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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.

Title: MULTI-SCALE AND MULTI-SITE RESAMPLING OF A STUDY AREA IN SPATIAL GENETICS: IMPLICATIONS FOR FLYING INSECT SPECIES Review round: 1 Reviewer: 1

Basic reporting: This manuscript had some grammatical errors, some jargon not defined, and colloquial English used in places (see General comments to author). It would benefit from being read by a native English speaker. I highlighted areas where additional references would be helpful in the General comments to author section. Experimental design: In general this study seems well-executed, but research questions need to be more specifically defined in the introduction. In the methods section the analyses that pertain to those questions need to be linked to those questions. Validity of the findings: Results need to be clarified at times (see general comments to author). Additional comments: This is an interesting study that investigates how varying the scale over which landscape genetic analysis is performed influences results. I do worry that the authors’ reliance on Mantel tests muddies the inferences that can be drawn from the results. In addition to acknowledging the limitations of Mantel tests, and performing other analyses to validate their results, the authors should discuss these limitations and the possible effects on their results in more detail in the discussion section. They do discuss how limitations of Mantel tests can lead to erroneous inferences in a specific case on line 498, but they should address caveats of Mantel tests more generally with regards to their results. At times I found the writing confusing. In addition to having a native English speaker read the manuscript, the authors need to take care to present the actual results and not make general statements like those on lines 356: “Best values were observed” or 374: “best values were obtained” (see additional examples below). One place that clarity must be improved is in the analyses described in the section “Multiple scales and multiple locations analysis”, because this seems to be the crux of this study. At first read I was left wondering exactly what the authors intended by these analyses. The authors need to clearly state which analyses pertain to which question, and what information would be gleaned from various potential results. The link between this paragraph and the results presented in Figure 3 was not immediately clear. The authors need to explicitly state what association they investigated, instead of using vague terms like “we tracked the evolution of” (line 280). The analysis presented in the second panel of Figure 3 is more straightforward, but it would be nice for the authors to set this analysis up in the introduction by telling us why it is important to investigate if correlation coefficients change over different geographic scales. In addition, the authors need to take care to put their results in the context of others’ work in the discussion (especially in the discussion of how Mantel correlation statistics change with scale). Specific comments are below: Introduction: I found the introduction interesting, and I think the authors could highlight the strengths of this study even more strongly by organizing them into their own paragraphs (for example one paragraph that explicitly focuses on scale and one that focuses on the value of replication across a large study area). These points come across as it is currently written but I think this would clarify the strengths of this study even more strongly. Line 58: The landscape genetic toolbox has greatly expanded in recent years, I’d remove “the landscape genetic toolbox is far from being established” from this sentence. Lines 93 - 95: needs citation as currently stated Line 100: remove “As a study system” Line 102: Write out the number Line 104: Move to methods Last paragraph: Instead of a description of the species (most of which could be moved to the methods section), it would be helpful for the authors to outline a few questions they focused on (for readers that are unfamiliar with LG analyses). Line 124: If the authors give specifics of localities they should refer to a map or table of locations. Line 124: remove “a sampling of” Line 125: give units. Also, the authors need to describe why 10 is relevant. Lines 127 & 129: do the authors need to refer both to figure/table and write “supporting information”? The names make it clear this is supporting information. Line 132: what publication are these loci described in Line 142: remove “already” Line 151: should be “allele” Lines 181-183: It would be helpful for the authors to explain what is a plateau. Line 815: It might be useful for the authors to also refer readers to Guillot et al. (2009, Molecular Ecology) here. Line 201 should read “temperature” Line 204: There is a typo. Lines 212-215: I found this confusing. It would be more clear if the authors said something like “M. galloprovincialis shows no preference for pine species. They will live in the dead wood of any species, which tend to include XXX on the Iberian peninsula”. Lines 216-221: This is repetitive, please make more succinct. Line 223 needs a citation Line 232 should read “mill” Line 244: Did the authors also test for colinearity when smaller areas were considered? Lines 272-299: It would be helpful for the authors to introduce this section by describing the question they are addressing, and then to describe what insight various potential patterns would provide. They say on line 280 that they tracked the evolution of areas. What does this mean? In the results they present data on how the number of areas with a significant Mantel result changes with size of area investigated. How does this address the question originally posed in the introduction of does genetic structure or the influence of landscape features vary spatially? Line 276: Please clarify what the sentence beginning “scale dimension” means. Line 277: replace “between” with “using” Line 280: The authors need to describe what unbalancing and a supported hypothesis mean. Line 281-285: I don’t understand how this gives the geographic distribution of areas, please clarify. Line 295: Do the authors mean “cumulative”? Line 313: If the authors are going to provide the delta k values they should explain what they mean in the methods section (I don’t think they are necessary). Line 321: The authors should describe to which analysis this refers It seems lines 326-329 belong in the discussion. Also, it would be helpful for the authors to describe what these and other variogram patterns would mean in the methods section. Line 333: what does “for a total of 116 and 102 alleles” mean/refer to? Line 334: Can the authors provide some evidence of this? This could also be moved to the methods section, and results presented only for 10x10. Line 343: The authors should move general statements like this to the discussion and focus on specific results here. The sub-headings throughout the manuscript are helpful but it would be nice for them to match between sections (where is Multiple scales and multiple locations analysis in the results?) Line 346: Give the actual percentages Line 350: The authors need to clarify what this sentence means and to which analysis this refers. Line 356: What is the “best” r? Please re-word. Also, there is a typo here. Paragraph starting on line 358: If the authors refer to specific places they should show them on a map. Line 368: The authors should keep their terms consistent, they use significant area, then non-supported area. Line 380: Why “common”? Line 383: This summary paragraph is helpful but the first sentence needs to be re-worded for clarity and cited. Line 387: The comment about environmental features appearing at different times can be removed. Line 391: The authors should use the words large and small (not low) Line 397: cite Figure 2 at the end of this sentence Line 403: “To gather a genetic structure” doesn’t make sense, please re-word. Lines 394-466 seem unorganized and should be re-structured to clearly discuss each point laid out in the results section. It would be helpful for the authors to add topic sentences instead of just continuously laying out points. For example, the paragraph beginning on line 408 should begin with a sentence about what it is about. Line 425: Please remove “deal with” and re-word Line 428: This is a clear topic sentence Lines 469-478: How does this relate to what others have found? Typo on line 495 (capitalization) Table 1: Abbreviations need to be defined in captions of other tables/figures (please take care to do that), so I do not think this table is necessary. Figure 2: The authors need to describe a semi-variogram in the methods section. Figure 3: The caption needs to clearly state what variable is on the y-axis. “Development” is confusing, please re-state this. Figure 5: The authors should change “sign” to “sig”, and define the abbreviations. Table S1: Where is the description of what the abbreviations mean?

