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: PLASTID TRANSIT PEPTIDES—WHERE DO THEY COME FROM AND WHERE DO THEY ALL BELONG? MULTI-GENOME AND PAN-GENOMIC ASSESSMENT OF CHLOROPLAST TRANSIT PEPTIDE EVOLUTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: no comment
Validity of the findings: no comments
Additional comments: In the manuscript by Christian et al. entitled, “Plastid transit peptides - Where do they come from and where do they all belong? Multi-genome and pan-genomic assessment of chloroplast transit peptide evolution”, the authors provide new insights into acquisition or loss of plastid targeting peptides during evolution using comparative genomics approach across 15 phylogenetically diverse genera and pangenomes from Arabidopsis and Brachypodium. It is a good piece of work however, it requires minor clarifications/changes. These are as follows:
1. All the legends need a relook. They need to be crisp, presently it appears as if the legends state results only.
2. Lines 132 to 150. Authors need to define the criteria chosen for significant difference.  While the authors place threonine (+11.1%) in a highly enriched category, but categorise asparagine (-28.6%) in a moderate depletion category. It would be better to adopt similar standards (percent) as ‘low, moderate and high’ for both enriched and depleted categories and subsequently reassign the amino acid residues.
3. Line 139. Authors note that phenylalanine was not highly different (-7.9%). This is surprising. Can authors comment on the significance of this in evolution?

4. In Table 1, please make ‘cTP’ uniform as ‘CTP’. Also expand the same in legend.

5. For prediction of protein localization authors have used Target P-1, but didn’t mention the specificity cut-off values or parameters utilized for candidate selection. Authors should repeat this prediction by using an updated version of TARGET P (TARGET P-2, http://www.cbs.dtu.dk/services/TargetP/) to ensure robustness of the previous analysis.

6. line 196, “only on only” does not make any sense. Please correct.

7. lines 316-319. Reference to Figure 6 comes before Figure 5 in text. Renumber and rearrange.

8. Figure 7B is not cited anywhere in the main text. Kindly include it at appropriate place.

9. Figure 8 is cited as one figure while it’s actually have three parts, cite all parts A, B and C.

10. Figure 10A and 10B are not cited in main text include these in appropriate section.


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: PLASTID TRANSIT PEPTIDES—WHERE DO THEY COME FROM AND WHERE DO THEY ALL BELONG? MULTI-GENOME AND PAN-GENOMIC ASSESSMENT OF CHLOROPLAST TRANSIT PEPTIDE EVOLUTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Literature references and sufficient field background/context is provided, with the following exceptions:
1. Origin of targeting systems and targeting signals: I think that the origin and evolution of the targeting itself should be covered in the introduction as well, with respect to the origin of the organelles, of the targeted genes, and of the targeting systems, and mitochondria should be mentioned (see https://doi.org/10.1016/j.tcb.2016.07.001 for an overview, and https://doi.org/10.1111/tra.12446 for an interesting hypothesis)
2. Role of miss targeting in evolution of metabolic pathway distribution: One interesting hypothesis to explain the maintenance of the cellular metabolism after gene transfer or targeting signal gain or loss is, that a low amount of “constitutional miss-targeting” enables survival of the cell, and further evolution of the targeting signals (https://doi.org/10.1098/rstb.2009.0252) I think that this is also relevant to the genes with recently acquired targeting signals analysed in this study.
(I am not an author of any of the cited papers, and I don't mind if these or other papers on the topics would be cited.)

The manuscript has a professional structure, figures, and tables, the raw data is shared. The text is on the long side, and the numbers of figures is high. As outlined below, I think that much of the data needs to be re-analysed. I think that some of the analyses might be difficult to maintain, because a part of the data might not easily be analysed on the within-genome level. However, I think that a revised manuscript with possibly shorter lenght/lower amount of data analysed would be interesting nevertheless.

Smaller things:

Line 48, sentence complicated, also sense unclear, maybe delete "in"?

Lines 63-70, the Apple part in the introduction seems unnecessary to me, I understand that Apple data was examined, but I do not understand the connection between the plastid types and the evolution of targeting peptides.

Line 101: I think that the term "nascent plastid-targeted proteins" has unfortunate potential for being confused with plastid targeted proteins that are in the process of translation at the ribosome (the "ribosome-nascent chain complex"). Furthermore, it's the genes that mutate, not the proteins.

Table 1 caption: I think that "subject and "query" labels make sense for the "Taxa Bias" columns, but they are irritating for the "Transit Peptide Bias" and "Prediction Bias" columns, in which different sets of sequences are compared (in my understanding).

Line 377, "all of cases", missing article?

Line 482, "radiated" is not a good choice of a word here; I think it might be confused with what happens in an "adaptive radiation" in evolutionary biology.

Line 807, "second", missing article?

The conclusion could be shortened to the key findings; here, especially I did not understand the sentence lines 707-709. What are "random substitution events"? Do the authors suggest that "simple substitutions, insertions and deletions, or alternative start sites" (earlier in the sentence) are not random?
Experimental design: The authors present a variety of multi- or pan- genome analyses, based on which hypotheses on the variability in plastid proteomes and the mutational processes that lead to their evolution are proposed. This concept is promising, however, the authors work with predicted protein sets from a variety of species, without accounting for the fact that mutations occur in the genes, not in the proteins, and without accounting for differences in the methods used to collect the protein sets. I think that such within-genome analyses are a necessary first step of the project in order to conduct further comparative analyses. A couple of mechanisms that have no biological relevance, but that do have an influence on the results of this study are:
1. The investigated genomes have been sequenced at different time points, the sequencing, assembly and annotation methods during this time improved, so in the "oldest" genomes, plastid targeted genes might not have been recognized simply because the projects at that time were more pioneering.
2. Pre-sequences are less conserved than mature protein sequences. So they are hard to detect via homology based gene modelling. And, depending on lengths and settings, a gene model that excludes an existing targeting signal might attain a higher homology score than one that includes a targeting signal. So the gene modelling itself has an influence, but also the choice of competing models for a non-redundant gene catalog.
3. Transcriptomics methods also changed with respect to sequence quality and coverage. Furthermore, due to cDNA synthesis with the often used oligo-T primers, there is often (but not always) a bias against the 5' end of transcripts.
I think that such possible biases in pre-sequence detection need to be standardized between the different investigated organisms/genomes.

Dual targeting (or multiple targeting) should be discussed more carefully, including estimations of numbers of dually targeted proteins.

Accounting for the gene structures and sequences would also allow to assess how existing 5' UTRs could be transformed into targeting signals, in all three reading frames.

Figure 2 might be condensed to sequence logos (http://dx.doi.org/10.1093/nar/18.20.6097) and/or frequency plots of amino acids. There is a fair amount of circular reasoning in the comparison of predicted transit peptides with the other sets, because the predicted transit peptides are already predicted to be transit peptides due to their sequence. I think this should be discussed more carefully/explicitly.
Sequence logos might be a good way to present the data shown in Figure 1 and 3, as well (although this would slightly change their meaning).
Validity of the findings: As mentioned above, I don't think that the results in the current form can be taken at face value, due to differences in gene modelling and model selection that led to the various investigated protein sets.
Additional comments: In their manuscript with the title "Plastid transit peptides - Where do they come from and where do they all belong? Multi-genome and pan-genomic assessment of chloroplast transit peptide evolution", Christian et al. investigate the evolution of plastid targeting signals in plants, especially with respect to genes of which the product has recently acquired a new targeting signal. I think that the topic is highly interesting and I would be happy to know more about the mutational processes that lead to modifications of intracellular targeting. In the present form, the analyses solely deal with protein sequences, without regard of the DNA sequence and gene structures of the investigated genes. However, I think that before multi- or pan- genome analyses can be done, within-genome analyses need to be completed. Please see the other sections for details.


