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: RELATIONSHIP BETWEEN ACROPORA MILLEPORA JUVENILE FLUORESCENCE AND COMPOSITION OF NEWLY ESTABLISHED SYMBIODINIUM ASSEMBLAGE
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study provides evidence for the role of coral fluorescence in affecting symbiont assemblages in early life stages of a recently metamorphosed aposymbiotic reef coral. While their results are preliminary, the results are interesting and informative and provide a framework that motivates further research. I find the manuscript well written with appropriate scholarship. My comments mostly consist of grammatical or stylistic suggestions or requests for clarity.

Abstract
Line 37: delete extra “as” here, in “acting as either as an…”

Introduction
The introduction paints a good picture of the literature and knowledge gaps, however, is there any room for the authors to address (perhaps briefly at Line 70) the role host FPs play during in bleached corals/during thermal stress?

Line 48: appears to be an extra space after “symbiosis”

Line 57: can the authors provide a citation for greater dispersal potential in brooded larvae

Line 93: recommend breaking this sentence in two for clarity, perhaps with period before “for example”.

Line 104: recommend incorporating “through variable fluorescent signal” above at Line 102. Feels redundant if retained at both Line 102 and 104.

Line 106: I commend the authors on this straightforward and pithy culmination to the introduction.
Experimental design: The largest issue I have is there is little information on the experimental design at the treatment level (see comment below). I would like the authors to address this concern and provide detail on the  bulk culturing/sedimentation treatment and the conditions the juvenile corals were maintained under during the 21d period.

Paragraph (2. Fluorescence…)
I do not have an issue with how color scores were determined, however, considering there are many published examples of color scoring, I feel a citation in necessary for at least some portion of this approach.  Can the authors provide such a citation?

Line 115: what depth were these colonies at? Could depth of parent affect the properties of offspring here—perhaps the parental effects your elude to in discussion.

Line 129: prior to exposure to sediment treatments but perhaps not wholly aposymbiotic? Could a few words be added here to clarify that ‘exposure’ references treatment exposure but not in situ exposure?

Line 138: there is limited information on sediment treatments consisted of. What were the conditions during these sediment treatments—ie., light, temp, flow, aquaria or well plates, in situ or in the lab? Were all symbionts assumed to originate in sediment or in seawater, was this natural seawater or filtered, were there statistical replicate treatments or a common garden…. Please clarify and provide more information on your treatment.

Line 170: errant “of” here at “…of OTUs”
Validity of the findings: The authors have interesting findings and are quite parsimonious in their interpretation—ie, testing for false positives and the discussion of correlation in lines 227.  I appreciate the conservative tone and agree this study provides needed insights for a larger discussion on the functional roles of the continuous distribution of the color and fluorescence properties of corals.

RESULTS
Line 204: need to make “Discriminant” lowercase

DISCUSSION
Line 221: recommend adding broadcast spawning in this description, since ‘highly dispersive’ is a not very specific

Line 239: perhaps mention that C15 can also dominate across substantial geography and depths in specific genera such as Porites (LaJeunesse, et al. (2004). High diversity and host specificity observed among symbiotic dinoflagellates in reef coral communities from Hawaii. Coral Reefs.)

Line 246: suggest “suppress” as opposed to retard

Line 258-269: could this ‘preference’ also be  referred to as photo-acclimatization potential? Is there evidence the authors could cite to clarify this point—I recognize it is speculative, but some clarity in the niche partitioning vs. acclimatization capacity would be informative.  i.e., how can this ‘preference’ influence coral performance? Suggest,
Ezzat, et al (2017). Carbon and Nitrogen Acquisition in Shallow and Deep Holobionts of the Scleractinian Coral S. pistillata. Frontiers in Marine Science, 4, 2273–12Line 261: here and elsewhere where species names for Symbiodinium types are referenced, I think you need to give full species name (Symbiodinium and not “S”) since these full specific epithets have not yet been introduced.

Line 269: missing a closing parenthesis here

Line 286: in which species? S. hystrix?

Line 279: this ‘bulk culture’ information needs to be detailed in the methods. It is unclear how this was performed.

Line 309-320: I agree with the authors here, and see a lot of opportunity to clarify these relationships in future studies. This is a very well constructed paragraph.

Line 319: Suggestion: Innis, et al. (2018). Coral color and depth drive symbiosis ecology of Montipora capitata in Kāne‘ohe Bay, O‘ahu, Hawai‘i. Coral Reefs, 1–8
Additional comments: A general comment: Was there any attempt to measure fluorescence at the end of the experiment? Could the same chlorophyll fluorescence subtraction (Line 128) have been done to determine if host fluorescence was maintained over time or during winnowing (as discussed in Line 307)? Could the authors provide any information on whether fluorescence may be fixed or variable during concurrently with ontogeny and symbiont winnowing?


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: RELATIONSHIP BETWEEN ACROPORA MILLEPORA JUVENILE FLUORESCENCE AND COMPOSITION OF NEWLY ESTABLISHED SYMBIODINIUM ASSEMBLAGE
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript was clear and easy to understand. The literature background/context was well supported, but see my comment below regarding a couple of additional citations I suggest including. The article structure, figures, and tables are professional, and raw data is available via the NCBI Sequence Read Archive. I did notice, however, that in the specified SRA entry, there were only 18 samples, compared to the 22 samples referenced in the text. Can you please clarify this difference?

Literature/Citation Suggestions:
I recommend mentioning the “Beacon hypothesis” by Hollingsworth et al (2005, Coral Reefs; 2006, Proceedings of the 10th ICRS) which demonstrated that motile Symbiodinium exhibit phototaxis towards green light. I think this should be discussed at least briefly, potentially when you mention that “This would test the hypothesis of host fluorescence acting as an attractant” (Lines 316-318), as it is directly relevant here.
You have done an excellent job of supporting your conclusions as well as proposing alternate mechanisms that may drive the patterns you observed. I will suggest one more alternate hypothesis – it is possible that the “green” juveniles are better at attracting more evolutionarily derived symbionts (i.e. C15), as the C15s may hone in on the brighter GFP, and be taken up by the coral. Since infection is attraction plus selective uptake (Yamashita et al 2014), perhaps those corals then do not need/want to uptake clade A Symbiodinium. It is also possible that “opportunistic” As stay away from bright green juveniles, and instead prefer the redder individuals (as you mention, because there is more light, and may be a better environment for them to compete with other Symbiodinium types/clades).
Experimental design: The research question was well defined and relevant. I was excited to see the authors investigating how juvenile fluorescence relates to symbiont uptake, as I believe it addresses an intriguing gap in our current knowledge. This manuscript and its conclusions would be strengthened by inclusion of parental information (i.e. relatedness of coral juveniles), but I acknowledge that is probably not possible at this point, and I think the authors did a good job of discussing this, proposing alternative hypotheses, and setting the stage for future research.
NMDS is good for visualizing multidimensional data, but I would like to see what the output of a constrained ordination looks like for these data. Specifically, you could use capscale in vegan to run this analysis with a call something like ord <- capscale(SymbioCommunity ~ redness) followed by anova(ord). Is this model significant?
Is the custom Symbiodinium database that you used (line 148) available somewhere for download? If so, please provide a citation for the database (I think the current citations are just for the NCBI database in general). If not, please include the database either with your code, and/or on GitHub, and/or somewhere else online. This will help with reproducibility of your study.
Validity of the findings: The findings of this study are valid, well supported, and alternative hypotheses are clearly presented.
Additional comments: Overall, I think this is an interesting manuscript that is worth publishing. I have included several comments below with specific suggestions for making the figures clearer and more intuitive.
Figures:
Fig 2a. The contour coloring would be more clear and consistent if the color bar went from red to green. This is because the color differences on the figure are a bit hard to distinguish. If you set a mid-line of 0.55 (as in Fig 1), with green (low) to red (high) this may be clearer. (Although check that it prints ok in black and white before making this change).
I find the use of randomly-chosen symbols for the different Symbiodinium types to be distracting. I acknowledge that this may be an inherent problem with so many types, but is it possible to try to make the symbols for each type within a clade, similar? For example, most of the Cs are somewhat box-shaped, except “C” and “C1*”, but D1 and Uncultured are also box-shaped. Perhaps you could switch those, so all Cs are box-shaped? I realize that this may seem picky, but as it is, it is fairly difficult to see the broader-scale patterns.
Fig 2b. For black-and-white printing, I’d suggest changing the shape of one of the juveniles, so they’re not distinguished only by color. I like that they are distinguished by color, but you could make one of them a box or a triangle for clarity.
Figure S1a. Both of these sub-figures should be truncated at 0 (i.e. the fitted model and confidence intervals shouldn’t cross 0, as a negative mean abundance isn’t possible). It also looks like the mid-line (yellow, between green and red) for your color bar is at 0.5. If possible, please change to 0.55, so it matches Fig 1.
Fig S1b. If you move up the second bar from the left on the bottom panel to the panel above it, you could expand the axes for the bottom panel (e.g. from 0 to 20) which would make the very low abundance symbionts easier to see.


