please provide a definition or use different language. For example, do you mean the valley of the Baker River?
Experimental design: The authors use robust and well-established methods. I have just one suggestion - I am not very familiar with the laboratory procedure involved in analysing otolith annuli but it strikes me that the authors should refer to a standard source(s) or a recent paper(s) using the method in the text around line 107. This will demonstrate clearly that a well-established procedure was used, which from my readings on the procedure appears to be the case here.
Validity of the findings: No comment
Additional comments: I foundd this to be a well-written and enjoyable paper to read. The methods used are robust and well-established and the Supplemental Files are comprehensive and relevant. The ethical statement is sufficient and the appropriate study permits are in place. There are a few, relatively minor ways I could suggest to improve the manuscript below.


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: OTOLITH MICROCHEMISTRY AND DIADROMY IN PATAGONIAN RIVER FISHES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The Introduction describe very weakly the reason of this study. How was the choose of fish?, why the author have exotic and native fish as models?.
Used the Hydropower development in Chile, but how is the policy in your country?.
Experimental design: This Ms do not has an experimental design clear, the authors used samples collected for another reason. Neither the fish species chooses for this study.

It can be a good thing to apply the rule of 3R, but in this case is not clear.
The statistical and method are ok.
Validity of the findings: The Otoliths results are interesting, but the movement or diadromy of Salmonid are clear, please to check the Carl Schreck  or Steve McCormick papers, and I was checking about Chilean Native species and quickly I found 3 paper the same group:
1.-Isolation Driven Divergence in Osmoregulation in Galaxias maculatus (Jenyns,1848)(Actinopterygii: Osmeriformes), Ignacio Ruiz-Jarabo PLOs ONE 2016
2.-Contrasting Genetic Structure and Diversity of Galaxias maculatus (Jenyns, 1848) Along the Chilean Coast: Stock Identification for Fishery Management Claudio González-Wevar, Journal of Heredity, 2015, 439–447
3.- Phylogeography in Galaxias maculatus (Jenyns,1848) a long Two Biogeographical Provinces in the Chilean Coast. Claudio A.González-Wevar PLOs ONE 2015

1.-This articles you can see that G. maculatus is a diadromy fish, with different osmoregulation response.

I think that authors need to improve the references revision and to include more studies.
Additional comments: Dear Author
Your Ms is interesting but need to improve the introduction, discussion and to improve the literature revision.
The Introduction describe very weakly the reason of this study. How was the choose of fish?, why the author have exotic and native fish as models?.
Used the Hydropower development in Chile, but how is the political in your country?.
The objective is not clear and method need to be more clear, Which is the real reason to do this study, or to they want to take advantage of the samples taken but without a prior reason.
Material and Method
Again, the fish choose, to mixture native vs exotic fish is interesting but here is not clear.
Discussion
Here the author to include the "Limitations and further studies" (line 291) I'm thinking that this paragraph is not necessary and it is better to include in the introduction-material and method. Also, to include "Conservation issues" (line 319) for what? to save an exotic fish? for this reviewer it is not clear.

References revision, I think that here the author need to do a better revision than the show us in this MS version,  I was checking about Chilean Native species and quickly I found 3 paper the same group:
1.-Isolation Driven Divergence in Osmoregulation in Galaxias maculatus (Jenyns,1848)(Actinopterygii: Osmeriformes), Ignacio Ruiz-Jarabo PLOs ONE 2016
2.-Contrasting Genetic Structure and Diversity of Galaxias maculatus (Jenyns, 1848) Along the Chilean Coast: Stock Identification for Fishery Management Claudio González-Wevar, Journal of Heredity, 2015, 439–447
3.- Phylogeography in Galaxias maculatus (Jenyns,1848) a long Two Biogeographical Provinces in the Chilean Coast. Claudio A.González-Wevar PLOs ONE 2015

1.-This articles you can see that G. maculatus is a diadromy fish, with different osmoregulation response.
I recommend to submitted this Ms at another journal as J.Fish Biology or Environmental Biology of Fishes, can be more suitable than Peer J.
I hope my suggestions are useful to improve your ms


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: OTOLITH MICROCHEMISTRY AND DIADROMY IN PATAGONIAN RIVER FISHES
Review round: 2
Reviewer: 1

Output:
Basic reporting: I was happy to re-review this manuscript. The authors have done a good job of responding to two reviewers’ comments. I can only suggest a few minor corrections. Once these are made, I believe the paper is suitable for acceptance.

Line 82-84: “Differing chemical composition of the otolith from the primordium (core) to the edge hint the different environments in which a fish has lived and allows for hypothesis tests related to patterns of fish movement.” – “Hint” is a little casual for a journal article, and should be followed by “at” in this context. More appropriate would be: “…is indicative of the different environments…”

Line 99-100: “This study sought to typify the potential diadromous characteristics from a wider diversity of native and exotic fish species in Patagonia…” – “Typify” is not quite right here. I suggest “characterize”.