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: PLASTID TRANSIT PEPTIDES—WHERE DO THEY COME FROM AND WHERE DO THEY ALL BELONG? MULTI-GENOME AND PAN-GENOMIC ASSESSMENT OF CHLOROPLAST TRANSIT PEPTIDE EVOLUTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: See comments to author.
Experimental design: See comments to author.
Validity of the findings: See comments to author.
Additional comments: The current manuscript describes a very comprehensive analysis of the evolution of plastid transit peptides in plant systems. The evolution has been studied at both intra- and inter-genera level. Residue level variations have been analyzed. In general the methodology is appropriate and results draw some interesting conclusions on the possible mechanism of the divergence of the plastid transit peptides. Thus in my opinion the manuscript is suitable for publication in the journal in its current form. There are only few points that the authors may address or clarify (as below).

Minor points/issues:

1. In conclusions, authors write “….peptides evolve via simple substitutions, insertions and deletions, or alternative start sites rather than from random substitutions events…” I am not able to understand the difference implied in the usage of the terms ‘simple substitution’ and ‘random substitution’. Authors may kindly elaborate.

2. Colored boxes (guides) in pie charts of figures 6a, 8a and 9a are not clear at all. Especially the shades of blue are not distinguishable. Maybe the size of the boxes can be increased and color more contrast.

3. Legends of figure 10c are not clear at least at the resolution available to me.


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: PLASTID TRANSIT PEPTIDES—WHERE DO THEY COME FROM AND WHERE DO THEY ALL BELONG? MULTI-GENOME AND PAN-GENOMIC ASSESSMENT OF CHLOROPLAST TRANSIT PEPTIDE EVOLUTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript by Christian et al. entitled, “Plastid transit peptides - Where do they come from and where do they all belong? Multi-genome and pan-genomic assessment of chloroplast transit peptide evolution”, has been suitably revised. The authors have addressed all queries in the present form.


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

Input:
Title: PLASTID TRANSIT PEPTIDES—WHERE DO THEY COME FROM AND WHERE DO THEY ALL BELONG? MULTI-GENOME AND PAN-GENOMIC ASSESSMENT OF CHLOROPLAST TRANSIT PEPTIDE EVOLUTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have satisfactorily modified 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: GROWTH PARAMETER K AND LOCATION AFFECT BODY SIZE RESPONSES TO SPATIAL PROTECTION BY EXPLOITED ROCKFISHES
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this study, the authors examine the effects of marine protection on body size of 12 species of rockfish, inside and outside rockfish conservation areas (RCAs) closed for up to 15 years, and the effects of life history and behavioral differences on the strength of that response. The authors find that fish are larger in body size in 2 of the 6 RCAs examined and that growth parameters may explain some of the differences in the strength of the response. They also report effects of topographic complexity and depth on body size, which varies in different ways based on mobility.

The paper was short and concise and generally well written. Raw data were shared.
Experimental design: I’m not particularly in love with the length anomaly metric as the sole means of providing information on the response of fish body size to protection. I think I would like it better if this approach complimented more traditional analyses using mean lengths, comparisons of size distributions, calculations of differences in lengths in vs. out, or response ratios. I understand why the authors used this standardization approach, because it allows them to compare across species that differ greatly in body size in the same analysis, but this was not articulated clearly in the manuscript.

The methods section needs to better describe how the length anomalies were calculated and then averaged and used in subsequent analyses. Did you calculate the mean anomaly for each species in each location pooled over all years and use that in the models?

If you have sizes of each RCA, how about conducting an analysis to test explicitly whether reserve size is associated with the strength of the length response measured? The same can be done with estimates of ocean productivity or fishing pressure. With 6 RCAs, the statistical power may be low, unless the effects are strong, but it would be interesting to see if the patterns match the qualitative descriptions of the likely drivers that the authors describe in the discussion. Are there differences in habitat complexity between any of the RCAs and their control sites that may help to explain why reserve effects were detected in some locations but not in others?
Validity of the findings: The growth parameter k is not necessarily a good measure of somatic growth. Instead, it reflects how quickly the asymptotic size Lmax is attained by a species, which may or may not correspond well with somatic growth. In the VBGF model, Lmax and k are often inversely related (higher Lmax results in a smaller k). However, species with a larger Lmax often attain larger sizes and can be bigger in size at the same age compared to species with a lower Lmax and higher k. Since they are larger at the same age, this means that actual somatic growth can be higher in cases of a lower k.

Goose Island and McMullin appear to share an RCA boundary (or they are very close), yet the responses to protection are very different. Is this just an artifact of habitat and which sites are the paired control sites? Some discussion is warranted.

Table 2 includes the parameter values for the GLSM, however nowhere in the manuscript were there reports of the test statistics, significance levels and p-values for the different factors included in the model. This is much more important than the parameter values and should be what is reported, especially because there were lots of factors and interactions included in the models. Most readers care much more about the significance of the model factors than the parameters that can used to create a predictive model of the expected length anomaly at any given location (which is what Table 2 is good for).

The body size anomaly effects of mobility class and topographic complexity need to be described in more detail and interpreted more in the discussion. The results are interesting that high mobility species are large in low complexity habitats while demersal species are larger in high complexity habitats. It is also interesting how the shifts in body size with depth are different between the two mobility classes.

Show size frequency distributions of each species, but plotted by samples inside RCAs vs outside RCAs. These can be overlapping and shaded with transparency to allow the reader to see whether fish are bigger inside MPAs in general for each species. Appendix S10 shows this for each species overall, but not broken down into inside vs. outside. It would also be good to test whether the distributions differ significantly for any species or locations.

The actual data points should be shown on the plots in Fig. 3 and Fig. 4, not just the model fit and 95% CI, so the reader can see the spread of the length anomaly data as a function of growth parameter k or topographic complexity or depth in each panel. It would also be nice to see the mean anomaly +/- SE for each point (which gives different information than the overall model fit and 95% CI). Would it be possible use a different symbol for each species, so readers can see which points correspond to the different species?

Why not also include the fish density data from the surveys to test whether abundances differ inside and outside the RCAs? Or the density and size data can be combined to calculate biomass and that could be used to test for MPA effects.

Are there any effects of time on the size differences detected inside and outside MPAs? The data from the first year could be compared to data collected in the last year or two of the surveys to calculate a change in size distributions and to test whether there is evidence that the size responses have increased over time inside the RCAs relative to the fished areas.
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: GROWTH PARAMETER K AND LOCATION AFFECT BODY SIZE RESPONSES TO SPATIAL PROTECTION BY EXPLOITED ROCKFISHES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the content of this manuscript is valuable to both the scientific community and ecosystem managers. The paper addresses the differences in how commercially and recreationally important fish species may differ in their response to spatial protection because of different life history strategies.

However, there are some issues with the writing, analysis, and reported information that should be addressed prior to acceptance.  The introduction seemed very choppy and is not constructed in logical progression. One simple change I would recommend is to combine the first and third paragraphs. The first paragraph discusses protected areas and how they may be beneficial, the third paragraph discusses how protection benefits are measured.  The fourth paragraph (line 62) is situated in the Introduction as if it was a summary of the literature, but in reality this paragraph contains a hypothesis that the authors are trying to test.  The Introduction should be re-written to identify the hypothesis that “interspecific variation in somatic growth rates and the age of spatial fishery closures should influence body size responses by exploited species to spatial protection (Fig. 1).” This is an interesting hypothesis, but is not sufficiently accepted or proven to be discussed as if it was fact.  With respect to Fig. 1, I would like to see the data generated by this project placed into this construct, it might lead credence to, or allow the rejection of, this unstated hypothesis.  Similarly, the authors correctly identify in the Introduction that habitat type affects life history characteristics of rockfishes, but don’t clearly describe how habitat fits into their conceptual model.
Experimental design: The work presented in this manuscript is appropriate for PeerJ. The research question is relevant and meaningful and the authors show how the research addresses an identified knowledge gap. The problem I saw, however, is that the authors mixed variables together in a way that makes it difficult to address their primary hypothesis.  They said they wanted to know if species life history parameters, habitats, and age of protected areas influenced body size responses (growth rates) of different species.  It appears to me, however, that they lumped all fished species together, didn’t include habitats specifically, and lumped different ages of protected areas. Also, I am concerned about the use of S. emphaeus as a control fish between the protected and fished sites. That species is small and probably shows less variation in body size compared to other rockfishes.  Also, I would like to see a more thorough explanation of the use of length anomalies. It is not clear why the authors chose to use length anomalies when they are trying to show that there is a difference in response of fast growing (larger) fishes vs slow growing (smaller) fishes.