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: COMPREHENSIVE CIRCULAR RNA EXPRESSION PROFILE IN RADIATION-TREATED HELA CELLS AND ANALYSIS OF RADIORESISTANCE-RELATED CIRCRNAS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The central topic of the paper and objective of performed experiments is clear.

The authors provide sufficient background and context which makes the paper a clear read.

The article is self contained.

A significant number of tables, raw data, and experimental methods need to be additionally provided.
Experimental design: To my knowledge the paper is original with a clearly defined question.

Methodological the paper is not yet fully sound and it will require additions:

- Line 168: If samples were RNase R digested how was GAPDH used as an internal control.
- Line 181-183: the cut-off to define a circle should be stated (eg. number or samples with circle, number of counts per circle)
- Line 211: missing description of the small RNA sequencing
- Line 216: Different type of correlations should be mapped on the network figure.
- Line 259: "selected for" is probably a typo and should be corrected.
- Missing raw data for small RNA sequencing.
- Missing counts for circRNAs as well as small RNA sequencing results.
- Missing supplementary table for miranda results was well as circRNA vs miRNA correlations.
Validity of the findings: The findings are linked to the original research question and seem valid but need more supporting information to be robust as described in "2. Experimental 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: COMPREHENSIVE CIRCULAR RNA EXPRESSION PROFILE IN RADIATION-TREATED HELA CELLS AND ANALYSIS OF RADIORESISTANCE-RELATED CIRCRNAS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The text is poorly written and need English revision.
Experimental design: Need more experiments.
Validity of the findings: Findings are not experimentally supported.
Additional comments: In this manuscript by Yu et al., the authors have performed RNA sequencing to study the effects X-ray on circRNA using HeLa cells. They found 76 upregulated and 77 downregulated. Based on this data, they performed several bioinformatics analyses to conclude that circRNAs may play a role in radioresistance in Hela cells and allow novel therapeutic approaches.

Comments
Although authors have acquired RNA seq data with respect to circRNAs, the study remains very descriptive and premature. Validation of circRNAs by RT-qPCR needs to be thoroughly performed. Authors have validated only few downregulated circRNAs. Validation of more circRNAs will strengthen the manuscript. Also, validation the sequences of the PCR products is essential to confirm the existence and junction sequences of the circRNAs. The assumption that circRNA activity was related to known functions of host linear transcripts is not experimentally supported. First, the levels of circRNAs could be different from mRNAs. Second, the abundances of both circRNA and mRNAs may be differentially altered upon radiation. Third, the biogenesis of circRNAs is dependent on backsplicing (totally different from mRNAs). Thus, authors need to experimentally support this assumption since it is the ground stone of the rest of the manuscript and the bioinformatics analysis. In addition, authors use another assumption that circRNAs and miRNAs will be correlated. Again this is not experimentally supported. First, mRNA levels are not assessed in this study. Second, the levels of miRNAs do not reflect their activity, which is not determined in this study. Third, not all circRNAs will regulate miRNA activity. Finally, it does not seem that the study  is representing clues about a possible role of circRNAs in radioresistance or novel therapeutic approaches.


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: COMPREHENSIVE CIRCULAR RNA EXPRESSION PROFILE IN RADIATION-TREATED HELA CELLS AND ANALYSIS OF RADIORESISTANCE-RELATED CIRCRNAS
Review round: 1
Reviewer: 3

Output:
Basic reporting: English needs to be improved. For example:

line#27 Malignant tumors are less sensitive or do not respond to irradiation because
#31 We sought to investigate the role of circRNAs in radioresistance in Hela cells
#37 Results: RNA-Seq allowed
#52 high mortality of cervical cancer patients
#62 that compose

similarly, minor corrections are required at several places to improve the English quality of this manuscript
Experimental design: no comment
Validity of the findings: no comment
Additional comments: circRNAs research is becoming increasingly valuable in patient's diagnostics and potential therapeutic interventions. Here, the authors analyze the differentially regulated circRNA in HeLa cells, which confer radioresistance. With some careful corrections in language flow and English writing mistakes, the manuscript may be accepted.


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

Input:
Title: COMPREHENSIVE CIRCULAR RNA EXPRESSION PROFILE IN RADIATION-TREATED HELA CELLS AND ANALYSIS OF RADIORESISTANCE-RELATED CIRCRNAS
Review round: 1
Reviewer: 4

Output:
Basic reporting: The basic reporting of the findings is adequate given that English is most likely not the main language of the authors. The literature is not entirely complete and authors may consider the recent findings by O’Leary et al 2017 given the radiation responsive circRNA KIRKOS 73 is highly expressed in the human cervix
Experimental design: There are a number is issues however with the experimental design. As the main aim of this study appears to be finding a basis for radiotherapy resistance – why use X-ray irradiation and not gamma ray? 10 Gy is rather excessive and it is surprising that the cells survived this level. At what time point was the RNA extracted following X-ray exposure?
RNase R treatment conditions – 20 units for 6 mg RNA – 1 unit will treat 1ug or 20 units will treat 20 ug. The subsequent reliance on the sample as representative of circRNA is incorrect given the low levels of enzyme added to the total RNA pool.
Please explain the ‘blank control’ in more detail – is this non-irradiated or sham-irradiated?
Selecting 8 circRNAs at random for verifying the RNA-seq results is perhaps not the best way – the study would have been better placed to select the top 5 upregulated and top 5 downregulated circRNA from the RNA-seq data and confirm the findings with QPCR (and ideally automated sequencing for the presence of the back spliced junction). The ‘tendency’ of the findings to confirm the RNA-seq is not very convincing. Also, table 4 is not a table and perhaps could be put in instead of histograms where no comparison is provided at present.
Validity of the findings: Validity of the findings: The authors state that ‘GO and KEGG biological pathway analysis was performed to predict potential activity of circRNAs’ yet GOseq was used to analyse the potential of linear transcripts (line 125). The authors should be aware that circRNA is regulated independently of linear counterparts. The authors even state that circRNA show expression patterns that are different from their linear counterparts (Line 243-244).
Rather than a circRNA having many binding sites for one miRNA, more emphasis has been placed here on many miRNA potentially binding to a circRNA (the former scenario would have greater biological relevance).

While 16,893 circRNA were indicated as being identified (although the numbers didn’t quite add up lines 181- 183), only ~ 1% were differentially expressed between groups.
Additional comments: This study undertook RNA-sequencing of X-ray irradiated HeLa cells and standard bioinformatic analysis with the view to extrapolating a role for circRNA in radiotherapy resistance of cervical cancer. Using linear counterpart information an interpretation was made for potential function and pathway alterations by circRNAs. Circular RNA is a newly recognised class of non-coding RNA with varying opinions on its ability to act as sponges for microRNA. To date there is very limited knowledge of the exact function of circRNA. While the study provides a potential bioinformatic source of circRNA for future research, the manuscript tends to lean towards over-conclusion and lack of depth.

Minor points:
Keyword: function – replace with some other term more specific to the study.
Line 52: reword
There are a lot of words jointed together in error throughout the text eg.thatcompose
Line 68: sentence too short, please elaborate a bit more.
Line 76-77: are pseudogenes regarded as a competing RNA group?
HeLa cells not Hela cells.
Line 83: should be ‘and to explore’
Line 106: mass?
Line 116: ‘fold change of the same circRNA in both groups’ – please rephrase for more clarity.
Line 141: cor.test () ?
Line 152: change retro to reverse
Line 174-175: rephrase and add more detail.
Line 178: reword the second part of the sentence – too general.
Line 235: why italics? and please provide references.
Line 264: linear
Line 298: absolute values close to 1/-1 – please explain?


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: COMPREHENSIVE CIRCULAR RNA EXPRESSION PROFILE IN RADIATION-TREATED HELA CELLS AND ANALYSIS OF RADIORESISTANCE-RELATED CIRCRNAS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The basic reporting has improved a lot upon revision.
Experimental design: This has been made clearer in the revised version.
Validity of the findings: The data is robust, statistically sound and is controlled correctly.
Additional comments: The manuscript is 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: DRINKING WATER AND RURAL SCHOOLS IN THE WESTERN AMAZON: AN ENVIRONMENTAL INTERVENTION STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper titled Drinking water and rural schools in the Western Amazon: an environmental intervention study is an interesting research work.
The authors have clearly highlighted the problem of study area.
Experimental design: The submission has clearly defined the research question, which is very much relevant and meaningful. The investigation  have been conducted rigorously and to a high technical standard. The research have been conducted in conformity with the prevailing standards in the study area.
Validity of the findings: The conclusions have been appropriately stated and have been connected to the original question investigated, and are result oriented
Additional comments: This is a meaningful research paper with a great potential to be published in this journal.