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.

Title: MULTI-SCALE AND MULTI-SITE RESAMPLING OF A STUDY AREA IN SPATIAL GENETICS: IMPLICATIONS FOR FLYING INSECT SPECIES Review round: 1 Reviewer: 2

Basic reporting: ## Clarity of writing, English In general the writing is clear. However, the English could be improved -- there are some dubious word choices and grammatical mistakes which hamper readability. Here are some examples: Line 38: what does "to track the variation of inference" mean? Line 42: should be "Detection of an effect of environmental features on gene flow" Line 54: "It allows which ... to be inferred" should be "It allows inference about which ..." Line 94: should be "...picture of the general effects of landscape on the dispersal..." Line 98 (also 532): don't you mean "variability" instead of "versatility"? Or do you mean "has the power to *increase* versatility" Line 402-404: what does "gather a genetic structure" mean? In general I don't understand this sentence. Line 405 (and other places): what does "development of the variation of frequency of areas" mean? Line 419: "important" doesn't seem like the correct adjective Also check for incorrect and missing pluralization throughout. ## Context, references The landscape genetic analyses used in the manuscript are typical of the norms for the field, and the references for the methods (as well as general analytical concerns specific to the field) are sufficient. Study-system-specific references and context are also sufficient. However, the method proposed in the paper is (at least superficially) similar to local regression methods and geographically weighted regression, both of which have been used in multiscale analysis (although not with pairwise data) -- in my opinion more attention could be paid to relevant statistical references outside the field of landscape genetics. A place to start looking might be: Brunsdon, Fotheringham, Charlton. 1996. Geographically weighted regression: a method for exploring spatial nonstationarity. Geographical Analysis ## Figures, tables The figures could be made more aesthetically pleasing by increasing resolution and reducing white space, but are generally clear ... although I suggest the following minor improvements for readability: Figure 2: decrease size of plot relative to axis titles, capitalize axis titles Figure 4: adding boxes around each panel would help with visual navigation. Also, a color scale would be informative -- what exactly do "low frequency" and "high frequency" mean? Figure 5: units on x-axis, shorten titles substantially ## Structure, data The general structure follows PeerJ standards, is self-contained, and the data (georeferenced microsat genotypes) is included in the supplement (as is the R code used to generate the results in the paper). Experimental design: ## Relevance, originality of research question The authors correctly identify that while environmental effects on animal movement are often scale dependent, yet few studies explicitly (or quantitatively) consider scale. The method presented here is novel to the field, to the best of my knowledge, and in my opinion contributes a useful idea about how to identify and describe scale dependence in isolation-by-resistance analyses. ## Rigor The insect sampling is extensive, and the field and molecular methods are sufficiently detailed and rigorous. ## Description of methods The landscape genetic analyses are sufficiently detailed; software and specific functions are referenced throughout. However, the goals of these analyses (and how they fit into the paper at large) were not clear until I read the results. I suggest that the introduction should contain the system-specific questions and hypotheses, and should lay a roadmap for the subsequent descriptions of analyses. For example, adding something to the effect of: "We first characterize the broad-scale genetic structure of the beetle across the study area. We then used the "isolation-by-resistance" (IBR) framework to model beetle dispersal as a function of temperature, elevation, and pine density. By subsetting the data to particular locations and spatial scales and repeating the analysis, we identify how the influence of environment on spatial genetic structure varies with spatial location and scale." On this note, the last paragraph of the introduction belongs in the methods. Including relevant hypotheses and the motivating questions in the beetle system is fine in the introduction, but detailed description of the study system should be moved to the methods. My main concern with the paper is that the methodological goal is not clear. There seem to be two intermixed goals. First, to determine what constitutes a sufficient sampling design (e.g. spatial scale) to detect an effect of environment on dispersal; in other words, what scale is sufficient to use in a hypothetical study to infer an effect. Second, to develop a method for investigating and visualizing scale dependence in the effect of environment on dispersal; in