One major concern I have is the statement that, “Total lengths were estimated visually with the aid of a 1-metre-long ruler attached to a pole; although these estimates have limited precision, their measurement error likely is consistent across treatments and species, not biasing results.” I would argue that this type of visual length estimate is very likely to be problematic unless the various scuba divers have trained together and evaluated their individual accuracy and precision biases.  In many places around the world, researchers using underwater visual census techniques have tested their accuracy and precision by estimating the lengths of targets of known size (e.g., plastic fish).  In one study I know of, observers systematically underestimated the lengths of large fishes and overestimated the lengths of small fishes, but not every observer was biased in the same direction.  I would like to see the authors address this issue more carefully.
Validity of the findings: I liked the creativity and analytical approach taken by the authors. I would like to see more of the author’s work in the results section rather than in the supplementary material. A major portion of the results discuss the percentages of fish captured at an RCA since implementation. However, time since RCA implementation was not a factor in any of the analyses presented and is confounded by the factor of location.

I think conclusions are well stated along with possible factors that could be influencing the results. The authors provided good recommendations about modifying RCA’s . One question I have is that while the authors go into detail about illegal fishing activity within the RCA and how that could be affecting their results, I am curious about the amount of fishing outside of the RCA’s in your reference sites. Could some reference sites be experiencing low to moderate fishing while others are experiencing high fishing? How could that affect the differences that you are seeing, or not seeing, between the protected sites and the adjacent fishes sites.
Additional comments: I like the questions you are asking about the differences in response of fishes with different life history characteristics to protections offered by MPAs.


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: GROWTH PARAMETER K AND LOCATION AFFECT BODY SIZE RESPONSES TO SPATIAL PROTECTION BY EXPLOITED ROCKFISHES
Review round: 1
Reviewer: 3

Output:
Basic reporting: This article is well written and generally well-presented. My main suggestions for improvements are:
(1) I think it is important to visualise the data (not just statistical fits) and some information presented in tables could be more usefully presented in figure form;
(2) Consider adding some nuance to the expectations for responses to exploitation/protection
(3) There are a few places where additional references could be added to better situate this work with relation to previously published work;
I expand on these 3 points and provide additional specific suggestions below. Please also refer to my comments in the "comments for the author" section for context.

1) visualising the data
- As a general principle, in studies based on primary data, in my view the data should always be visualised (ideally in a way that directly relates to the statistical models being fit) so that the reader can visually assess the patterns. Just showing the statistical fits can be a bit misleading. I acknowledge that you do present the diagnostic plots in the supplementary materials, and that interested readers can grab the data and make plots themselves, but I still think it is preferable to show the data in the main manuscript. In this case, ideally points would be added to figs 3 and 4 showing the raw data that the models are fit to.
- The information presented in table 2 could be more usefully presented in the form of a "parameter plot". Most importantly, parameter plots allow for easy comparison of the magnitude of parameter estimates and whether CIs around estimates include 0.

2) expectations for responses to exploitation/protection
- it struck me that the expectations explained from lines 62-72 and in figure 1 might be a bit of a simplification, as the expectations for responses of fast growing species in particular will be dependent upon the intensity of exploitation (along with how fast growing the species is). For the fastest-growing species, it might be expected that there would not be any appreciable impact on body size distribution until exploitation reaches a very high intensity. It would be good to add this nuance to the explanation. It would also be helpful to make it clearer in lines 62-71 that the explanation is in terms of relative (not absolute) body size. This is apparent in figure 1 and later in the main text, but not clear at lines 62-71.

3) framing with respect to the literature:
- line 59: consider adding papers that directly coniser impacts of exploitation on size structure of reef fish e.g.
Zgliczynski, B.J., Sandin, S.A., 2017. Size-structural shifts reveal intensity of exploitation in coral reef fisheries. Ecological Indicators 73, 411–421. https://doi.org/10.1016/j.ecolind.2016.09.045

Dulvy, N.K., Polunin, N.V., Mill, A.C., Graham, N.A., 2004. Size structural change in lightly exploited coral reef fish communities: evidence for weak indirect effects. Can. J. Fish. Aquat. Sci. 61, 466–475. https://doi.org/10.1139/f03-169

- line 88: consider adding papers led by indigenous authors at line 88. e.g.
Jones, R., Rigg, C., Lee, L., 2010. Haida Marine Planning: First Nations as a Partner in Marine Conservation. E&S 15, art12. https://doi.org/10.5751/ES-03225-150112

Jones, R., Rigg, C., Pinkerton, E., 2017. Strategies for assertion of conservation and local management rights: A Haida Gwaii herring story. Marine Policy 80, 154–167. https://doi.org/10.1016/j.marpol.2016.09.031

- lines 100 -- 111: It would be good to provide some context for expectations around the effects of habitat on size structure given that this features prominently in analyses. e.g.
Trebilco, R., Dulvy, N., Stewart, H., Salomon, A., 2015. The role of habitat complexity in shaping the size structure of a temperate reef fish community. Mar. Ecol. Prog. Ser. 532, 197–211. https://doi.org/10.3354/meps11330

- line 301: Trebilco et al 2016 demonstrated increasing trophic position with body size among rockfish prior to the article by Olson et al. cited here.
Trebilco, R., Dulvy, N.K., Anderson, S.C., Salomon, A.K., 2016. The paradox of inverted biomass pyramids in kelp forest fish communities. Proc. R. Soc. B 283, 20160816. https://doi.org/10.1098/rspb.2016.0816

# additional specific suggestions and corrections
Abstract
- line 26: consider rewording the sentence beginning "For 12 species" to "Here, using a visual survey data collected from inside and outside Rockfish Conservation Areas on the Central Coast of British Columbia". This could also note that these data were collected by the Central Coast Indigenous Research Alliance, in collaboration with First Nations. This is a really outstanding aspect of this work.
- line 40: consider rewording "frameworks can predict and help interpret..." to "theory provide a useful lens for framing and interpreting..."

Discussion:
- line 222: consider adding "better" with "more fully"
- line 228: add "mature" after "most"
- line 242: "exclusive" missing after "mutually"
- line 248: consider replacing "controlling for key habitat variables" with "accounting for habitat structural complexity"
- line 252--257: consider adding some mention of potential importance of algal cover and kelp canopy structure.

Tables:
- consider adding RVI (relative variable importance) values to table 2 to give some indication of how consistently variables were included in the best-supported models, without having to refer to the supplementary materials. Also see my comment above --- this would be better as a figure.

Figure 1
- consider adding "with respect to growth rate" to main title.
- add definition of k parameter to the caption
- consider making labelling on the plot axes more readily interporable (e.g. change "length anomaly" to "length anomaly (observed size relative to species average) and "lower (smaller)" and "higher (larger)" instead of just "lower" and "higher")

Figure 2
- are points not shown for site locations for a particular reason? If not, I'd suggest showing them. Open circles could still work if points overlap.

Figures 3 & 4
- Invoke Ram Myers' 3 golden rules of data analysis: Plot the data, Plot the data, Plot the data.

Throughout
- be consistent with a convention for presenting common names followed by latin names in parentheses or vice versa. At present it's a bit haphazard.
Experimental design: The experimental design is appropriate and sample sizes are impressive given the logistical challenges of data collection.
Validity of the findings: Findings are valid and conclusions are well stated, with clear links to supporting results. The statistical analysis is rigorous and intelligent.  Interpretations and speculation are articulated appropriately.
Additional comments: I enjoyed reading this manuscript and think that it will be a valuable addition to the literature. It is well-conceived and well-written, the statistical analyses are rigorous and appropriate, and the authors do an exemplary job of making the analyses transparent and repeatable.

I think the manuscript is at a publishable standard in its current form (with a few minor typographical corrections), but I also offer some suggestions for improvement that I would encourage the authors to consider under 'basic reporting'.