Line 170 – “Phyton scripts” should be “Python scripts”

Line 275 – “self-sustaining coho salmon populations” – You should give the scientific name in parentheses as this is the only time you refer to this species by its common name. Alternatively, just refer to the scientific name.
Experimental design: No further comment
Validity of the findings: No further comment
Additional comments: 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: OTOLITH MICROCHEMISTRY AND DIADROMY IN PATAGONIAN RIVER FISHES
Review round: 2
Reviewer: 2

Output:
Basic reporting: Dear Author
Your Ms is interesting and after the revision, I can see that the MS is improved and for me I don' t have more revisions or comments.
Experimental design: It is ok for me after revision.
Validity of the findings: After the revision the Ms had improved.
Additional comments: Dear Author
Your Ms is interesting and after the revision, I can see that the MS is improved and for me I don' t have more revisions or 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: MODULATION OF CORTICOMOTOR EXCITABILITY IN RESPONSE TO DISTAL FOCAL COOLING
Review round: 1
Reviewer: 1

Output:
Basic reporting: Line 45-60: The authors described “there is still very limited information regarding the neural basis underlying such facilitatory effects.”
The introduction attempted to give a background on this study but overall, the descriptions on neuropathway from sensory stimulation receptor to motor cortex are lacking. In addition, a definition of what is meant by thermal stimulation would be good and the range of temperatures of thermal stimulation should be described.
Experimental design: Method
Line 97-99:  “As described previously, all participants were considered healthy and were screened to ensure that their sensations were normal and that they had no contra-indications to TMS.”
I think it would be better list the inclusive and exclusive criteria items.

Line 129-135: How many minutes to spend in completing TMS testing during thermal stimulation protocol?  Please clarify the short intervals between 1-min and 5- and 10-min post-cooling during the cooling application whether enough time to measure TMS parameters.
In addition, the authors did not state any past experience regarding safety of the procedures. Has the safety issue been studied? It would be better to clarify the reason using a pre-cooled gel pack at~10 °C.
Validity of the findings: Results
Line193-195: It would be better to add the citation reference for the statement “In fact, when individual responses were classified in terms of either inhibition (MEP<10% BL), facilitation (MEP>10% BL) or no modulation (MEP ±10% BL), two distinct patterns emerged”

Discussion
Regarding the resting motor threshold (rMTs), MEP amplitude and latency, the authors should state the clinical implication for these parameters.
The description in discussion section is too long. Can you make it more succinct? However, it would be helpful to address the limitation section in the revised manuscript.

Conclusion
Line 339-341: “Such observations, notably with regards to spatial summation effects and repeatability between sessions, seem critical for future therapeutic applications of cooling stimuli for rehabilitative purposes in neurological populations.”
Only healthy adults (n=22) were recruited in this study and thus the results of the present study cannot support the above-mentioned statement. I suggest the authors delete the statement.
Additional comments: This cross-sectional study is an interesting and practical research project. The research entitled “Modulation of corticomotor excitability in response to distal focal cooling”. The aim of this study was to examine whether an increase in stimulation area from single-digit (SD) to multi-digit (MD), four fingers could elicit more consistent modulation. Skin temperature and motor evoked potentials (MEPs) elicited with transcranial magnetic stimulation were measured at baseline (BL, neutral gel pack at 22 ◦C), at 1-min during the cooling application (pre-cooled 10◦C gel pack) and 5- and 10-min post-cooling (PC5 and PC10). The results highlight that responses to cooling stimulation in terms of either depression or facilitation of corticomotor excitability tend to be fairly consistent in a given individual with repeated applications. However, the generalizability of the results with regards to spatial summation effects and repeatability between sessions for rehabilitative applications in neurological populations were limited.


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: MODULATION OF CORTICOMOTOR EXCITABILITY IN RESPONSE TO DISTAL FOCAL COOLING
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study addressed the impact in thermal stimulation has been proposed to facilitate motor recovery in neurological populations, such as stroke. The authors extended their own previous work with single digit cooling, and extended it to multiple digit cooling based on their goal of determining whether an increase in stimulation area could elicit more consistent modulation of motor evoked potentials elicited by Transcranial magnetic stimulation (TMS) directed to the motor area of the first dorsal interosseous (FDI) muscle.
The study is clearly written and the English is of a high standard. The literature review is well referenced and contextualizes the background to the study well. The figures are clear and well labelled and the raw data is supplied.
Experimental design: The authors extended their own previous work with single digit cooling, and extended it to multiple digit cooling to address their stated research question of determining whether an increase in stimulation area could elicit more consistent modulation of motor evoked potentials elicited by Transcranial magnetic stimulation (TMS) directed to the motor area of the first dorsal interosseous (FDI) muscle.  The research fills an important gap about the possible mechanisms by which cool influences cortical excitability.
The methods are described in detail and important additional measures such as skin temperature to confirm that cooling indeed took places were included. The work was performed to a high technical and ethical standard.
The overall group results were not significant but the authors observed that half of participants showed post cooling MEP facilitation while the other half showed post cooling MEP depression. Further they showed that the tendency to either inhibit or depress MEPs in response to cooling was consistent with participants who participated in their previous single digit cooling experiment and the current multi-digit cooling.
This is the biggest strength of the work. There is a growing trend to determine if MEP responses to interventions show a differential response to a given intervention, this work clearly does.