other words, given a single large-scale dataset, to determine at what scales an effect is present. While these goals are superficially related, they require different presentations, and in my opinion, different statistical support (details below). It should be clear from the outset which goal (one, the other, or both) this paper is addressing. The first goal (study design re: spatial scale) is suggested by passages like the following (not a comprehensive list): Lines 42-43 (Abstract): "Detection of environmental features on gene flow generally increased with an increasing scale of study" Lines 394-400 (Discussion): "We observed a notable influence of scale on the detection of supported IBR hypotheses ... This correspondence suggested that at this range of scales, dissimiliarity between individuals was often not appropriate to show a significant effect of environmental features on gene flow" Lines 476-478: "our results show a tradeoff between the sampling of small areas where effects of environmental feastures are strong but scarcely detected and the sampling of large surfaces, where this effect is weaker but often detected" In my opinion, statements regarding optimal design with regards to "detection" of effects need to be backed up by a simulation study. There are three main reasons for this: (1) it is impossible to say if an effect is detected unless one knowns a priori whether it is true or not (clearly only possible with simulation). (2) With the current setup, sample size is confounded with spatial scale: smaller scales will contain fewer individuals, and a trivial consequence is that inference will be more "noisy" and less powerful. In a simulation study, one can keep sample size constant across spatial scales. (3) On any given dataset, it's difficult to say to what degree the results are artifacts of the particular spatial distribution of samples. I'm not saying that the real-world dataset used in the paper is useless for this goal (it's an excellent worked example), but I think the addition of a simulation study is necessary if the authors want to make statements about "detection" of effects with regards to study design. However, if the paper is simply trying to illustrate an idea for a new exploratory statistical method, then I don't think simulation is necessary (although it would certainly add credibility). On this note, the goal of the method as stated in the abstract isn't very clear: "a method for resampling of study areas at multiple scales and multiple locations (sliding windows) to track the variation of inference in spatial genetics". Based on the rest of the paper, it seems like the method is designed to determine what scales/locations exhibit a significant effect of the environment on spatial genetic structure, and I'd suggest rewording along these lines. In general, the methodological goal should be made much clearer in the abstract. Validity of the findings: Appropriate care seems to have been taken with regards to assumptions underlying the molecular data (linkage disequillibrium, HWE, null alleles, etc.) The authors have been careful to employ multiple methods and address already published concerns about typical landscape genetic analyses. One concern I have with regard to the proposed resampling method: no mention is made of controlling for Type-I errors, and how these might interact with sample size to influence the results. The goal is to determine if there's an effect of environment on dispersal across a set of locations and spatial scales. Effectively, this is accomplished by subsetting the data, but because the subsets of data across locations/scales overlap, null hypothesis tests across these will not be independent. If trying to aggregate these results over space to get a sense of what effects are most important at what scales, regardless of geography (as is done in figure 3A), then inflation of Type-I error will bias the percent of areas with significant isolation-by-resistance upward. The result could be very misleading if Type-I error varies systematically with spatial scale (for example, because tests at larger spatial scales share more of the same sampling locations). Also, how might edge effects bias this method at locations along the margin of the study area? In general, interpolating effect size (ie. Mantel r) onto maps might be more informative than interpolating frequency of rejected null hypotheses. One way to show both is to use color for interpolated effect size, and contour lines to show regions wherein p<0.05. Finally, I'd suggest that the authors restructure the discussion: currently, it reads like two different papers (with the method-specific discussion in the first half, and the system-specific discussion in the second half). It would be more clear and more effective, in my opinion, to integrate the two sections; so as to show how the proposed method gives biological insight into the long-horned beetle system that would not obtained otherwise. Additional comments: No additional comments