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

Input:
Title: DRINKING WATER AND RURAL SCHOOLS IN THE WESTERN AMAZON: AN ENVIRONMENTAL INTERVENTION STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is based on the study of a sanitary intervention in rural areas of Brazilian western Amazon. The main outcome of the study can be considered as not really significant or without a clear perspective of an impact since it dedicates too much effort just in order to prove a predictable reduction of coliforms in water supply after the implementation of a simplified chlorinator. Besides this particular hypothesis the paper refers to such intervention as a social technology, but instead of treating such issue as an inter/multidisciplinary concern the paper does not explore the challenge of implementation which could bring a more contextualized and rich study considering the peculiar conditions and socio-environmental constraints in the rural settlements. Therefore, dealing with the complexity of implementation of a social technology would be a task for a different study design.
Experimental design: Insufficient to deal with sanitation as a social technology.
Validity of the findings: Findings are not relevant.
Additional comments: The simple confirmation of coliform reduction can be considered as a matter of how sanitation and health concerns have been dealt as a surpassed paradigm, but of course, it is not something really solved, indeed there is a need for interdisciplinary approaches to bring new outcomes and solutions.


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: DRINKING WATER AND RURAL SCHOOLS IN THE WESTERN AMAZON: AN ENVIRONMENTAL INTERVENTION STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The document has a clear intervention study and its evaluation and perspectives.
The review presented is well fitted to the subject supporting the proposal and in the identification of comparative papers to evaluate the results.
About the structure:
•	An adequate abstract presents background, methods, results and discussion.
•	The methods are well described with:
o	Study design, studied area and population, intervention process, water parameters and data analysis.
•	The results are clear and
•	The discussion relates the results obtained with other studies and important references.
•	The conclusion presents the findings of the intervention study, could include the future directions.
The figures are well labelled and described:
•	In Figure 1 - Infographic of the environmental intervention study, it would be interesting to indicate the initial data collection as a step in the environmental intervention study.
•	Figure 2 – Indicates the area of study, it would be interesting to present some spatial reference (coordinates, name of location or limits).
•	Figures 3 to 5 present a synthesis of the Results obtained before and after intervention.
The Raw data supplied is consistent with the paper presented.
Experimental design: The proposed research scope is well defined as an intervention investigation. The method is clear and pertinent, with an emphasis on the application and its replicability.
Validity of the findings: The data included corroborate the findings demonstrating the improve in the school’s water quality using the intervention proposed. The literature arguments are consistent. The descriptive statistics is clear, and the graphics contribute in the presentation.
Additional comments: Suggestions:
The English language could be improved to make the text more precise. Some examples are indicated.
•	Line 24 “the water quality of the schools” - schools water quality
•	Line 32 “about three out of ten people (2.1 billion)” -  worldly about three out of ten people (2.1 billion)
•	Line 40 “As the rural population remains living in adverse conditions and deprived of drinking water,” - As most rural population remains living in adverse conditions and deprived of drinking water

Figure 1 - Infographic of the environmental intervention study, it would be interesting to indicate the initial data collection as a step in the environmental intervention study.

Figure 2 – Indicates the area of study, it would be interesting to present some spatial reference (coordinates, name of location or limits).


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: DRINKING WATER AND RURAL SCHOOLS IN THE WESTERN AMAZON: AN ENVIRONMENTAL INTERVENTION STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The suggestions proposed were considered and the improvements realized. The document is clear and unambiguous. The references are presented and the research fits into the field of knowledge. The article structure is properly organized. Raw data is available and clearly presented. Figures and table are relevant and contribute to the understanding of the paper.
Experimental design: The paper presents a research project with environmental intervention for the treatment of water for human consumption, in public schools.
The methods are presented, the proposal is consistent, and the meaning of the water quality control and measures are significant. Results obtained support the analysis and the procedures are understandable and could be replicated.
Validity of the findings: The data presented is clear and the related analysis consistent.
The presentation made contribute to the literature by showing the environmental intervention for the treatment of water for human consumption and the relation to literature contributing to the set of studies applied in the area.
.
Additional comments: I suggest that you improve the writing at lines 41- 47, to make it clearer.


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 USE OF CHLOROPLAST GENOME SEQUENCES TO SOLVE PHYLOGENETIC INCONGRUENCES IN POLYSTACHYA HOOK (ORCHIDACEAE JUSS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors cite one enrichment method (i.e. Anchored Hybrid Enrichment), their citations could be enriched by including other published studies applying this method to other plant genera. Also, authors should notice that the Anchored Hybrid Enrichment method targets nuclear loci and that a revision of equivalent techniques focused on chloroplast markers is missing in their introduction.
Experimental design: 1. I have some concerns about the logic behind the subsampling schemes tested. First, it is not clear to me why the authors having sequenced and annotated 40 full chloroplast genomes they limited their search of potentially fast evolving regions to two previously published conventional markers. There is no explanation about why the authors expect that analyzing multiple copies of the same region will have a positive impact on phylogenetic reconstruction. Even if economic resources are limited, researches make an effort to include as many independent loci as possible. With the data that the authors have on hand they could being offering a more meaningful subsampling scheme where different regions (e.g. genes, introns and intergenic spacers) are ranked according to their phylogenetic informativeness (see Townsend 2007 method) before designating subsets of “fast evolving” regions. This procedure would allow the comparison of several independent fast evolving regions for a better evaluation of how many of these data is necessary to obtain a phylogenetic tree with comparable resolution and support to that obtained from the analysis of the full data set. Second, fast evolving sites or regions tent to provide support for shallow nodes, whereas slower evolving regions can provide support for deeper nodes. Therefore, excluding slower evolving regions can result on low resolution and/or support for deep nodes. Third, unusually fast evolving regions or sites can introduce phylogenetic noise which can decrease the resolution and/or support obtained. This latter possibility is not considered or discussed in the manuscript.

2. Inversions were reduced to a single randomly selected site within them to avoid overweighting this evolutionary event during the phylogenetic analyses. The authors should consider that the primary homology assumption for that selected site is still incorrect (i.e. they shouldn´t be aligned at the same position) and that in the case that a substitution event followed the inversion, the potential groupings resulting from their analysis can be confused.

3. Taking into account the approximate average and range of PP for comparing the randomly sampled matrices is a way of comparing them, but it does not consider the topology.

4. For probe design the plastome sequence of Phalaenopsis aphrodite subsp. formosana was divided into 360 bp long blocks, please specify if they were or not overlapping blocks.

5. Two species are cited as outgroup, however Phalaenopsis aphrodite was used for rooting the trees. Why P. aphrodite is not cited as outgroup?
Validity of the findings: 1. In the conclusion section: you only generated 40 Polystachya specimens full plastome sequences, not 48.
Additional comments: The authors generated and analyzed a comprehensive amount of plastid DNA data (fully sequenced genomes) for improving our knowledge on the phylogenetic relationships of a lineage within the orchid genus Polystachya. They tested different subsampling schemes of presumably fast evolving regions in order to estimate how much of these data is needed to obtain a well resolved and supported phylogenetic hypothesis and compared this against their tree resulting from the analysis of the full data set. The probe capture strategy applied is valuable as they demonstrated that a full chloroplast genome could be assembled from the retrieved regions.

In addition to these general comments I have some particular comments that can help the authors improving their manuscript.

1.	I suggest to narrow the title down to the genus Polystachya since even within it they only targeted a single lineage.
2.	Otherwise indicated by the journal rules, all names under the genus level must be written in italics, this includes names of genus sections.
3.	Generally, gene names are written in italics.


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 USE OF CHLOROPLAST GENOME SEQUENCES TO SOLVE PHYLOGENETIC INCONGRUENCES IN POLYSTACHYA HOOK (ORCHIDACEAE JUSS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Although no easy to read, the author used the correct English expression in manuscript. And I can't found mistakes in other parts including reference, figs and tables. The result is self-contained.
Experimental design: Some doubts should be resolved, but no so fatal. The details listed in General comments for the author.
Validity of the findings: no comment.
Additional comments: L73-74: more reference should be add here since this number isn't the latest one.
L135: I don't think that is complete one, "nearly complete" would be better.
L161: Please explain here why use that as outgroup（ex. "a section far away based on XX's taxonomic treatment"）.
L162-165: Actually I found your outgroup should be Phalaenopsis, isn't it? You use the sequence from that to root the tree so phalaenopsis was the outgroup indeed.
L179-180: Why seven?
L255-265: I am confused why you used these two regions and the P. estrelensis8 as representative? And only psbD-trnT and matK have been estimated here, I don't think it can be the representative of chloroplast genome.
L266: As the reference is Phalaenopsis, the non-coding regions are no credible in Polystachya based on your method since these regions are highly influenced by leading reference. Moreover, these regions are various among species especially in different taxonomic tribe. Any confuse non-coding regions without steable estimation should be removed from the downstream phylogenetic reconstruction.
L415-418: The P. bicolor was the outgroup species, it was inpossible to nest within ingroup as soon as you root. Also it is not suitable to use the species in confusion as outgroup.
L432-433: Paraphyletic group is not a evidence for high species diversity.
L436: Hybrid origin is not a correct word since no evidence to distingush that from incomplete lineage sorting, alternatively the reticular evolution could be used.


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 USE OF CHLOROPLAST GENOME SEQUENCES TO SOLVE PHYLOGENETIC INCONGRUENCES IN POLYSTACHYA HOOK (ORCHIDACEAE JUSS)
Review round: 1
Reviewer: 3

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: 1. The name of genus Polystachya should better be added in the title of the article for a clear expression.