Their conclusions were fair, related to the original research question  and did not overstep the mark. Their correlative analysis of MEP modulation between sessions for SD and MD cooling found reasonable within-subject repeatability over time on repeated applications of cooling. This suggests that the tendency for MEPs to either inhibit or facilitate in response to cooling is consistent within a given individual, providing validity to their decision to report those who facilitated and those who inhibited in response to cold as two separate groups.  Their conclusion that this work on the impact of spatial summation effects and individual repeatability between sessions, is important work needed to validate “future therapeutic applications of cooling stimuli for rehabilitative purposes in neurological populations.”
Suggestion: if possible, include 2 or 3 references from previous TMS work where a bimodal response in terms of MEP inhibition or facilitation has been observed. This is not essential but it would strengthen their decision to compare the “inhibitors” and “facilitators” as two separate groups.
Validity of the findings: The study has in important impact because thermal stimulation has been proposed to facilitate motor recovery in neurological populations, such as stroke, and yet little is understood regarding the physiological mechanisms by which thermal stimulation impacts neural function. In this study, neural function was assessed by measuring changes in motor evoked potential (MEP) amplitude and latency at varying time points during and after multi-digit cooling. The data is robust (averaging 20 MEPs at each time point), and the statistics are appropriate for the research design.
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: MODULATION OF CORTICOMOTOR EXCITABILITY IN RESPONSE TO DISTAL FOCAL COOLING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The author has made an appropriate response.
Experimental design: The author has made a satisfactory revision for the comment.
Validity of the findings: The author has made an appropriate response.
Additional comments: The author has made an appropriate and satisfactory response.


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: SPECIES TURNOVER IN PLANTS DOES NOT PREDICT TURNOVER IN FLOWER-VISITING INSECTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Simaika et al. investigated whether flowering plant diversity can be used as a surrogate for the diversity of flower visiting insects in the Cape Floristic Region, South Africa. They found that plant diversity was a poor predictor for anthophile diversity although there was similarly strong spatial turnover in species assemblages.

The manuscript is in all parts well-written and well-structured. The literature cited is adequate and the results are relevant to the research questions. I just have few minor suggestions to improve the structure of the manuscript (for comments on the structure of the discussion see “Validity of findings”):

L43-59 I think this paragraph could be shortened, because it focuses on plants and insects in general while the paper is focused on flower visiting insects.

L91-93 It could be better explained why the spatial scale is important for the research question and conservation planning.
Experimental design: The research questions are interesting and the experimental design is valid and meaningful. Additionally, the methods are generally well described and provide important details. However, some questions remain about the sampling and methods:

L147-154 Here you should describe how you measured flower cover. Additionally, you sampled the plots twice during a three month period, but flowering status was only recorded once, before the insect sampling? Could the flower cover have changed dramatically from the vegetation to the insect sampling? Is it also possible that other herbaceous plant species appeared later in the season that were not recorded during the plant sampling?

L.168-171 Please, provide more details. How many models were conducted? Which were the response variables? Did you account for the nested design of your data?
Validity of the findings: I have no experience with some of the statistical methods used such as cluster analysis, seriation and RDA, but the authors’ explanations are convincing to me.

However, for the simple linear regression testing the relationship between plant and insect diversity the nested design of the study (with several plots per site) should be taken into account, because several plots from the same site are not independent. Mixed models would be appropriate in this case.

The discussion addresses all important issues, but I would prefer to structure the discussion according to the two research questions stated at the end of the introduction. Additionally, I would be more interested in how the spatial scale is important. The title states that spatial scale matters when flowering plants are used as surrogates for anthophiles suggesting that plants can be used as surrogates at certain scales, but not at others. However, this question is not addressed in the manuscript. You should change the title or focus more on the importance of the spatial scale for the usage of plants as surrogates for anthophiles.