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.

Title: MULTI-SCALE AND MULTI-SITE RESAMPLING OF A STUDY AREA IN SPATIAL GENETICS: IMPLICATIONS FOR FLYING INSECT SPECIES Review round: 1 Reviewer: 3

Basic reporting: This manuscript reports the genetic structure of Momochamus galloprovincialis in the Iberian Peninsula. The authors detected that the effects of environmental features differed among spatial scales, suggesting that the combination of a resampled study area at multiple spatial scales across various locations provide a more general picture of the effects of environmental feature. I highly evaluate the authors’ motivation for contributing the landscape genetics study, but dataset of M. galloprovincialis seems difficult to be discussed in context of this study. Kawai et al. (2006) showed that new mutualism system between Monochamus species and the pine wood nematode destroy original genetic structure formed by historical and environmental effects. I don’t know exact damaged areas of pine wilt disease in the Iberian Peninsula, but invasion of the nematode should affect the genetic structure of the beetle, i.e. they should not be in genetic equilibrium in the damaged area. Artificial movement of the beetle due to damage protection is also problematic to study the genetic structure. In such a situation, discussion of the land scape genetics must be done in more careful manner, at least divide the dames from damaged from undamaged areas. Experimental design: Collecting the beetles throughout the Iberian Peninsula meets the aim of this study to show the gene flow there. Many of the individual numbers from one dame were not enough for population genetics analyses, but resampling method could make up the results. This method sounds adequate. Validity of the findings: Authors adopted well considered and sophisticated statistical methods. The results are well stated if the effect of invasion the nematode can be set aside. Additional comments: -I think the main aim of this study is measuring gene flow of the beetle, not presenting a method for resampling of study areas at multiple scales and multiple location. -Figure captions are not kind to readers. For example, what do you indicate by colors and ellipse of Figure1C? They should be more concise. - “el al.” must not be in italics.