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: GROWTH PARAMETER K AND LOCATION AFFECT BODY SIZE RESPONSES TO SPATIAL PROTECTION BY EXPLOITED ROCKFISHES
Review round: 2
Reviewer: 1

Output:
Basic reporting: See comments on previous review. General comments are below.
Experimental design: See comments on previous review. General comments are below.
Validity of the findings: See comments on previous review. General comments are below.
Additional comments: The authors have generally done a good job revising the manuscript to address my comments and those of the other two reviewers. The changes to the introduction and discussion in particular have helped to streamline and improve the clarity and focus of the study. In addition, the parameter plot (new Fig. 2) makes it much clearer which factors are important in explaining size differences of fish assemblages inside and outside of MPAs. I am a bit confused though because on line 238 of the results and later in the discussion they state that “The effects of spatial protection on the body sizes of exploited species depended on growth parameter k and RCA location, but not on behavioral class (Fig. 2; Appendices S13a, S14a).” Yet, if you look at Figure 2, behavioral class appears to be a highly significant model parameter, with a positive coefficient score and 95% CI that do not cross zero. Doesn’t this mean that there are different body size responses for the high mobility-benthopelagic group and the low mobility-demersal group? If so, which group showed a stronger body size response and why?

I was also unable to view any of the supplementary information and figures that were submitted. The file appeared to be corrupted. I tried to download it multiple times and it would not open. Thus, I was unable to evaluate the relevance of the supplementary material.

I also recommend that the authors closely read through the manuscript to make sure they are not overstating their conclusions about the effects of the British Columbia MPAs on body size responses of fish. Only 2 of the 6 MPAs showed differences in body size, meaning the majority of their sites did not show a difference. I’m not sure if this is included in the supplementary, but it would be good to investigate whether the body size differences at the 2 sites showing change were present at the time the MPAs were implemented (or as near that as possible) or whether these differences appeared more recently after the MPAs had been in place for up to 15 years. Are those body size differences pre-existing or not?


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: GROWTH PARAMETER K AND LOCATION AFFECT BODY SIZE RESPONSES TO SPATIAL PROTECTION BY EXPLOITED ROCKFISHES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised manuscript is clear and easy to read.
Experimental design: The research question is clearly articulated, the experimental design is clearly stated along with potential limitations, and methods are describe with sufficient detail.
Validity of the findings: The authors have done a good job of describing how their data fit the predictions and discuss reasons why results may have differed from expected results.
Additional comments: This manuscript is greatly improved from the version I reviewed. The authors addressed all the concerns I had.


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: GROWTH PARAMETER K AND LOCATION AFFECT BODY SIZE RESPONSES TO SPATIAL PROTECTION BY EXPLOITED ROCKFISHES
Review round: 2
Reviewer: 3

Output:
Basic reporting: Please refer to general comments to authors
Experimental design: Please refer to general comments to authors
Validity of the findings: Please refer to general comments to authors
Additional comments: As I noted in my previous review, my assessment was that the original submission (prior to revisions) was of a publishable standard, but I offered several suggestions for improvement. The assessment of the other reviewers was that more substantial revisions were necessary.

I commend the authors for undertaking a comprehensive and considered revision of this article, which has been substantially strengthened. I thought their responses -- both to my comments and those of the other reviewers -- were thorough and well reasoned.

I support the publication of this manuscript in its current form.


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

Input:
Title: WHY PEOPLE SELECT THE OUTPATIENT CLINIC OF MEDICAL CENTERS: A NATIONWIDE ANALYSIS IN TAIWAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: The reference style needs to be checked and matched with the Journal's style.
Experimental design: Methods is not described with sufficient detail & information to replicate.
Validity of the findings: The low response rate inherent in Internet-based survey could affect the validity of the findings.
Additional comments: The manuscript reported the results of a nationwide Internet-based survey on the factors associated with patients’ visiting the outpatient clinic of the medical center for an illness without a referral. Multiple logistic regression analysis showed that age, past experience in primary clinics, consider copayment is important, have regular family physician, living area, and two factors (image and reputation & facility and medication) obtained from an exploratory factor analysis were significantly associated with the outcome variable. The conclusion appeared to be supported by the results, but a number of clarifications need to be made, particularly regarding the study methodology.

1. Line 103: Please provide more details on how the survey was advertised on the three social media platforms. Is there a link to a study survey website, such as Google Form?

2. Line 109: Was the deduplication protocol based on only identical age, occupation, and answer options? Were IPs checked for duplication? Is there a survey report generated by the survey website that allows one to roughly identify the geographical distribution of the survey respondents? If so, this will add support to the “nationwide” coverage of the survey.

3. Line 119: Please provide more information on the focus group, including the demographic characteristics of its member and the main questions used.

4. Line 120: “expert validity” should be referred to as “content validity”. Was Content Validity Index (CVI) measured? If so, this should be used to replace the sentence (line 129) “it exhibited a satisfactory level of content validity”.

5. Line 142 and 187: A p-value of less than .001 should be presented as < .001 rather than .000.

6. Line 143: While the results may not be different, principal axis factoring rather than principal components method should be used for the factor analysis because it appears that the aim of the authors were trying to assess a set of latent variables that cannot be directly measured with a single variable.

7. Line 145 and 190: “Multiple logistic regression” rather than “multivariate logistic regression” is the correct term to use here.

8. Line 156: The age distribution should be described. In addition to the mean and SD, please provide the minimum and maximum age of the participants.

9. Line 191 and Table 4: The authors used hierarchical (nested) regression models, which should be first described in the Methods section. Please explain why this approach rather than stepwise regression approach was used.

10. Line 241: The low response rate should also be mentioned in the limitations.

11. References: They are a number of formatting errors, such as missing page number (Line 287), and the use abbreviated journal title (PeerJ style is journal title in full form) (Line 273, 275, 277, etc.).

12. Table 4: The columns for beta and standard error of beta should be deleted and replaced by columns showing p values and 95% confidence intervals for the odds ratios.

13. Table 4: Income was represented by a categorical variable with five levels in Table 1. However, it was represented by a single variable “income degree”. Are the five levels treated as a continuous variable? Unless there are special reasons for this, it should be analyzed as a categorical variable.

14. Table 4: The models should be compared formally with likelihood-ratio Chi-square test. It is not clear whether Model 3 is significantly “better” than Model 2.


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: WHY PEOPLE SELECT THE OUTPATIENT CLINIC OF MEDICAL CENTERS: A NATIONWIDE ANALYSIS IN TAIWAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: - The manuscript is presented clearly and unambiguously, using professional English. The text is easy to understand.

- Bibliographic references follow the APA style, according to the instructions for the journal's authors. However, errors have been detected in the citation in the text. For example, "Chen et al. 2006” on line 65. According to the regulations, up to 3 authors must be determined, while if you have 4 or more authors, you must specify the first followed by et al. Authors are recommended to review APA citation style and make relevant changes to the text.
Despite the fact that the references are current and fit the theme of the study, the introduction needs more bibliographic support. Suggest that you quote other authors on lines 61-62, 72-74, and 86-92 to provide more justification for your study:
"In most countries, primary care physicians act as 'gatekeepers' to health care by providing initial medical interventions and referring patients to additional specialists." You must comply with this statement with bibliographic references.
"Studies have reported that people in developed countries visit a doctor 5 to 6 times a year, while in Taiwan, the average frequency of visits is 13." You must comply with this statement with bibliographic references.
"Although changes in NHI co-pay policies have slightly reduced the use of high-level healthcare facilities, studies have indicated that the effect of medical prices on people's medical behavior is very limited (Lee et al . 2018).
Factors affecting patient selection in high-level healthcare settings have not been fully identified. Cheng et al. reported that patients tend to base their judgment on the quality of the hospital on the medical team (Cheng, 2015). "Please note that this explanation seems somewhat contradictory when mentioning the influence of medical prices and the quality of hospitals in the choice by the patients, once they mention that the factors that completely affect this question have not been completely identified This precise fragment is scientifically clarified and supported.

- The structure of the manuscript and tables is understandable and adjusted to the specific ones. We suggest that in the materials and methods part the authors elaborate a sub-section on ethical and legal aspects for lines 112-113 “No rewards were given to the participants. This study was approved by the Institutional Review Board of the Taipei General Veterans Hospital (2017-07-009AC). "In addition, we recommend that you expand the information on the legal guidelines that the authors followed to protect the confidentiality of the data.