Next generation sequencing commonly indicates more than one technique, the Next generation sequencing technique is just a tool, that enabled faster collection of sequence data, and did not contribute  more  information than the cp genomes, furthermore, the Next generation sequencing technique is often used for indicating several high through-put sequencing methods, actually only information from chloroplast genomes directly helped the authors realize their research
aims.
Therefore, it is not necessary to emphase "the Next generation sequencing" in the title of the article.
A more clear and proper title of the article might be
Solving phylogenetic incongruences in genus Polystachya of Orchidaceae Juss using chloroplast genome sequences
or
The use of chloroplast genome sequences solving phylogenetic incongruences in genus Polystachya of Orchidaceae Juss
or something like these.

2. If sequencing results were compared using different methods for confirmation when the cp genomes were sequenced using the method mentioned in the 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: THE USE OF CHLOROPLAST GENOME SEQUENCES TO SOLVE PHYLOGENETIC INCONGRUENCES IN POLYSTACHYA HOOK (ORCHIDACEAE JUSS)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Fulfilled
Experimental design: Fulfilled
Validity of the findings: Fulfilled
Additional comments: The authors properly addressed each of the comments raised in the previous revision by complementing they literature search and by explaining in much more detail several points not clearly explained in the previous manuscript text.


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 EFFECT OF RECENT COMPETITION BETWEEN THE NATIVE ANOLIS OCULATUS AND THE INVASIVE A. CRISTATELLUS ON DISPLAY BEHAVIOR
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors investigated whether human-mediated introduction of Anolis cristatellus to the distribution area of native Anolis oculatus changed communication behavior of these two closely related species in wild. The result clearly showed that invasive A. cristatellus changed display behavior in sympatry with the congener compared to that in allopatry, whereas A. oculatus did not change its display between sympatric and allopatric population. Although the data did not fully demonstrate the change in behavior was caused by interspecific interaction, this provide a rare example of early response of an invasive species to recent secondary contact with a closely related native species.
Experimental design: Although this is a well-written paper that presents interesting data in general, I believe authors can analyze the data more; to consider whether the difference of display between contexts was induced by interspecific interaction, correlation between degree of habitat displacement and behavioral displacement in each site should be also checked. These displacements would positively correlate if both change were caused by interspecific interaction. Comparison of body size and BMI may be also informative to evaluate presence of interspecific competition. In addition, boxplots showing habitat characteristics would be useful for readers to know how different average and variation of habitat characteristics between species and context.
Validity of the findings: All in all, the findings drawn from the data presented was enough valid. The only concern is it was unclear whether social function of dewlap extension and push-up is fully same. This information would be important to interpret the change of the proportion of time spent displaying.
Additional comments: It will be of interest to readers of this journal, PeerJ, particularly researchers in evolutionary ecology and behavioral ecology. However, in the Discussion section, the authors mostly stated about the results and previous researches on anoles. I think the authors should refer to other studies about behavioral response of introduced or native organisms when they contact secondarily.


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 EFFECT OF RECENT COMPETITION BETWEEN THE NATIVE ANOLIS OCULATUS AND THE INVASIVE A. CRISTATELLUS ON DISPLAY BEHAVIOR
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, the authors compare display behaviors of a native and introduced anole species at different sites in Calibishie, Dominica to identify if display characteristics change when the species are in allopatry versus sympatry. The authors have a great system to study this question with the introduction history of the invasive species well known and recent. The manuscript is well-written and an interesting read.
Experimental design: I suggest that the research question is more hypothesis driven. After the introduction, I was left wondering why the display differ with increased competition, especially if the two species have diverged in microhabitat? The authors offer some hypotheses in the discussion, but I think the introduction could be made stronger if there was more of a prediction to help lead the reader along.
Validity of the findings: I am curious why the authors decided to focus on the dewlap extensions and not the push up behavior. Push ups are actually thought to be more conspicuous and used for long distance communication compared to dewlap extensions (e.g. Fleishman 1992, Ord and Stamps 2008) – the opposite of what the authors speculate (lines 175-177) – so if A. cristatellus increased push up time in sympatry, could this be a sign of more aggressive behavior? Some discussion of the push up part of the display would strengthen the paper.

This paper could also be strengthened by some acknowledgement that anoles use different types of displays (Type A, Type B; Jenssen 1977) presumably in different contexts (e.g. “challenge” versus “assertion” displays, Carpenter 1967). It seems that the authors would have captured a variety of different displays in different societal contexts - could the differences between sympatric and allopatric sites be due to the anoles using one type of display more often than another?

I also would like to see Figures 2 & 3 as a strip chart so all the data points are seen. It would be helpful to see how disparate the displays really are among the different sites, rather than just the mean and standard error. Along these lines, I would have liked a better sense about the different sites. For example, how far apart are the different sites? Some detail in the text or, even better, a map of the sites would be useful. Also, how much variation is there among the different sites? If the authors present Fig. 2 & 3 as a strip chart, they could even show the data points from different sites by representing the data points as different symbols.
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: A NEW MINUTE ECTOSYMBIOTIC HARPACTICOID COPEPOD LIVING ON THE SEA CUCUMBER EUPENTACTA FRAUDATRIX IN THE EAST/JAPAN SEA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a nice and interesting contribution to the diversity of harpacticoid copepods of the family Laophontidae from the littoral zone of the East Japan Sea. The manuscript is relatively well-written, but in my opinion, the authors need to revise the general style.

I’m not an English native speaker but took the liberty to suggest some corrections to improve the general style.

It would be good to let an English native speaker read the manuscript to further improve the general style of the manuscript.
Experimental design: The manuscript fits the subjects of the journal, the question addressed by the authors is well-defined and relevant.

The authors describe a new genus and species of Laophontidae of the subfamily Laophontinae associated to a holothurian. This is interesting since only few symbiotic Laophontinae copepods have been reported associated to holothurians, filling an important gap to better understand the ecological radiation of laophontids. The investigation performed by the authors were conducted rigorously and meet the standards in harpacticoid taxonomy. The methods were described with sufficient detail.

The authors included the most relevant literature. However, in the list of references I detected that one of them is probably wrong (Huys R. 2009. On the junior subjective synonyms of Coullia Hamond, 1973 (Copepoda, Harpacticoida, Laophontidae): an update and key to species and related genera. Zookeys 5:33-40). I think the reference here is “Huys, R. 2009. Unresolved cases of type fixation, synonymy and homonymy in harpacticoid copepod nomenclature (Crustacea: Copepoda). Zootaxa 2183: 1-99”. I also suggested to include some other references that might help to improve the general content of the manuscript.

The figures included in the manuscript are very informative and of excellent quality. I suggest including a figure (map) showing the type locality, as well as the places where the species of Microchelonia have been found. I think this is important since the authors claim a sister group relationship between the new genus, Vostoklaophonte, and Microchelonia.

The general structure of the manuscript meets the requirements of the International Code of Zoological Nomenclature, the type material has been deposited in a well-known collection and is available for future inspection. However, I noted that some material was deposited in a collection whose acronym seems to be “Me”. I’m not sure if this acronym corresponds to the Zoological Museum of Lomonosov Moscow State University. This should be clearly stated in “Materials and Methods”.
Validity of the findings: The data are robust, and the results and conclusions are, in my opinion, sound. However, I still do not understand why the authors included the 18S-rDNA data for all the harpacticoid species available from GenBank. The information in Table 1 and in Figure 8 were not discussed in detail and, in my opinion, are not informative or relevant to the general content of the manuscript. Some comments on this and on other subjects were also included in the revised word document of the manuscript.
Additional comments: I have no general comments for the author(s) other that those in the revised word version of the manuscript.


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

Input:
Title: A NEW MINUTE ECTOSYMBIOTIC HARPACTICOID COPEPOD LIVING ON THE SEA CUCUMBER EUPENTACTA FRAUDATRIX IN THE EAST/JAPAN SEA
Review round: 1
Reviewer: 2