Conclusions: The conclusions should focus more on the results of this manuscript. You should repeat the main findings of your work and what we learned from this study. Additionally, you could also mention how your results affect the conservation planning in your region. Is the current conservation management good? What could be improved to promote flower-visiting insects? Should new conservation schemes be developed that target insects directly?
Additional comments: Other minor comments:
L.23 This sentence suggests to me that the diversity of plants was related to the diversity of anthophiles which was not the case. Consider rewording.
L60-61 Should not plant diversity predict insect diversity?
L83-84 The cited paper is only on herbivores. So maybe better write “established plant-insect herbivore diversity relationship”?
L120 Here you could already mention Appenidx S4 (In the document peerj-28146-Appendix_S4_Distance_table_for_all_plots.docx the title is Appendix S1?).
L.162-163 Species richness of plants and anthophiles? I think you don’t need to repeat that insect data was pooled across transects per plot.
L.218 This table should be mentioned in the methods section.
L.241 “Clear pattern” regarding which question?
L.253 Do you mean “plots”?
L.267-270 Why are the results of Fig. 2b not in the same paragraph as Fig. 2a?
L.313 According to which statistical test?
Fig.2 I think you don’t need to repeat the letters in parentheses: (a) and (A).
Appendix S1: It would be interesting to sum the abundances of each insect species to see which taxa were dominant.


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: SPECIES TURNOVER IN PLANTS DOES NOT PREDICT TURNOVER IN FLOWER-VISITING INSECTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The Authors compared insect diversity and abundance with plant diversity at six locations (16 plots) in protected areas of Cape Floristic Region, South Africa. The purpose of the analysis was evaluating the reliability of plant diversity as a surrogate for insect diversity/abundance. This has relevance for conservation (both in the region and in general), since plant diversity is often better known than insect diversity. The Authors claimed to have performed investigations at different spatial scales (from 0.5 to 80 km). Their main findings are that: 1) plant diversity is not correlated with insect diversity and abundance, and hence does not constitute a useful proxy to be used in conservation study; and 2) plant and insect turnover is similar, and strongly affected by ‘spatial scale’, in terms of pairwise distance between localities. I have a few minor comments I have annotated while reading the paper, and a major concern about the Authors’ conclusions.
Looking at the map of sampling locations, it is apparent that the Authors focused in fact on three distinct areas (A1 = circles + triangles + diamonds; A2 = crosses + stars; A3 = squares). It is true that the total area of the analysis is not extremely large, but it is also true that the region is particularly rich in species, and that differences are expected to emerge at relatively small spatial scales. The distribution of sampling sites makes the results in Fig. 3 (which seems to me as the core result of the paper, also motivating the title) not very surprising; in fact, the graph shows simply that the turnover within area groups (A1, A2, A3) is lower than the turnover between A1 and A2 (which are at approximately at 20-30km of distance), and that the highest turnover is recorded between A1+A2 vs. A3 (which are at a distance of 70-80km). Now, that is far from being surprising, being simply an additional confirmation to the commonness of distance-similarity decay relationships. The fact that this applies to both insects and plants has no clear, straightforward implications for conservation.
Thus, in general, although I think that the dataset collected by the Authors is valuable, and the paper is relatively clear and well written, I am not convinced it offers some novel insights regarding turnover patterns.
Yet, although a negative result, the lack of correlation between insect diversity and plant diversity is a valuable result (even if the number of plots, considering also their geographical clustering, is a bit small to draw general conclusions). My advice would be that of reworking the paper by focusing on this aspect, also putting emphasis on all possible caveats.
Also, I found the method section not particularly clear. The Authors provide many technical details on the statistical procedures, including the software tools used to perform the analyses, which is appreciable. However, the reasons why each analysis was chosen is, in most cases, not provided. Authors should better explained the goals behind their choices, i.e. clarify a priori the questions addressed by each analysis. Considering my general advice above, I would recommend to simplify the analytical approach; many of the analyses presented in the text seem a bit out of the main goals described in the introduction.

66 specify the level of endemism
144-146 needs clarification
167 remove comma before the bracket
167-176 How seriation is computed could be better explained
178-179 What is the point of computing shared species between plot pairs? This quantity is not independent from plots’ species richness (which is affected by factors likely independent from overlap itself), and thus does not provide an informative measure of overlap. I would have expected to see the quantity compared with some null expectation obtained by randomly reallocating species in plots.

228 In general, I have no problem in the use of simple linear regression as a robust approach to detect relationships; however, in the case of the comparison between insect and plant richness, it would make sense to check at least for rank correlation, especially considering that the dependence between plants and insects, and the likely asymmetric specialization between the two groups could possibly lead to different trends in species richness across sites.
Also, I think that readers (at least myself) would like to see a figure where insect diversity and abundance is compared with plant diversity across sites.

Scale of x-axis in fig 3 is missing (is it km x 10-4?)
Discussion, lines 287-289: “Interestingly, in plants, turnover decreased with greater distance between plot pairs, from local to regional. In contrast, with insects there was high turnover at both the local and regional scale, with a tendency to increase across distance groups.”
This is not apparent from fig. 3, and a bit in contrast with the positive relationship claimed elsewhere in the manuscript.