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.

Title: MULTI-SCALE AND MULTI-SITE RESAMPLING OF A STUDY AREA IN SPATIAL GENETICS: IMPLICATIONS FOR FLYING INSECT SPECIES Review round: 2 Reviewer: 1

Basic reporting: Lines 35 & 48 should read "gene flow" The introduction, methods and results are much clearer. A few comments on the discussion: Line 410: This is confusing, please re-word. Lines 420 & 450: Please state this as cause and effect. "A genetic structure" doesn't make sense, and line 450 is repetitive. Line 436: "geographic scales" would be better than "study scales" Line 456: "causes" would be better than "origin" Line 468: This is confusing, please re-word. Line 495: affected how? Line 510: "We speculate that" or "It is possible that" would be better than "we suggest". Experimental design: no comment Validity of the findings: no comment Additional comments: This manuscript has greatly improved.

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.

Title: MULTI-SCALE AND MULTI-SITE RESAMPLING OF A STUDY AREA IN SPATIAL GENETICS: IMPLICATIONS FOR FLYING INSECT SPECIES Review round: 2 Reviewer: 2

Basic reporting: The English is improved from the first submission, and the meaning is clear although there are still a few confusing word choices: I have some minor suggestions on word choice/grammar, listed line-by-line under "comments to authors". Experimental design: The authors have substantially clarified the goals of this study. I have a few minor comments/questions about methodology that are listed line-by-line under "comments to authors". Validity of the findings: No additional comments ... I am satisfied by the clarifying rewrites to the discussion and the authors' responses to my questions Additional comments: Some minor comments/questions/suggestions, line-by-line: 65: What is meant by "method"? "Discipline" or "framework" seems more appropriate 66: "optimal" not "optimum". And what exactly is being optimized? 82: "limited" in what sense? Do you mean "incorrect inferences"? 109-110: Clarify ... "_spatial_ resampling methods". Also a statistical method is not a "perspective", per se. How about something like: "Spatial resampling methods (...) provide a technique to examine variation in inferences across geographic subsets of a single sampling design ..." 160: Language is a little atypical, instead consider something like: "To check for convergence of the Markov chain Monte Carlo algorithm implemented by STRUCTURE, we ran ten independent Markov chains for 500,000 iterations each after an initial burn-in period of 200,000 iterations" 251: I'm confused by "values were set as negative". Do you mean reciprocal instead of negative? Assuming that edge weights (resistances) are calculated by averaging values of adjacent cells, then negative cell values would mean negative transition rates in the random walk, which doesn't make sense. 285: "Mantel" not "Mantels" 285: "areas of various spatial scales" doesn't make sense here; how about: "conducted across various spatial scales and locations" 293 (and elsewhere): "landscape analysis" is vague. Specifically, you are referring to the tests of IBD/IBR? 294: "generated sampling areas" not "sampling areas generated" 319: delete "tested" 328: Sentence is a bit garbled, use something like: "In the STRUCTURE analysis, the optimal number of admixed clusters was two (Figure S3)" 334: What's the test here? ANOVA of the first PCA axis as a function of cluster identity? 359: By positive effect, do you mean positive effect size (like a positive Mantel r)? Or do you mean a significant effect? The wording doesn't make sense as a hypothesis can't have an effect 361: "plateau" of what? The meaning of this sentence isn't clear 372: Does "low effects" mean a low effect size, or a low frequency of significant IBR tests? 373: "_spatial_ distribution of supported hypotheses" 391: Remove "context" 395: How about "dramatic variation in landscape features" instead of "dramatic landscape changes" 396-397: This sentence is vague ... What does "successfully" mean in this context? Is "success" finding significant IBD/IBR? I think what you mean is something like "across different inference methods, we successfully detected variation in the effects of environmental features on gene flow across spatial locations." 402: Change "could be explained by" to "are consistent with" 403: "by _known_ artifacts of the sampling design" 403-404: What does "known changes in the landscape structure" mean? Do you mean systematic spatial variation in the landscape variables? 407-408: "Detection of supported hypotheses" sounds strange and is unclear. I think you mean that patterns that were evident at large spatial scales/sample sizes (and so presumably real) were not found at small scales/sample sizes. You can reword to make this clearer. For example, wording like "Support was scarcely detected on the smallest spatial scale" could be changed to "Hypotheses were rarely supported at the smallest spatial scale". 410: "larger sampling" should be "larger sample size" 413: "the inference of the hypothesis tested" should be "inference about the tested hypotheses" 448-449: Check grammar here 450: "an effect" is too vague in this context. Effect of what? 457: "It is suggested" should be "We suggest" 460: "as well as _influencing_ the _continuity_ and fragmentation of pine tree cover ..." 468: "genetic differentiation of the species _due_ to evolutionary history" 479: as for line 468 505: Do you mean "_the statistical support for_ the Pr hypothesis _in this study_ suggests that ..."