- The data presented is consistent and fully presented to interpret the results. The tables are clear and expand the information without repeating the data.
Experimental design: - The research is within the objectives and scope of the journal.

- We suggest that the authors restate the objective of the abstract study as "The main purpose of this study was to explore the factors contributing to patients' selection of the outpatient clinic of medical centers without a referral." We recommend that the authors change the verb “explore” (mostly used in qualitative studies) to “identify or evaluate”, since they use a Likert scale questionnaire and not interviews or open questions.

- The authors must write a short justification of the study together with the objective at the end of the introduction.

- They should briefly identify the knowledge gap being investigated and statements should be made about how the study contributes to filling that gap.

- Also, keep in mind that the hypothesis proposed must be clearly defined, relevant and significant.

- We suggest that the authors expand the information in relation to the elaboration of the questionnaire that allows its correct interpretation, as well as its reproducibility.

- Also, it is recommended that the authors express more clearly based on which criteria the group of experts were selected and how they were recruited for the identification and review of the factors expressed in the questionnaire.

- In general, keep in mind that the methods must be described with enough information so that another researcher can reproduce them.
Validity of the findings: - It is important to note that the questionnaire was previously tested on 20 patients and was valid and reliable to measure the factors that influence the choice of the medical center.

- Despite the fact that the data seems reliable and statistically sound, the authors are encouraged to provide information on who was responsible for managing and performing the analysis and interpretation of the data, and how this procedure was carried out.

- We recommend the authors to move the first paragraph of the conclusions to the discussion section, since due to its content it is more appropriate.

- The conclusions must respond to the stated objective and should be limited only to those supported by the results expressed in the manuscript.
Additional comments: We want to thank the authors for considering the journal PeerJ - The Journal of Life and Environmental Sciences for the publication of their manuscript entitled “Why do people in Taiwan select the outpatient clinic of the medical center? A nationwide analysis ”.
The topic addressed is important for the development of graduated medical care, and even more so if it is a first foray into it to give priority to the primary medical system.
Despite the importance of the topic covered and the aspects previously discussed, some aspects of the manuscript require review by the authors for clarity and reproducibility.

Other aspects that we suggest to the authors are the following:

- We recommend that the authors add the study design in the abstract.

- Regarding the keywords, we suggest that they change "national health insurance" to "national health programs" and "survey" to "health care surveys" to improve the presence of the manuscript in the databases.

- In addition, we recommend reviewing the rest of the keywords and, if possible, replacing them with appropriate MeSH terms according to the topic of the study.

- Finally, it is important that the authors mention in the manuscript the reporting guidelines they have followed according to their type of study, with the aim of improving the quality and transparency of their research. If you have questions, you can consult the instructions for the authors of the magazine or https://www.equator-network.org/


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: WHY PEOPLE SELECT THE OUTPATIENT CLINIC OF MEDICAL CENTERS: A NATIONWIDE ANALYSIS IN TAIWAN
Review round: 2
Reviewer: 1

Output:
Basic reporting: No further comments.
Experimental design: No further comments.
Validity of the findings: No further comments.
Additional comments: The authors have satisfactorily responded to all my comments and made the necessary changes 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: WHY PEOPLE SELECT THE OUTPATIENT CLINIC OF MEDICAL CENTERS: A NATIONWIDE ANALYSIS IN TAIWAN
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Dear authors,

We thank you for the new submission of your manuscript 47877v2 entitled "Reasons Why People Select the Outpatient Clinic of the Medical Center: A National Analysis in Taiwan."

Changes made after reviewers' comments have improved the clarity and reproducibility of their manuscript, as well as their understanding by readers.


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: IN SITU SEM/EDS COMPOSITIONAL CHARACTERIZATION OF OSTEOCYTES AND BLOOD VESSELS IN FOSSIL AND EXTANT TURTLES ON UNTREATED BONE SURFACES; DIFFERENT PRESERVATIONAL PATHWAYS MICRONS AWAY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some sentences could use rephrasing to improve their ease of readability and to provide better clarity on some of the methods, results, or intended meaning. I’ve noted where changes would be most helpful with comments, and have provided small in-text suggestions that could augment the text’s readability.

‘Groups’ should instead be referred to as ‘clades’.

Do the grouped citations each need to be rearranged into chronological order?

The author might consider adding a couple citations to a published doctoral thesis (Boles 2016) that also explores preservation and composition of cells and tissues within fossil turtle bones via demineralization and EDX.

Many of the figure captions could use clarification/further details concerning labeling, explanation of color uses and symbols in the figures, and further explanation of how to interpret them. I may know how to read/interpret most of them visually, but not every reader will, so adding a few details would be beneficial. I’ve attempted to identify questions that can be answered through such (short) additions, and to provide suggested edits to improve reader comprehension of them.
Experimental design: A bit more detail on the treatment, transfer, and imaging protocols used with demineralization-isolated cells would be helpful to less-informed readers looking to try this type of study themselves. See my Comment #27 in the attached PDF for details.
Validity of the findings: Since no biochemical methods have been used to rigorously test the endogeneity of the recovered microstructures and identification was guided instead by morphology and gross elemental composition, osteocytes, osteoblasts, and blood vessels should all technically be referred to with either single or double quotation marks around them, or with the addition of a ‘-like’ term after the word (e.g., ‘osteocytes’, or osteocyte-like microstructures). This is standard practice for this topic, and is a requirement until they are shown via more advanced biochemical methods to truly be endogenous in molecular structure/composition.

In a couple spots in the Discussion the statements seem to be written in a bit too strong of language. It’s not over-interpretation of results, but rather how the verbs are used in a few sentences. It would be appropriate to simply dial back the statements applying toward other microstructures recovered by previous and/or future studies, such as using “may vary” instead of saying it simply “varies” (line 256), or using “may also be involved” rather than “is involved” (line 261). Those statements might be true with the samples studied here, but might not be universally true concerning similar microstructures identified in other previous and future reports. So, I think it would be better to use more conservative language. My Comments #41, 51, and 56 in the attached review PDF note similar instances.
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: IN SITU SEM/EDS COMPOSITIONAL CHARACTERIZATION OF OSTEOCYTES AND BLOOD VESSELS IN FOSSIL AND EXTANT TURTLES ON UNTREATED BONE SURFACES; DIFFERENT PRESERVATIONAL PATHWAYS MICRONS AWAY
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: For all specimens, especially the Mongolian ones, were all permits obtained?
Validity of the findings: No comment.
Additional comments: In this manuscript, Cadena examines non-demineralized turtle bones from a range of ages and regions around the world to examine elemental composition of soft tissue preservation. This study fills a gap between elemental characterization of soft tissues from demineralized fossil material and clearly shows the restricted nature of the elemental composition leading to preservation.

Please make sure that all figures are colorblind accessible. For example, Figure S2 has multiple areas with both red and green figures. If there are questions of accessibility, https://www.color-blindness.com/coblis-color-blindness-simulator/ works quite well.


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: SPATIAL DISTRIBUTION OF MICROBIAL COMMUNITIES AMONG COLONIES AND GENOTYPES IN NURSERY-REARED ACROPORA CERVICORNIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is well-written.

Some additional background is needed in the introduction:
it is confusing that you start off reference florida when the study takes place in the cayman islands.

I would also mention why cervicornis is especially easy to grow like this.

A discussion of Gignoux-Wolfsohn et al. 2017 should be added as they found a strong effect of presumed genotype (referred to as colony in the paper becuase they did not actually genotype) on A. cervicornis microbiomes. Although, many of those corals were dominated by Endozoicomonas. This difference and implications would be good to discuss as well.
S. Gignoux-Wolfsohn, F. M. Aronson, S. V. Vollmer, Complex interactions between potentially pathogenic, opportunistic, and resident bacteria emerge during infection on a reef-building coral. FEMS microbiology ecology 93,  (2017).
Experimental design: The sampling strategy and methods are well-described.

A few comments on methods:
104- should be and yellow

106- were these wild colonies?

159: Did you do one analysis with all 3 of your factors? or separate?
Validity of the findings: Findings are well-described.

Comments on the results:
Missing figure legends?