Output:
Basic reporting: A new genus and species of harpacticoid copepods is well described and illustrated. However, Discussion and others must be greatly revised. If SEM materials are not designated as paratypes, descriptions based on it should not be included in the descriptive parts. These are separately mentioned. Generally, a description of a new taxon must be based on type series (ICZN). Indicate what types were used for the illustrations. A molecular analysis should be reconsidered. In the descriptive parts, articles can be deleted for simple telegraphic style.
Experimental design: Terminology should be reconsidered. Since “The East Sea” is not internationally and officially recognized at the moment, it should not be used here. This is a scientific paper, but not a political one.
Validity of the findings: The definition of the new genus should be more cleared in comparison of other genera belonging to the subfamily Laophontinae. The new genus is compared with Microchelonia and others, but still problematic (see General comments).
Additional comments: See above also (Basic reporting, Experimental design, Validity of the findings).
Specific comments
1)Abstract. Laophontinae should be used here rather than Laophontidae (see Discussion).
You focused the subfamily rather than the family.
2) L41, 47. ..on the ...coast.
3) L49-52. This paragraph abruptly appears. Reconsider the context.
4) L65. Why did you use 10 % ethanol?  If you followed previous reports, cite them.
5) L68, 69. This paragraph should be moved to somewhere in L80 to 98.
6) L77. Delete “All the...of”.
7) L104, 107. What article in ICZN?
8) Diagnosis. Some are not diagnostic to the new genus, but to Copepoda!
9) L135. You may use “by monotypy”.
10) L153. Citation is OK?
11) L156, 240, 242. Indicate what types were measured. Those of the holotypes should be mentioned separately.
12) L164. What type for 0.78?
13) L172. What do you mean by “Each of P6”?
14) L174. Delete “, but...unclear”.
15) L177. What type(s) for 0.93 and 0.88?
16) L188-192. Reconsider the description of the antenna. Exopod seems to be 2-segmented.
17) L195. Can you identify an outer seta as a remnant of the exopod?
18) L198, 199. The homology of the maxilla is OK? The conditions are highly reduced, your understanding of the homologues must be careful.
19) L200. Space is needed in front of Maxilla (new paragraph).
20) L204. ...drawn out into...
21) L218. Not plumose.
22) L251. Not only the antenna but also other mouthpart appendages should be mentioned in comparison with those of the female.
23) Phylogenetic position. Using numerous taxa, you said only one thing. Reconsider the analytic results and discussion.
24) Discussion. This part must be greatly changed for better context. The following steps may be better: (1) why can the present new genus belong to the subfamily?; (2) listing symapomorphies, closely related genera are compared to the new genus, and then differences among them must be stressed for the establishment of it; (3) some kinds of morphoclines throughout the symbiotic evolution of the subfamily can be discussed. L295-301 and L345-352 are strange considering the context. L326, 327. The new genus and Microchelonia are not properly compared here. L330. P3 and P4. L342. Don’t use “swimming”. L358. Microchelonia glacialis was found from Laminaria (L296).
25) Table 1.There are careless mistakes. Sp. is not italic; period of et al.
26) Fig. 1. This is an ovigerous female. So the egg-sac and eggs should be mentioned in the text.
27) Fig. 4D. This may be abnormal seeing that of the male. Check other types.


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: A NEW MINUTE ECTOSYMBIOTIC HARPACTICOID COPEPOD LIVING ON THE SEA CUCUMBER EUPENTACTA FRAUDATRIX IN THE EAST/JAPAN SEA
Review round: 1
Reviewer: 3

Output:
Basic reporting: General things for English and sentences throughout the MS:
Some places are not available. Comma and space are not used proper. Hyphen and n-dash are also not utilized properly. Moreover, the unity of writing is not taken at all in the Descriptive section (e.g., three-segmented or 3-segmented). There are also many careless mistakes.

Line drawings:
Several points should be redrawn.
Experimental design: The main frame of this study is simple, and thus it is no problem.
Validity of the findings: The most important thing of the MS is to prove the validity of the new genus. However, this MS is inadequate for its explanation. Together with the Basic reporting, the MS requires to be polished before publishing.
Additional comments: The manuscript is including a description of new genus of symbiotic Harpacticoid. Descriptive part was prepared to explain the adequacy of the new genus, and subsequent phylogenetic analysis helped the result. However, there are several problems which have to be resolve before publishing.

Also, as I mentioned in the Basic reporting, there are many careless mistakes which I marked on the PDF file.


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

Input:
Title: A NEW MINUTE ECTOSYMBIOTIC HARPACTICOID COPEPOD LIVING ON THE SEA CUCUMBER EUPENTACTA FRAUDATRIX IN THE EAST/JAPAN SEA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This is the second review of the manuscript "A new ectosymbiotic harpacticoid copepod living on the sea cucumber Eupenctata fraudatrix in the East/Japan Sea". The authors did a good job with the "Introduction", "Materials and Methods" and "Systematics". I am not an English native speaker but took the liberty to suggest some changes in order to improve the manuscript. Also, I have only some minor comments. For example, I noted that the authors included in the abstract a new record of Microchelonia koreensis, but nothing is said about this in any other part of the manuscript. I still don't understand why the authors included all the harpactioid species in Table 1 and Figure 8. None of these species were discussed in the manuscript. Also, I suggest to check carefully the journal's format. For example, I'm not sure about the use of "1", "2", "3", "4", etc., instead of "one", "two", "three", "four", etc., "1-segmented" instead of "one-segmented" (the authors used both formats), etc.; also I suggest the authors to check that the authority of genera and species are given in full only the first time they are mentioned in the text, and to check the proper use of hypehns, n-dashes, and m-dashes (I assume that the use m-dashes should be kept at a minumum).
On the other hand, I think that the Discussion needs some extra work to improve the general content and structure of the manuscript. I took the liberty to suggest some changes. I am particularly interested in the possibility of (1) defining the new genus and species based on autapomorphies rather than on the combination of a set of characters, and (2) defining a monophyletic group of symbiotic genera (Vostoklaophonte and Microchelonia) based on synapomorphies and supported also on molecular evidence given by the authors. I think this could add some extra value to the manuscript.


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

Input:
Title: REPRODUCTIVE ECOLOGY OF THE ENDANGERED BEAL’S-EYED TURTLE, SACALIA BEALEI
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English used is clear, I made many comments on ms for a better English style.  There is no problem with the text, except they make several untrue statements, I have cut out much of the text whcih talks about conservation issues and poaching, this article is about reproduction, and does not need to have and discussion about humans trafficking this species.
They do not describe the type of transmitters used. They should have a photo of their string trailing device.  I do not know why they mentioned radio telemetry if they did not report the data.  Also they did not report if they followed the female turtles after they returned to the river.  These turtles could have had the opportunity to nest several more times during the season.  No mention was made if they followed the turtles until hibernation or not or if they exrayed them again or not.  If they had used infrasound they could have detected enlarged follicles as well as oviducal eggs, which would suggest future clutches of the same year. Many format errors in Lit Cit.
Experimental design: There was no mention of turtle activity after they returned to the water, the turtles should have been followed with radio telemetry for at least 2 months and captured and palped, placing a finger into the inguinal cavity will allow one to detect the presence of shelled eggs in turtles, even species such as Kinosternon acutum, a much smaller species where nesting females have a mean crapace length of 97 mm and mass of 117 g, they can be palped, and they froduce 2-4 clutches of 1-3 eggs during a season.
Validity of the findings: The nest descriptions, number of eggs per clutch, incubation temperatures, haching and female sizes are all important findings.

But, as i said above, since they did not follow the females after they nested they can not validate that they only nest once a year

Also they do not know if the eggs that did not develop were fertile or not, they did not open the eggs and inspect the membranes for dead sperm.

But fecundity does not seem to be a problem in this study
Additional comments: I think you did a wonderful job writing this article in English.  You should report the size of the females that nested in the results in the text, the measurements of the hatchlings should be in the text and the temperature data as well delete all of the tables, and it would be informative to have a photograph of a turtle with the trailing device attached.  If you have data with radio telemetry after the turtles nested you should report on it.  You should also mention when they entered hibernation.
I think your data are worthy of publication, heed my remarks and rewrite it.  The allusion to seaturtles and larger freshwater turtles making flask shaped nests is not relevant, very small freshwater turtles also make flask shaped nests,  I do not think bringing a discussion of flask shaped nests has anything to do with your study.  You do not talk about sex ratios in nature, so do not mention it.  This article is about nesting and incubation in nature.  Leave all of the material about the pet trade out of the article.


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

Input:
Title: REPRODUCTIVE ECOLOGY OF THE ENDANGERED BEAL’S-EYED TURTLE, SACALIA BEALEI
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting of the paper is fine, except for the noticeable lack of figures. Since PeerJ publishes free color this is an easy way to greatly enhance the reporting of this paper.

Line 47 (and elsewhere) the authors discuss spool and line tracking, they should show images of what this is. How it looks in the field.

Lines 57 and 58 describe the vegetation, the authors should supplement the short written description with images. Show the stream and and nesting sites.

Line 138 show camoflauged nest and fishing line

Lines 160-164 they give description of hatchlings that is begging for photographs.

Lines 175 and elsewhere they discuss the nest shape, they should show pictures.

Optional images would be of the adult species.
Experimental design: I think the experimental design is fine. Maybe there are other approaches, I don't know, but they added many new data and inisghts about this poorly know species.
Validity of the findings: I think their conclusions are well stated and connect to the literature.
Additional comments: This paper definitely needs some photographs.