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.

Title: MULTI-SCALE AND MULTI-SITE RESAMPLING OF A STUDY AREA IN SPATIAL GENETICS: IMPLICATIONS FOR FLYING INSECT SPECIES Review round: 2 Reviewer: 3

Basic reporting: I found this resubmitted manuscript by Haran et al. to be well improved. I recommend it to be accepted. Experimental design: This study seems well executed. Validity of the findings: This study sounds. Additional comments: No additional comments

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.

Title: REGULATING THE UAS/GAL4 SYSTEM IN ADULT DROSOPHILA WITH TET-OFF GAL80 TRANSGENES Review round: 1 Reviewer: 1

Basic reporting: This is a good idea that introduces a new method for the temporal regulation of GAL4/UAS based transgene expression in Drosophila. The potential for this method is enormous in my opinion. I believe that this new approach to temporal transgene control will be of great utility to many fly researchers, but still need to present more “specific” GAL4 lines to proof this idea. However, the paper should be significantly edited; really they should re-write it entirely. Experimental design: As describes, the results of UAS-grim lethality test reveals that it’s unstable lethality ratio displayed not only in each independent experiment but also under copy number of DJ146 or DJ147 (tubulin 1alfa and actin 5C promoter). The applicability of lethality caused from grim gene been concerned. Improving grim function or change lethal gene in here are suggested. The author notices that insufficient Gal80 maybe cause by tTA uniformly distributed, molecule number of Gal80 (driver copy number), not overall overlap between Gal80 and grim expression region which under driver controlling. But it is interesting about that grim cannot blocked effectively by Gal80 even in the same tissue or same cells although Gal80 expression level is more far than the expression level of grim. Would you refer possible reasons to explain it? I'm sure that the field would like to know how long it would take to turn expression off again by removing the drug from the food. Validity of the findings: Does the "tet-GAL80 inducible system” by rtA-tetracycline showed more efficacy precisely expression? I suggest that more specific GAL4-driver confirm is needed. Would be better supported these evidences if the GFP fluorescence results were quantified and if the specific criteria used for the comparisons were defined, when compared with the conventional TARGET system by tub-GAL80ts under permissive temperature? The stereotyped nature of the olfactory lobes and mushroom bodies should make objective quantification of reporter in the relevant glomeruli/neuropils feasible for these assays (Kuo et al., PLoS One. 2012; 7(12):e50855). But even if the contention that TARGET system gave higher expression levels would be a most welcome addition to this study. The authors should report specific P-values for all statistical tests, either in the results section or the figure legends. Additional comments: The writing is confusing and the organization of the data is not very good either. I suggest that they fully re-write the paper starting with a results section that describes the reagents carefully, which does not need to be long but clear. The set of data is coherent, but too dense. The message they want to convey will be clearer after a serious trim showing a synthesis of the principal results. This ms should be rewrite or significantly edited.