Figure 4: Would be nice if this were all rickettsiales asvs. Given that there are only 11 I dont think it would significantly detract from your point and would allow the reader to make more clear comparisons between figure 3 and figure 4
Its also unclear if the y axis on this figure refers to total abundance or abundance within the rickettsiales?

177-184: its confusing that you are discussing most abundant asvs but referencing a figure where everything is grouped by order.

228-230: Would you expect rerunning the pipeline to produce different results? how much variation was there?

The discussion needs a little more work to place your findings in the context of the previous literature.

281: Also, they sampled differently, there could be differences you aren't capturing using syringe sampling. This sampling strategy should be discussed.

303: But Gignoux-Wolfsohn 2020 used V6 and found high abundance of Rickettsiales.

288-304: There is a lot of discussion of A. palmata that seems out of place given that this was not the objective of this study.

It is also interesting that you are sampling them right next to each other removing the environmental variation that confounds natural studies of genotypes. It would be interesting to have more of a discussion of  the effect of site as found in Chu and Vollmer and Gignoux-Wolfsohn and Vollmer 2014

The discussion needs another paragraph focused on your finding of genotype-specificity of the dominant strains of Rickettsia as seen in Figure 4, espeically the finding that all samples across colonies of the green genotype were dominated by the same ASV. This is an interesting finding! To me it suggests a closer relationship between these rickettsia and the coral host. You can delve into more of a discussion of microbial symbionts of corals (both beneficial and pathogenic). What does this tell you about when these corals are aquiring the rickettsia?
see  M. J. Neave et al., Differential specificity between closely related corals and abundant Endozoicomonas endosymbionts across global scales. The ISME journal,  (2016).
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: SPATIAL DISTRIBUTION OF MICROBIAL COMMUNITIES AMONG COLONIES AND GENOTYPES IN NURSERY-REARED ACROPORA CERVICORNIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this study, the Authors analyzed the microbial community composition of a keystone and endangered coral species, Acropora cervicornis, after sampling colonies that were reared in a Caribbean ocean nursery. Based on 16S rRNA gene metabarcoding, the Authors identified that bacterial communities did not display significant spatial heterogeneity across single colonies, but that they were influenced by the host genotype (with one bacterial genus dominated most samples). This work provides interesting information about the microbial communities associated with A. cervicornis and is a useful baseline for future studies.

Overall, the manuscript is clear, well written and structured, and the language is professional. Figures are relevant; raw data as well as scripts used for analysis are available.

The introduction flows well and is appropriately referenced, but could benefit from some additional information and minor revisions:

Lines 71-73: The allusion to coral microbiome manipulation comes a little bit abruptly and is not really the scope of the present study. While still useful to mention it, it would be interesting to expand on the roles of the coral microbiome and justify why it is important to characterize it (which would emphasize the value of this study).

Line 79: The Authors refer to coral “microbial communities”, which would actually encompass not only bacteria and archaea (assessed here), but also other microbes. As for example Symbiodiniaceae communities were not investigated, it would be more accurate to refer to “prokaryotic communities” or “bacterial communities” (when relevant) throughout the manuscript.

Lines 79-86: Either here or in the discussion, the Authors could emphasize why it is important to investigate spatial heterogeneity of bacterial communities in corals. If the communities are homogeneous, it would enable to collect fewer samples across the colony and hence reduce the stress of sampling. If the community composition varies across the colony, then it would be important to collect a sufficient number of samples from different locations on the same host, otherwise results wouldn’t be representative and within-colony differences could bias interpretations.

Line 86: It could be relevant to mention that some studies have reported a strong association between the coral host genotype and its bacterial community composition (for example: Glasl et al. 2019 PeerJ; or Hester et al. 2016 ISME - a review in which the authors listed studies having found an effect of coral species or environment on coral-associated bacterial communities).
Experimental design: Research questions are well-defined and most methods clearly explained, however the points outlined below require further explanations. Importantly, the Authors haven’t specified how they dealt with potential bacterial contaminants (as they didn’t seem to have done DNA extraction controls). If that is the case, this shortcoming should be acknowledged. Also, it is not indicated whether read counts were normalized prior to statistical analyses, which could bias the results given that samples had varying sequencing depths.

Line 115: What was the purpose of the agitation with the syringe? Was it to clear off the tissue from potential debris, trigger mucus release, or else…?

Line 120: Seawater was excluded from the samples by decanting, however it is unlikely that microorganisms present in seawater were entirely removed from samples. It would have been interesting to sample the seawater as well, and assess which part of the bacterial microbiome was coral-specific vs derived from the environment. This could perhaps be commented on?

Line 124: Was an equal amount of each sample used for DNA extraction (if so, how much)?

Lines 133-134: Negative PCR controls were performed, however there is no mention of DNA extraction controls. This is an important procedure to identify laboratory/reagent contaminants and is becoming a critical inclusion in microbiome studies (see Slater et al. 2014 BMC Biology; de Goffau et al. 2018 Nature Microbiology; Davis et al. 2018 Microbiome). If the Authors did not perform blank DNA extractions (and sequenced them alongside samples) to identify and remove contaminants, this should be explicitly acknowledged.

Lines 151 and further: Was any variance stabilization method (such as with DESeq2 or  rarefaction) applied on the data to account for the different read counts across samples? Not normalizing the read counts could greatly impact and bias the results (especially since samples fell within a large range of counts, as outlined on line 168).

Line 160: Please specify that ANCOM stands for “Analysis of Composition of Microbiomes”
Validity of the findings: Results are nicely reported and discussed, and the conclusion clearly summarizes the findings of the study. Could the Authors please address the following points?

Lines 174-175: Was a pairwise comparison performed to check whether the difference in microbiome composition between the Y and the G/R was statistically significant?

Lines 209 and further: Did the Authors try to conduct the same analysis pipelines by removing these very abundant Rickettsiales from the samples? Sometimes rare members of the microbiome play important roles. The Authors specified on lines 232-234 that differences between coral colonies of different genotypes were based on microbes with low relative abundance. Such an analysis (with Rickettsiales removed from the dataset) would therefore be relevant to enable patterns to emerge that are based on low-abundance bacteria.

Lines 272-273: As the Authors outlined on the previous lines, it is interesting that the microbiome seems relatively uniform within A. cervicornis colonies and if so, stress of sampling could be reduced in future studies by collecting fewer samples while still obtaining a representative microbial community. However, the conclusion needs to be tempered, as patterns observed in one or two species cannot be directly extrapolated to all acroporid corals. This study in particular considered nursery-reared corals, which were hung on a structure and not growing on the reef as natural colonies would be. This in itself might lead to different microbial signatures and it would be relevant to repeat a similar study with wild A. cervicornis corals to confirm the present results. Moreover, other acroporid species (some of which have different morphology/physiology, or individuals sampled from another location) might possess microbial structures that are not consistent with those observed in this study. Hence, lack of spatial heterogeneity should not be assumed for most acroporid corals, at least without further evidence.

Lines 284-285: Maybe the Authors could suggest that more studies on spatial heterogeneity of microbial communities need to be undertaken across numerous coral species to verify these conclusions. If colonies are large enough, taking more than 3 samples per location on the colony would be a more robust design.

It could be useful to suggest that future studies should investigate the microbial community composition of corals transferred to the reef and compare how they relate to nursery-reared communities. Perhaps the success of individual corals could be linked with the composition of their microbiome, or some detrimental shifts could be identified in colonies that perform less well?
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: EXTRACELLULAR VESICLES IN PATIENTS IN THE ACUTE PHASE OF PSYCHOSIS AND AFTER CLINICAL IMPROVEMENT: AN EXPLORATIVE STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Here, in this paper, authors found five biomarkers in peripheral blood-derived extracellular vesicles (EVs), which can be used to diagnose psychotic patients. In addition, they found physio-chemical differences of EVs between healthy controls and patients. These findings will impact on further development of diagnostic tool. I think this paper can be accepted with minor revisions. My comments follow in the sections below.