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: STRUCTURAL INSIGHTS AND CHARACTERIZATION OF HUMAN NPAS4 PROTEIN
Review round: 1
Reviewer: 1

Output:
Basic reporting: This report focuses on the activity-dependent transcription factor NPAS4. The authors attempt to provide structural insight and to predict proteomics information regarding this protein. The goals of this study are clearly stated. However, this manuscript does not provide sufficient information for this topic. This reviewer lists the following 4 concerns:

1. There are grammar errors throughout the manuscript. There are also terms that need clarification. For instance, in line 50-51, the "activity dependent genes" is confusing.
2. The authors did not provide sufficient background information. For example, the authors mentioned that there is no structural homologues for homology modeling of NPAS4. However, in Protein Data Bank (PDB), at least a crystal structure of the heterodimeric NPAS3-ARNT complex with HRE DNA is available. Or the authors should have describe that why this crystal structure is irrelevant for NPAS4 modeling.
3. Except the figure 9, none of these figures contain proper labels to be informative. The structure model is not further analyzed to provide insights.
4. The set of results is meaningful. However, the data is too preliminary. The results generated in this report are not sufficient to facilitate future studies.
Experimental design: The authors clearly stated the absence of structural information for NPAS4. However, solely
relying on web servers, the authors have not developed any methods or designed any experiments to generate results that contain unique insights. Moreover, as mentioned above, the authors did not mention why homology modeling using the crystal structure of NPAS3 is infeasible. It is difficult to put this manuscript into the category of Research Articles or Literature Review Articles, the only two types of articles PeerJ considers.
Validity of the findings: Since all the results reported in this manuscript were generated by web servers, they are too preliminary and thus difficult to validate.
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: STRUCTURAL INSIGHTS AND CHARACTERIZATION OF HUMAN NPAS4 PROTEIN
Review round: 1
Reviewer: 2

Output:
Basic reporting: In their manuscript submitted to PeerJ, Fahim et al present an in-silico study of an important human neuro-transmission Npas4 protein. They have reported a tentative tertiary structure and analyzed physio-chemical properties of Npas4 protein along with study of its closest phylogeny and identified the interaction partners of the protein.
Introduction has been well-written with adequate references emphasizing on the importance of Npas4 and the objective of this research work. However, the paper needs rephrasing at several places as mentioned below to make the comprehension more easily understandable:
•	L61: “predict the structural insight” should be replaced with “provide structural insights into”
•	L69, L73: “orthologus” spelling is incorrect
•	L77: “forecast” should be replaced with “calculate” or “predict”
•	L94, L97: “Accession No.” is misleading
•	L100: “,” should be replaced with “.”
•	L106: Line needs rephrasing
•	L141: “.” should be removed after “potential”
•	L144: “are” to inserted before “present”
•	L148: correct spelling as “revealed”
•	L157: “,” to be replaced by “and”
•	L160: “are” to be inserted before “shown”
•	L187: Replace “,” with “.”
•	L189, L194, L195, L196: “Npas4” should be replaced with “Npas4” as only gene names are written in italics.
•	L199: replace “affect” by “are affected by”
•	L213: “phosphorylation,” to be replaced by “phosphorylation.”
•	L221: Line needs rephrasing
•	L252: the word “elaboration” is confusing
The manuscript has been drafted in accordance with PeerJ standards and all the figures with the exception of Fig 3 are of high quality. Some additional suggestions that might benefit a reader are: 1) adding a color legend in Fig 4 that explains the red bar and purple straight lines, and 2) adding the sequence length of each domain along with highlighting the N- and C- terminal regions in Fig 7.
The raw data has not been shared adequately. UniprotID corresponding to the Npas4 protein sequence should be mentioned in the first section of Materials and Methods. Also, the parameters used for running ClustalX and Mega7 are lacking in the manuscript. Tuning the parameters always changes the output results. So mentioning parameter values or mentioning default parameters in Experimental section is appreciable.
Experimental design: The research problem as posed by the authors is very relevant in the absence of sufficient literature regarding structural insights and characterization of human Npas4 or its homologues. But, their experimental design and approach to address their goal of research lacks technical expertise and is not up to the scope of this journal.
The main drawback of the manuscript is the ambiguity in the results obtained by running various bioinformatics servers used by the authors. Uniprot database provides a detailed analysis of Npas4 protein (Uniprotid: Q8IUM7) with a list of similar proteins from phylogenetic analyses clustered on basis of sequence identities, yields information on interaction partners of this protein from STRING and other databases and also provide models of this protein from various 3D structure databases. Its therefore, crucial that the authors provide a comparative explanation of how their results are different or superior than those provided by Uniprot.
As with all prediction servers, it’s also highly recommended to assess the statistical significance of the results, which the authors have failed to address in the current manuscript. For example, while discussing the results of tertiary structural prediction, authors should provide more details of I-tasser output, like the values of C-score for 5 models and templates selected by I-tasser to generate such models. It’s equally important to analyze the templates and check alignment accuracy of the template with Npas4 protein (Tm score). Also, there is no mention of how clusters were generated from the output of STRING database to determine interaction partners.
Validity of the findings: The results as reported by authors need several edits as suggested below:
1)	L105: Authors mention using Multiple Sequence alignment but there is no accompanying figure of the alignment highlighting the conserved residues.
2)	L108: Supplementary Table S1 is missing.
3)	L109: Use of taxonomic nomenclature is preferred instead of the generic animal names like chimpanzees and monkey.
4)	L109: Please check spelling of rodents like mramrt.
5)	Fig3: Resolution of the plots needs to be greatly improved.
6)	L115-137: Authors have reported values of several physiochemical properties of Npas4 but it will be interesting to see how these values are correlated to the structure/functioning of the protein. For example, does the flexible amino acid residues from position 204 to 295 as identified by flexibility index correlate with flexible region in the modelled structure obtained from I-tasser.
7)	L153-155: While predicting secondary structure from GOR4, PsiPred and JPred3, it will be helpful if the authors provide a consensus secondary structure figure representing which residues/regions of Npas4 correspond to helix, sheet and coil.
8)	L156-160: Authors have chosen the highest C-score model from I-Tasser to be the best model. However, they haven’t provided any C-score values nor provided the statistics of ProSA, PROCHECK and Pymol to compare these 5 models. It will be also good to compare the templates used by I-tasser and those used by 3D structure databases as in Uniprot to have better confidence about the tertiary structure of Npas4 protein model.
In the discussion section of the manuscript, the physicochemical property values and study of posttranslational modification sites is interesting but mostly based on prediction without any correlation to the tertiary structure. The significance of different interacting protein clusters of Npas4 as obtained from STRING database is well documented but authors are encouraged to discuss the input parameters used (like maximum number of interactors to be shown in 1st and 2nd shell) to explain the analysis. A few corrections needed are:
1)	L185: Correct the reference author name as Adachi et al 2014.
2)	L187: The reference (Katoh et al) doesn’t match with the paper cited.
3)	L222: STRING is a database and it’s advisable to write in capitals so as to not to confuse readers with word “string”.
Additional comments: Overall, the authors have well highlighted the scientific importance of Npas4 and the purpose of their study. However, their results fail to provide detailed structural insights of human Npas4 protein as claimed in their manuscript title and hence, needs further research work and statistical analyses, before Acceptance.


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: STRUCTURAL INSIGHTS AND CHARACTERIZATION OF HUMAN NPAS4 PROTEIN
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thank you for providing me the opportunity to re-evaluate this manuscript.

The two major concerns I had for the previous manuscript were: 1. the lack of unique insight provided by the authors to facilitate future studies; 2. the authors did not address why homology modeling is infeasible.

I recommend acceptance of the revised manuscript for two reasons: 1. the authors actively provided reasonable response to my concerns. 2. After more investigation of this topic, I better understand that Npas4 protein is not only important, but also very unknown at structural level. Even the results author provided does not suffice to lead clear direction, they encourage more studies in this topic.


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: ESTABLISHMENT OF A 12-GENE EXPRESSION SIGNATURE TO PREDICT COLON CANCER PROGNOSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript requires some editing of English language, grammar and phrasing to improve clarity of meaning. For example correct application of singular and plural rules.
Please explain abbreviations at first use in manuscript e.g. TNM, TCGA in abstract. Some explanation of the GSE datasets should be added to the abstract to clarify.
Appropriate introduction and background provided to the topic, but note comment above on English language use.
Figures are relevant, but issues of resolution should be addressed in the high density figures e.g. Figure 2. Text resolution in Figure 3 plot is poor. Further description and explanation of terms should be added to figure legends e.g. abbreviations used, axis labels. Raw data files are provided. The statistical plots and test results displayed in the figures need further explanation for those not familiar with their use.
Experimental design: The use of available data from TCGA and GEO is described in detail. There appears to be rigorous validation using patient data. However, full assessment of the statistical approaches applied is beyond my area of expertise.
Validity of the findings: The impact and novelty of the 12 gene signature is clearly outlined and potential application and benefits explained in colon and other cancers. Identification of potential genes implicated in progression and metastasis provide useful preliminary information for focus of further study of the factors influencing prognosis, cancer progression and metastasis.
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: ESTABLISHMENT OF A 12-GENE EXPRESSION SIGNATURE TO PREDICT COLON CANCER PROGNOSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No Comment
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: In this manuscript “Establishment of a 12-gene expression signature to predict colon cancer prognosis” from Dalong Sun et al., the authors outline a bioinformatics approach to identify poor prognostic group from a general colorectal cancer population. This is an area of importance currently, given the availability of transcriptional and molecular data, in this regard the author add to the already well-established literature in the development of prognostic biomarkers for colorectal cancer. The author have used multiple data sets for discovery and validation, in addition to an extended collection of non-CRC cohorts to demonstrate the prognostic power of their classifier.
The manuscript presents a clear methodology that combines a number of well established approaches to prognostic classification, I do however have a number of points concerning the clinical and pathological status of the patient cohorts selected, which could potentially undermine the approach taken by the authors.