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.

Title: REGULATING THE UAS/GAL4 SYSTEM IN ADULT DROSOPHILA WITH TET-OFF GAL80 TRANSGENES Review round: 1 Reviewer: 2

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: In this manuscript, the authors describe a new approach to regulate gene expression in Drosophila using a tet-off Gal80 system. It complements the currently available tools such as Gene-switch or temperature sensitive Gal80 to control the temporal activity of Gal4. They provide detailed analyses on the new system in the result and supplementary sections. The tet-off Gal80 system requires multiple transgene (>2 copies) to obtain high level of Gal4 inhibition, which makes the system not very practical in its current state. However, it provides a proof of concept and a foundation for the research community to improve and optimize the system. There are some general concerns and suggestions to the authors that they may find useful. General concerns: 1) Does the chronic tetracycline treatment have any detrimental effect? Do the authors have any information about the dosage effect on lifespan or behavior (e.g. minimum effective dosage)? 2) Do the author have any information about the efficiency of the CMV1-TetO promoter in pDJ146/147? The reported low efficiency in suppressing Gal4 using these two constructs comes a little bit surprising. 3) The authors may want to consider rearranging some of the supplementary and result figures. In fact, some of the supplementary results are more informative (e.g. SFig 13, 14 – demonstrate the effect of inducer; STable 15 – effect on various Gal4 lines) than result figures (e.g. Fig 2 - not very informative unless you also show the expression pattern with pDJ146/147, with/without inducer). Specific comments: 1) The layout of Figure 1A is a little confusing. The authors could simplify the figure by showing progenies on one side (tet on) and the other side (tet off). “Hexagon” in the figure text should be “pentagon”. 2) It is very helpful for the authors to provide figures to every step in the construction of the transgenes. The authors could consider simplifying them by removing some of the irrelevant restriction sites but provide the final, full sequence information of the constructs pDJ146/147. The author may also want to consider using Gibson assembly to generate complex transgene constructs next time. 3) It is quite surprise to see pDJ147, with actin5c promoter, has no expression in the CNS (fig 6)? Could it be a positional effect specific to this transgenic line? 4) In figure 8, does the flies have UAS-grim? If not, it should be removed from the figure text? 5) In supplementary tables (e.g. STable 9,10), it would be better to show the actual measurement and highlight the one in bold to show the significance. Showing the P-value only without the actual number is not very informative. 6) The background of Figure 6 is too dark in some panels. It would be helpful if the authors can fix it.

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.

Title: REGULATING THE UAS/GAL4 SYSTEM IN ADULT DROSOPHILA WITH TET-OFF GAL80 TRANSGENES Review round: 2 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: This is a brilliant study that introduces a new method for the temporal regulation of GAL4/UAS based transgene expression in Drosophila. The potential for this method is enormous in my opinion. Authors replied all the points that I asked and showed reasonable results. I think this manuscript is ready to be published in PeerJ. I believe that this new approach to temporal transgene control will be of great utility to many fly researchers. It is a significant and welcome contribution to the field.

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.

Title: CD4 COUNT AND TUBERCULOSIS RISK IN HIV-POSITIVE ADULTS NOT ON ART: A SYSTEMATIC REVIEW AND META-ANALYSIS Review round: 1 Reviewer: 1