Page numbers were not provided, even though line numbers were, so I will write main text’s section name. For detailed comments like typos, I strongly recommend to provide page number next time. Because the PDF file I got have three additional pages at front added by the editor, I cannot be sure that the page number in Adobe Acrobat will be the same page number that authors see in their own manuscript.
Experimental design: 1. Were brain related proteins significantly enriched in this EV proteome? 45 out of 1853 seems to be not that significant and I cannot find how you defined “brain-related” with specific cutoff of Human Protein Atlas. Please notify how you defined “brain-related” and if it is not statistically significant, please note this clearly on the main text.

2. Washing step between LC-MS/MS sample runs can be specified in “Proteomics of isolated EVs” section of “Materials and Methods”.
Validity of the findings: 1. PXD016293 in PRIDE database cannot be searched or viewed, because authors kept it private. For revision process, they have to share reviewer ID and password so that reviewers can check their data quality.
Additional comments: 1. Instead of UniProt accession, it will be better to provide gene symbol in the main text for smooth reading.

2. References should be formatted properly. For example, some paper titles use capital letter for each word not others use it for the first letter of whole title. Please check page number format (117-122 vs 105-11 / adding p. even though it is not page number but article number, etc) and journal name format (adding period if you use abbreviation, etc).

3. Figure 1 is too wordy and not essential for this paper. This figure seems to be “graphical abstract” that authors might have used for submission to other journals. Authors can summarized it to one figure panel if they want to keep this figure.

4. Figure 2B – For each patient, did you use the exactly the same number of EVs to test size? If not, it will be fair comparison if you change y axis to “relative percentage” not the actual number of EVs. If you use the exactly the same numbers for each sample, please write it down in main text or figure legend.

5. Figure 2A 2C 2D and 4 – I guess, you can see statistical significance because you used “paired” t-test. Please provide the information about sample pairing or use paired ratio (patient/control) to visualize your data more significantly in the figure. It seems not that significant graphically in the figures.


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

Input:
Title: EXTRACELLULAR VESICLES IN PATIENTS IN THE ACUTE PHASE OF PSYCHOSIS AND AFTER CLINICAL IMPROVEMENT: AN EXPLORATIVE STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript is relevant for initiating studies of extraceular vesicles in patients with  psychosis . The study group is small, however through the experimental methodology of analyzing  proteomic EVs , at least the study allows two interesting objectives such as seeing proteins associated with synaptic regulation and also seeing if there are possible markers in brain-derived EVs circulating in periphery blood.
The greatest weakness of the manuscript is the way of working with samples and with the isolation of extracellular vesicles. The authors do not follow the recommendations given by the ISEV international society of extracellular vesicles that regulates most works in this area.
Experimental design: The sample collect should be made with citrate and not with EDTA a calcium chelating necessary for the production of EVS. The sample should be worked immediately for  purifications of EVs and maintained  a 4 C  and not a -80 C
Unfortunately the comparisons of size , amount of protein and kind of EVs are wrong  because to keep at -80 C generate aggregates and break of vesicles. The recomendation from ISEV include more steps and the centrigugations to
eliminate debris and to obtain small and large EVs .
Validity of the findings: My recommendation is to consider total extracellular vesicles and work proteomically with the 45 unique brain molecules and with the putative markers of Vesicules of  brain detected in EVS for bioinformatics analysis and with a greater discussion of the relevance of markers and as they could be validated through an experimental  design
 Re write the manuscript , removing the EVs analisis and to concentrate in proteomic findings .
Additional comments: Due to the current situation of the pandemic and being a study with patients, the authors should make a effort to rewrite with a better rational, shorten the manuscript and dedicate efforts  to highlight the importance of the molecules obtained and how it could be validated in the future


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: EXTRACELLULAR VESICLES IN PATIENTS IN THE ACUTE PHASE OF PSYCHOSIS AND AFTER CLINICAL IMPROVEMENT: AN EXPLORATIVE STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: No further comment for this secondary revision.
Experimental design: No further comment for this secondary revision.
Validity of the findings: No further comment for this secondary revision.
Additional comments: The authors carefully revised their manuscript, including omission of Fig. 1, which strengthens the manuscript. I want to congratulate the authors to their work and suggest to go ahead with 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: IDIOSYNCRATIC LIVER PIGMENT ALTERATIONS OF FIVE FROG SPECIES IN RESPONSE TO CONTRASTING LAND USE PATTERNS IN THE BRAZILIAN CERRADO
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general, the manuscript "Idiosyncratic liver alterations of five frog species to land use changes in the Brazilian Cerrado" is correctly written, in professional English.

I suggest to check the references. There are citations that appear in the text that do not appear in the References section.

Please check text citations: "et al." should be in italic

Figure 1: It is important that the map include a clear scale, to see the distances between the study sites. It would also be useful to observe the location of the ponds on the maps.
Experimental design: The title and objective of the manuscript do not correspond to the experimental design. It is mentioned that the change in land use will be evaluated, but two different scenarios are proposed: a protected site (pristine area) and a site with agricultural activity. If one wants to study changes in land use, one should evaluate the same site before and after a change in land use. These are long-term field studies, which include continuous monitoring. But not having previous information on the studied sites, I suggest to change the way of stating the objective of the work, title and phrases associated with evaluation of changes in land use.
It seems more likely that the study evaluates how melanin, hemosiderin and lipofuscin pigments vary depending on land use.

Materials and Methods Section is correctly written, but some issues must be improved.
Some detailed suggestions:

(Lines 128-130): What are the distance between the ponds in each region? Why did you select three ponds in one region and only two in the other? I suggest you including this information in the text

In lines 139-141, it is not clear how do you structure the campaigns to collect in both sites. Did you collect both sites in the same day, week, month, year? I suggest to clarify this issue, it is important for comparisons.

Its important to know also the conservation status of the selected species (lines 141-144). I suggest you incorporate it.

Lines 157-158: Why did you collect only one sample from only one pond on each region? If you collected samples from each pond.

Lines 161-162: You report results in Materials and Methods section. I suggest to move the data to the Results section.
Validity of the findings: The results are relevant, and very interesting, but, there no do not respond to the stated objective. I suggest restructuring the objective and title of the manuscript.

In addition, there are some results that must be clarified. (Line 241) There is a lack of information about how the occurrence of the species was evaluated. In the Materials and Methods section there is no description of how the monitoring of species was done, or how the occurrence was evaluated.

In line 33, you mention: "Our results add a new perspective to the influence of land use changes on environmental health by highlighting the effect of environmental changes on internal morphological aspects of animals". The experimental design does not allow evaluating the effects of the change in habitat use, but rather the effects observed in frogs that inhabit areas with different land use. Although this results are relevant when evaluating a possible change in land use, and even carrying out environmental risk assessments, it cannot go outside the scope of speculation and great care must be taken when talking about the effects produced by a change in land use.
Additional comments: In general, the manuscript "Idiosyncratic liver alterations of five frog species to land use changes in the Brazilian Cerrado" show relevant results for the ecotoxicology field. The intention of the study is interesting, but many aspects are missing to understand how the research was carried out. And there are some issues regarding the title, objectives and specially Materials and Methods that must be improved.

1- Your most important issue is to check the objectives, title and experimental design and restructure the study so that the experimental design answers the research question being asked
2- More detailed Methology must be introduced. For example, Sampling in just one pond, without knowing the distances between the ponds in the same region, or the dimensions thereof, generates more questions than answers. It is probable that the sample taken is not representative of the region, nor of the pond in question. According to what methodology the water samples, storage, etc. were taken? these are relevant data when validating the results.

Finally, some suggestions are detailed below:

Lines 260-262: please check the wording of that phrase.

Lines 263-279: Could there be a relationship between the amount of liver melanin and exposure to UV radiation due to the lack of refuge sites in the planted pastures? It is interesting if you include this in the Discussion section, taking into account that planted pastures eliminate the Natural refuge sites, such as shrubbery.

Lines 307-310: It would be interesting to give a phylogenetic look that also sheds light on these interspecific differences.