Phase I and Phase II patient cohorts:
Can the authors outline details in a table to clarify the stage of the patients in the training prognostic groups; is it that the poor prognosis group are all late stage, with the good prognosis group being early stage?

As it stands, the classifier is potentially a signature of tumour stage, which is an already well established prognostic indicator as detailed by the authors in their introduction. Again further detailed information needs to be explained here in both Phase I and Phase II.

Can the authors define in a table if these patient received treated at any stage of their disease, and if so was this adjuvant, neo-adjuvant or in the metastatic setting, or have they received surgery alone; factors which have strong bearing on disease outcome. The patient sets used at this stage needs to be detailed in a clear table, a link to the Cancer Genome atlas of GEO is insufficient.

Patient cohort selected.
The authors state in the first 2 lines of their abstract that - “Robust and accurate gene expression signature is essential to assist oncologists to determine which subset of patients at similar TNM stage has high recurrence risk” – also on line 54-56 in the introduction the authors again highlight the importance of this “However, due to the high heterogeneity of disease, the patients at similar stage behave differently in terms of recurrence and response to chemotherapy often differs.” – based on these key points, the author correctly describe that the generation of a signature that can distinguish prognosis within a defined TNM stage is therefore essential (stage II only or stage III only or stage II/III), and not just identify stage which is already defined by TNM.

In order to align this study with the key points raised by the authors within their manuscript, can the authors focus on a single stage, or indeed combine stage II/III and carry out their prognostic analysis; ie does this signature distinguish poor prognostic patients from within a similar stage?

In Figure 4 the authors clearly show that the signature is associated with worst outcome. As discussed above, if this was developed using a defined TNM stage and then tested in an independent cohort of the same specific TNM stage, it would strengthen the validity of the findings. Line 37-39 detail that “Interestingly, advanced stage patients were significantly enriched in high 12-gene score group (Fisher’s exact test p=0.0003).” indicates this may be the case.

The signature generated:
In the first 5 lines of the abstract the authors state: “Here we applied two steps of supervised machine-learning method and established a 12-gene expression signature to precisely predict colon adenocarcinoma (COAD) prognosis by exhaustively using expression of all genes and associated clinic information of TCGA COAD patients” – it is rather misleading to suggest the clinical factors have been included in the machine learning approach to develop the signature – please clarify this sentence or else combine in the clinical information into the machine learning approach.

From figure 1 (which is the extended signature) it appears that it is increased gene expression that is associated with high-risk – is this true for the 12 genes? Is the signature upregulated or down regulated when identifying good or poor prognostic patients?

Functional classification of the signature:
The author’s state in their conclusion, line 278, that “Functional classification” of the genes was carried out, which is not detailed in this study, please include these results in detail.

The 12 gene signature is not listed in the results, apart from being discussed; as this is the key piece to this study more focus needs to be put on the genes themselves – have they been previously associated with prognosis, what is their underlying biology.. etc. Functional classification will require potentially pathway analysis or other methods to characterise the biology.

In addition, if this is an “immune” signature, how does this overlap with the CMS1 subtype identified by Nat Med. 2015 Nov;21(11):1350-6 or other well established immune signature in CRC?

Validation cohorts:
The study is strengthened by the addition of multiple validation cohorts, although similar points raised in the training cohort are present in these cohorts.
For GSE39582; the 566 patients in this cohort again span all stages with mixture or treated and untreated patients – to address the points above the use of a specific stage with a similar treatment schedule (perhaps untreated only) would address concerns.

Furthermore, surprisingly the authors use only the pMMR sub-set of this cohort; if showing this analysis it is also required to show the dMMR (and combined) data also for comparison. Similar comments for GSE17538. In both cases, patient set used at this stage needs to be detailed in a clear table with clear indication what the characteristic are of the entire cohort alongside the identified high- and low-risk group.

Other non-CRC cohorts:
The authors should be commended for applying their signature to other disease types, where they find prognostic value outside of COAD – given the presence of 7 immune genes in their signature, these results are reflective of he importance iof the immue system in the prognosis of cancer – discussion of these points to include Nature Medicine volume 21, pages 938–945 (2015) should be included here.

Introduction lines 50-53 describe classical genetic markers of CRC; as this study is focussed on transcriptional approaches to subtyping cancer, the authors will need to include the CMS Nat Med. 2015 Nov;21(11):1350-6. transcriptional classification system in the introduction and discussion.


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: ESTABLISHMENT OF A 12-GENE EXPRESSION SIGNATURE TO PREDICT COLON CANCER PROGNOSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have revised the manuscript and have satisfactory addressed the major points raised during the initial review. If possible (within the word-count limitations) if a small concise description of the specific genes/biology could be included in the discussion this would be welcome.


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: CRUCIAL BIOLOGICAL FUNCTIONS OF CCL7 IN CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is an attractive and well written review. The figures are well designed and the article covers an important field. However, the survey methodology appears to have left certain key elements in need of additional discussion. The information on the expression of key CCL7 receptors (CCR1, -2, -3) on tumor cells and CCL-7 mediated signaling responses mediated within the tumor cells is too sparse. Brief mention is given to CCR1 expression on breast cancer cells.  However much more recent information is available on CCR1 expression not only by breast cancer (Cao Faseb J 2017, Shin Oncotarget 2017), but colorectal cancer (PMID 26383527), and lung cancer (PMID 18972130) for instance. Another area that is lacking involves therapeutic options: How do the authors propose targeting CCL7 and which methodologies hold more promise (e.g. peptide, shRNA, antisense oligo, receptor aptamer etc)? Do the authors believe that, and how should CCL7 antagonists be developed for cancer treatment?
Experimental design: As previously mentioned, the survey methodology appears to have left certain key elements in need of additional discussion.
Validity of the findings: No comment
Additional comments: No comment


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

Input:
Title: CRUCIAL BIOLOGICAL FUNCTIONS OF CCL7 IN CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This is a comprehensive review that summarized both pro-tumor and anti-tumor roles of CCL7 derived from a variety of tumor and tumor-associated cells. While the authors aimed to review all available literatures on this topic, it would be important to connect every piece of observation in a logic way. Some issues need to be addressed.

Specific comments:
1. The gene symbol for human CCL7 should be capitalized (CCL7).
2. Line 45: “the first to be discovered” should be “the first have been discovered”.
3. Line 56: “and under pathological conditions (tumor cells)” should be “and in tumor cells under pathological conditions”.
4. Line 57: Please specify “immune-related cells” as all examples are leukocytes.
5. Line 60-63: Please explain briefly how CCL7 is related to these diseases.
6. Line 94-105: This paragraph is to describe the regulation of CCL7 expression. The first sentence “Chemokines often have shared and exclusive chemokine receptors.” is actually irrelevant and it may be moved to the paragraph of “CCL7 RECEPTORS”. In addition, the content of the text here and that of Figure 1 are completely different. How is CCL7 induced or suppressed by cytokines/stimuli? Which responsive elements in its promoter/enhancer are involved? Which cells are examined? All of these information together with references should be included in both the text and Figure 1 consistently.
7. Line 98-100: Please revise this sentence “The upregulation of CCL7 is associated with IL-1β, IFN-α, IFN-β, and measles virus in peripheral blood mononuclear cells (PBMC) (Menten et al. 1999).” to clarify its meaning.
8. Line 108: “subpopulation” should be “subtype”.
9. Line 141: “CCL7 also may affects” should be “CCL7 may also affect”.
10. Line 161: Please revise this sentence “CCL7 greatly impacts both antiviral and anti-bacterial immune responses.” as the role of CCL7 in antifungal immunity is discussed later in the same paragraph.
11. Line 166: Please describe “Tip-DC” briefly.
12. Line 176-186: The anti-tumor role of CCL7 should be mentioned in this paragraph.
13. Line 203: “the ERK-JNK signaling pathway” is parallel. Both ERK and JNK are MAPK.
14. Line 206-216: The regulation of CCL7 by microRNA Let-7d should be discussed in the paragraph of “THE STRUCTURE AND REGULATION OF CCL7”.
15. Line 219: “TGBβ1” should be “TGF-β1”.
16. Line 223: What is the relationship between CCL7 secretion and IL-1β?
17. Line 306: “do BM-MSCs” should be “BM-MSCs do”.
18. The paragraph of “CCL7 and clinical prognosis” should be moved after the paragraph of “Antitumor effects of CCL7”.
19. In Figure 2, the MAPK signaling pathway should appear inside tumor cells since the other arrows around indicate different tumor-associated cells. Please also correct the MAPK-ERK-JNK pathway in this figure.
20. In Figure 3, please clarify the anti-tumor role of CCL7 in detail.


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: CRUCIAL BIOLOGICAL FUNCTIONS OF CCL7 IN CANCER
Review round: 1
Reviewer: 3

Output:
Basic reporting: Please find attached PDF file.
Experimental design: Please find attached PDF file.
Validity of the findings: Please find attached PDF file.
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: POPULATION DIVERSITY AND RELATEDNESS IN SUGARBIRDS (PROMEROPIDAE: PROMEROPS SPP.)
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: These seem appropriate, although I would like to see some statistical comparisons between the diversity metrics of the two species as I expect they are not significantly different.
Additional comments: This is a straightforward paper that compares diversity measures from one population of each sugarbird species. A secondary contribution, are the publication of two new mtDNA primers that appear to be of broad utility to PCR-amplify the NADH2 locus across birds.  I have only relatively minor comments that I hope the authors will find useful in revising their paper.

Introduction
1)	I would move the first paragraph to the end of the intro. First lead with the information pertaining to sugarbirds, then on to the markers to be used and approaches to be adopted.
2)	Line 5 – make mention that not only does mtDNA have a fast mutation rate, but it has a faster coalescence time relative to nuclear DNA due to its smaller effective population size.
3)	Line 7 – a more bird specific ref may be useful, for a southern African example see Ribeiro et al. 2012 Mol Ecol 21: 662-672.
4)	Line 11 – hanging sentence “… and thus serve as effective population genetic markers…FOR WHAT…”
5)	Lines 28 & 29 – provide latin names for the two sunbird species mentioned.
6)	Line 30 – the use of the wording “mating opportunities” in the sentence structure is confusing as it implies that sugarbirds and sunbirds are competing for mating opportunities – i.e. with each other, which is certainly not the case.
7)	Line 35 – Proteaceae should not be in italics
8)	Line 38 – language “…, and IT is thought…”

Methods
1)	Details of the Animal Use Committee (IACUC) permissions with reference to a protocol number should be provided.
2)	Lines 64, 66 and various other places in the manuscript – I do not think Free State or Western Cape should be hyphenated.
3)	Line 69 – Cape Nature Conservation is now called CapeNature
4)	Line 120 – what is “Noah’s Ark”?  This seems unnecessary. Why not just say xx species, spanning a large extent of avian diversity and then cite a general bird phylogenetics paper such as Hackett et al. 2008 Science, Prum et al. 2015 Nature.
5)	Line 159 – I do not think Jack-knifing is should be in caps.

Results
1)	Line 95 – drop the “Noah’s ark”
2)	Drop Figure 2, it does not add to the paper.
3)	Line 218 – full stop is missing.

Discussion
1)	line 233 – add latin name for sunbird
2)	lines 230-233 – you could extend this section by adding data on Zosterops species as another nectivorous bird – see papers by Graeme Oatley for southern African species.
3)	line 235 – “…inferred BY bird…
4)	Line 230 – make clear that you are talking about genetic diversity
5)	Line 261 – measures of diversity – can these be compared statistically.  Many of the values are very similar and I expect that they do not differ significantly, which would suggest that in the core of their respective ranges the two sugarbird species retain similar levels of genetic diversity.
6)	Line 266 – provide a ref in support of the statement that these measures are “robust to variation in sample size”.
7)	Lines 302-304 – provide refs to support these statements
8)	Lines 312-315 – remove, this is repetition of the results
9)	Line 329 – extent the sentence to say why Bayesian approaches were not adopted – nature of the data?

Figures and tables

Figure 1 – be more specific than Cape Town.  Provide co-ordinates in the figure legend.

Figure 2 – can be removed from the paper.

Figure 3 – Specify what kind of network this is in the legend. In addition add the percentage uncorrected sequence divergence to the figure above the cross-ticks separating the two species.

Table 3 – Add species common names to the figure.

Rauri C. K. Bowie
March 2018
bowie@berkeley.edu


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: POPULATION DIVERSITY AND RELATEDNESS IN SUGARBIRDS (PROMEROPIDAE: PROMEROPS SPP.)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is clear, unambiguous, and the text is technically correct. The article does include sufficient introduction and background of how the work fits into the broader field of knowledge. The work is also appropriately referenced and figures and tables are easy to follow.
In summary, two species of sugarbird are found in different geographical regions, where one Promerops cafer is in a region with much greater diversity than the other P. gurneyi. The authors therefore propose that the latter may have lower genetic diversity and greater inbreeding so the objective is to provide phylogeographic and conservation assessments for P. gurneyi because it may be on the decline. To do this the authors make use of two genetic markers.
Experimental design: The submission describes original primary research which builds on previous ecological studies of the very same samples. The knowledge gap being investigated is identified, as the authors cover the differences in genetic diversity, inbreeding and relatedness, and effective population size in good detail and the study contributes to filling that gap.
The investigation has been conducted rigorously: For the microsatellite genotyping the authors checked consistency of fragment assignment by replicate amplification. Although only six microsatellite loci were used, they were all highly polymorphic and effective in assigning parentage. The ND2 primer design and authentication across a “Noah’s Ark” of birds is a neat addition to the paper. The mitochondrial gene sequencing was performed using both forward and reverse primers to confirm sequences and increase coverage.
All the methods are described with sufficient information to be reproducible by another investigator.
The research was conducted in conformity with prevailing ethical standards in the field and permits were obtained from the relevant provincial conservation agencies.

I could not find the GenBank accession numbers: MG972851 to MG972879,  however, they are probably embargoed until publication.
Validity of the findings: The data on which the conclusions are based are provided. The conclusions are appropriately stated and are connected to the original question investigated in that the authors characterize and compare genetic diversity and relatedness in populations of P. gurneyi and P. cafer.
Additional comments: List of suggested corrections
Abstract
In the abstract I advise changing Cape Town to Helderberg Nature Reserve
Introduction
I think the Introduction would flow better if it started at Line 12.
Lines 2 -11 are out of place and should be included later in the introduction after the bird species have been described and introduced. So move to Line 56.
Line 13 and line 53  southern with a small letter ‘s’.
Line 20 Explain the use of the word “abundance” in this line.  Sentence is not clear.
Line21 The sentence does not make sense and is incomplete ..used by this species how?
Line 22-23 (Taylor, Peacock & Wanless, 2015; ….)
Line 30 Change the sentence starting on the line to help with English flow:
Taking into account that Proteaceae utilized by ……, and that territory size …..
line 36 to line 37 new paragraph is slightly disjointed ….
Line 56 new paragraph Add lines 1 -11 in here and then go on to the microsatellite mrakers as is from line 56.
Line 57 McFarlane (capital F) and elsewhere in the manuscript.
Methods and materials
Sample collection and DNA extraction
No sample sizes are given except that one individual was caught in Sani Pass. It would help to include the sample numbers for the two populations here
Line 63 µl not ul
Line 64  ….. National Park with a capital letter
Line 65 in Sani Pass
Line 66 Helderberg Nature Reserve,
Line 68  …2003). Permits were obtained from the relevant
Line 83 roadkill not killed
Microsatellite genotyping
Line 97 …used multiplex PCR
Line 98 McFarlane
Line 115 Table 3 not 2
Line 108  …. for both species. (not for either species)
Line 120 not Giving.  Should change to …. resulting in an expected….
Line 152 trial runs
Line 159 …with jack-kniffing across loci - small letter j
Results
In Line 178 to 190: Please would the authors explain why there is a reduced sample size amplification for Pro86 and Pro66 in both P. gurneyi and P. cafer. i.e. in Table 2 sample size for P gurnyei is n=49 for all loci except Pro86 (n=24) and Pro66 ( n=31), and for P. cafer  n=63 for all loci except Pro86 (n=37) and Pro66 ( n=61),
Line 178 six
Line 196 Sanger with a capital S
Lines 207  The authors write “despite a smaller sample of the former”.   This does not make sense to me because P. cafer had the larger sample size of n=63 compared to P. gurneyi having n=49
Line 215 Cervus 3.0.7 (Kalinowski, Taper & Marshall, 2007)

Discussion
Line 231 and 273 McFarlane
Line 235 …has been inferred in bird monitoring….
Line 284 ….may be higher abundance….
Line 287 six
Line 291 This may reflect more first and second order…
Line 298 south is small letter
Line 328  linkage-disequilibrium (small letters)
References
All cited and listed and vice versa
except for Line 347 Altshul et al 1990 is listed but not cited.
Line 412  extra-pair
Line 416  Journal of Primatology


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: INCREASED CHRONIC KIDNEY DISEASE DEVELOPMENT AND PROGRESSION IN DIABETIC PATIENTS AFTER APPENDECTOMY: A POPULATION-BASED COHORT STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is written in clear English and is very understandable for readers. Introduction and background is sufficient, structure is appropriate. Raw data are available.
Experimental design: The research question has been answered. IRB reviewed. Impressive and stuffiest number of participants was included: 10383 patients and 41532 healthy people. Statistical analysis is appropriate.
Validity of the findings: This is an intrigue paper with new finding of link between appendectomy and development of CKD and ESRD, and particularly in patients with DM. DM is one of the leading causes of ESRD, and idea of close follow up after appendectomy for CKD development is clinically relevant.
Additional comments: The significant funding of interaction between appendectomy and DM for the risk of CKD/ESRD development are interesting and clinically relevant.

I was not sure about the duration of the study. In the abstract authors mentioned 2000-2012, but in the text: 2000-2013.
There are repetitions in Discussion section, which were already addressed in Results. There is no need for that. It should be corrected.

Table 1 needs a bigger font and not all abbreviations were spelled out (DM).