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: IDIOSYNCRATIC LIVER PIGMENT ALTERATIONS OF FIVE FROG SPECIES IN RESPONSE TO CONTRASTING LAND USE PATTERNS IN THE BRAZILIAN CERRADO
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript examined liver’ melanomacrophages and their metabolic substances including melanin, lipofuscin and hemosiderin to evaluate the land use changes inducing environmental changes. As stated by authors, there are many laboratory studies in this topic but area studies are very important. Contaminants can alter vital functions of internal organs and affect the viability and persistence of species in the environment. To that end liver cell physiology is an important endpoint to determine the effects of land use changes.
Introduction
This part is good organized and clearly emphasize the purpose of the study.
In line 89, …….xenobiotics (…). Here is a literature made by Çakıcı 2015 should also be added to mention melanomacrophage increases. This study revealed an increase in melanomacrophage number of liver tissue after exposure to a pesticide (carbaryl) in an important anuran species, Bufotes variabilis.
(Çakıcı Ö 2015. Histopathologic changes in liver and kidney tissues induced by carbaryl in Bufotes variabilis (Anura: Bufonidae). Experimental and Toxicologic Pathology 67(3), 237–243)
Material and methods
This section is comprehensive and enough to justify the results of study.
Results
There are important findings and these findings are the results of well designed experiment. However, there are no detailed histologic figure. Only one image was found in graphical abstract. And there is no information about which species the liver figure belongs to. Therefore, these histological photographs should be added, if necessary.
Discussion
In this part, results are logical evaluated. And as stated by authors, melanomacroges are important biomarkers showing alterations in the liver in response to land use changes.
Language
English should be checked by a native speaker. In some places, there are grammatical errors.
For example In line 312. Therefore, MMCs seem to be an efficient biomarker that indicated (indicating) alterations in the liver in response to land use change.

Best Regards
Experimental design: This section is comprehensive and enough to justify the results of study.
Validity of the findings: There are important findings and these findings are the results of well designed experiment.
Additional comments: Dear Authors,
This manuscript has valuable findings. As you said, melanomacroges are important biomarkers showing alterations in the liver in response to land use changes. However, I have some suggestions.
-In introduction part: In line 89, …….xenobiotics (....). Here is a literature made by Çakıcı 2015 should also be added to mention melanomacrophage increases. This study revealed an increase in melanomacrophage number of liver tissue after exposure to a pesticide (carbaryl) in an important anuran species, Bufotes variabilis.
(Çakıcı Ö 2015. Histopathologic changes in liver and kidney tissues induced by carbaryl in Bufotes variabilis (Anura: Bufonidae). Experimental and Toxicologic Pathology 67(3), 237–243)
-There are important findings and these findings are the results of well designed experiment. However, there are no detailed histologic figure. Only one image was found in graphical abstract. And there is no information about which species the liver figure belongs to. Therefore, these histological photographs should be added if necessary.
-English should be checked by a native speaker. In some places, there are grammatical errors.
For example In line 312. Therefore, MMCs seem to be an efficient biomarker that indicated (indicating) alterations in the liver in response to land use change.

Best Regards


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: IDIOSYNCRATIC LIVER PIGMENT ALTERATIONS OF FIVE FROG SPECIES IN RESPONSE TO CONTRASTING LAND USE PATTERNS IN THE BRAZILIAN CERRADO
Review round: 2
Reviewer: 1

Output:
Basic reporting: I believe that almost all the points to be improved have been improved, or failing that, the limitations have been explained.
Experimental design: The entire article is much clearer and all my concerns have already been answered.
Validity of the findings: Although the article has some limitations, it is novel and has great potential. The results are relevant in the field of ecotoxicology.
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: IDIOSYNCRATIC LIVER PIGMENT ALTERATIONS OF FIVE FROG SPECIES IN RESPONSE TO CONTRASTING LAND USE PATTERNS IN THE BRAZILIAN CERRADO
Review round: 2
Reviewer: 2

Output:
Basic reporting: New title "Idiosyncratic liver pigment alterations of five frog species in response to contrasting land use patterns in the Brazilian Cerrado" is more suitable.

Contaminants can alter vital functions of internal organs and affect the viability and persistence of species in the environment. Liver cell physiology is an important endpoint to that reflects important reactions to environmental alterations. This manuscript examined liver’ melanomacrophages and their metabolic substances including melanin, lipofuscin and hemosiderin to evaluate the contrasting land use patterns in five Anuran species.
Experimental design: This section is well designed.
Validity of the findings: Findings are well stated and linked to aim of this study.
Additional comments: In this revision, all corrections were made by authors.


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: REVIEWS ON COMMON OBJECTIVES AND EVALUATION INDICATORS FOR RISK COMMUNICATION ACTIVITIES FROM 2011 TO 2017
Review round: 1
Reviewer: 1

Output:
Basic reporting: I appreciate the opportunity to review this study. Risk communication is both an important and timely topic.  I believe this manuscript has a good potential for publication, provided that revisions are made, primarily those contained in other sections of this review.

The manuscript does a reasonably good job of introducing the topic of risk communication, although in general I find the manuscript lacking in relevant examples to engage the reader. The quality of the writing is good overall (some minor suggestions are made in the “additional comments” section).  It does not appear that similar work has been done recently, making a review paper appropriate.

The paper’s abstract and introduction places considerable emphasis on evaluation of risk communication strategies, but does not appear to delve deeply into the results of these evaluations – that is to say, any assessment of which risk communications strategies that the reviewed papers found to be more or less effective. In my opinion this is a significant oversight, as at present the paper only describes some evaluation methods and notes their frequency, but does not discuss their effectiveness.
Experimental design: The study design in this manuscript generally appears sound. However, it is difficult to evaluate many of the author’s choices because they are presented only as fact (for example, in noting that the years 2011 to 2017 were chosen for evaluation) without providing insights on why these decisions were made.  As examples:

1. Is there some important event that took place in or before 2011 that made that year to be the early cutoff? Or was this simply a resource issue?

2. What is the significance of the particular academic search engines used for this study? Given that PubMed (a comprehensive database, but focused exclusively on medical research) was one of the search engines, it is no surprise that most of the articles were medical in nature.  Was any consideration given to other academic search engines?

One of the main findings of the manuscript is that the lack of academic research appearing through these particular search parameters in many fields means that little risk communication is happening in those fields (see for example, lines 608-610). It does not appear that the authors have explored alternative reasons why those fields did not show up in their search.  For example, could the databases possibly not contain those fields of study? Is it possible that those fields of study use a term other than “risk communication” for this activity? Or some other reason?
Validity of the findings: This study seems to lack substantive conclusions. At present, the conclusions section is mostly a series of generalities that could easily have been included in the introduction section.  Only a few sentences (around 600-610) are truly informed by the work.  I’d recommend that the authors find ways to better express the results of the study within the conclusions section, regardless of what those conclusions are.

The authors also have not clearly explained why key decisions were made (see “study design”) which at present is a substantial impediment to evaluating the manuscript.
Additional comments: General suggestion: are there any ways that the authors could create more graphical information for this manuscript? It is currently table-heavy (understandably given the subject matter) and some additional informative figures could make the article more engaging for readers.

Line 27: Risk communication is used both in “academic fields” as well as through various types of practitioners, which is acknowledged in the paper but not in the abstract.

Line 31: The use of “search engines” in the abstract implies the use of popular search engines (i.e. Google or Yahoo) as opposed to academic or scholarly search engines.

Line 79-90: Given the mention of H7N9, it would seem prudent to provide any relevant insights related to COVID-19 in the revisions.  Being an all-encompassing topic for much of the world, any relevant linkages would be beneficial to bring forward.

Lines 84-87: This is a very long run on sentence.  I recommend splitting this into several different thoughts, which will be easier for the reader to understand.

Lines 114-116: Although this is a succinct statement of what was done, the reasons for the various choices are not clear. For example, is there some significance to the year 2011? Why these particular search engines?

Lines 146-154: This is an extremely long and complex sentence which is very confusing. Recommend splitting this into several different thoughts.

Lines 164-166: It is not clear how the codes were chosen.  Given that many of the codes are fairly general, it’s unclear how bundled or unbundled the codes were meant to be, and how they were developed.  This is key context for the reader.

Lines 277-278: Is this referring to “Likert Scales” or “Likert-type data”? Scales are made from aggregating more than one Likert-type data point. The terms appear to be mixed here.

Line 424: The abstract and introduction place considerable emphasis on the evaluation of risk communication methods, but here it implies this was only a development of “methods by which it is practiced.”