Basic reporting: The paper is well written, but can be improved with minor copy-editing. Experimental design: The study objective and rationale for this study and study design methods are well stated. Validity of the findings: The analysis is sound, but the findings have to specifically stated that they are applicable only to HIV positive adults (>= 15 years of age) not on ART, rather than generally to all people living with HIV not on ART, since this can be misunderstood to mean both children and adults. Additional comments: The authors have conducted a systematic review and meta-analysis to quantify the risk between CD4+ T cell count and tuberculosis among HIV-positive adults (>= 15 years of age) who are not on antiretroviral therapy. This is a valuable study that quantifies this risk in a systematic manner, and adds the the current evidence illustrating the exponential increase in TB incidence with declining CD4+ T cell count. Here are my specific comments: 1. Lines 1-2 -- Title: Change the “people” in title to “adults”, since this study is specific to HIV-positive adults (>= 15 years of age). You can change the title to “CD4 count and tuberculosis risk in HIV-positive adults not on ART: A systematic review and meta-analysis” 2. Line 4 -- Short title: Change short title to “CD4 count and TB risk in HIV-positive adults not on ART” 3. Lines 30-32: Add “among HIV-positive adults (>= 15 years of age) not on ART” to the end of the sentence, (i.e), change sentence to “Our meta-analysis estimated a 1.43 (95% credible interval: 1.16 – 1.88)-fold increase in TB incidence per 100 cells per mm3 decrease in CD4 cell count among HIV-positive adults (>= 15 years of age) not on ART” 4. Line 33-35: Add “among HIV-positive adults (>= 15 years of age) not on ART”, (i.e), change sentence to “Our analysis confirms previous estimates of exponential increase in TB incidence with declining CD4 cell count among HIV-positive adults (>= 15 years of age) not on ART, emphasizing the importance of early ART initiation to reduce TB risk. 5. Similar to comments 1-4, please change any reference throughout the paper from people to make it specific to adults. Thereby, the analysis and findings in this paper will not be misunderstood to be applicable to all people, including children and adults. 6. Line 43-45: Be clear there are a total of 1.8 million deaths from TB, of which 1.4 million deaths are among HIV- people and 0.4 million deaths among HIV+ people. 7. Line 154-156 and Figure 4: Why is the upper CD4 T cell count of 1000 cells/mm3 used? Can’t the curve be projected/extrapolated using the available data? 8. Line 293-294: Add “except for one study (Wolday et al. 2003)”, (i.e), modify the sentence to “BCG vaccination status was not reported except for one study (Wolday et al. 2003), and may itself have confounded results, either through variation in coverage or variation in efficacy.” 9. Figure 1: Update the box “Studies included in qualitative synthesis (n=7)” to “Studies included in qualitative and quantitative synthesis (n=7)”. 10. Figure 2 legend: Change “people” to “adults (>= 15 years of age)”. 11. Figure 3: Try the plot with X axis in linear scale instead of log scale for “Increase in TB incidence per 100 CD4 cells/mm3 decrease” -- This is likely to represent this data better for understanding. If the current plot (log scale) is a better visualization of this result, than leave it as is; if not replace it with the linear scale plot. 12. Table 1: Split the “Study description” column to “Study description” and “Conclusion”. That is, add a separate column for “Conclusion”, and move this corresponding text from the “Study description” column. 13. Table 1: Monge, 2014 study -- age range is > 13 years. Is the overall systematic review representative of >=15 years of age or >=14 years of age? 14. Table 1: Nicholas, 2011 study -- Name all the 6 countries in the “Country” column.

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.

Title: CD4 COUNT AND TUBERCULOSIS RISK IN HIV-POSITIVE ADULTS NOT ON ART: A SYSTEMATIC REVIEW AND META-ANALYSIS Review round: 1 Reviewer: 2

Basic reporting: in line 109 they say they excluded studies where the majority of HIV infections are known to be not HIV-1; I think it should be clarified to the reader what HIV-1 means. In line 194 it says "The studies used previous analyses" while I think it should say "The studies used in previous analyses" Experimental design: I think the purpose of the study is clearly stated. Methods are explained in such a way that allows other researchers to reproduce or further explore the topic. Validity of the findings: Theay clearly state how their findigs relate to previos results in literature, explaining where their findigs confirm what was reported before, and highlighting new possible paths for further understanding the topic. Additional comments: No additional comments

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.

Title: UNRAVELING THE STRUCTURE AND COMPOSITION OF VARADERO REEF, AN IMPROBABLE AND IMPERILED CORAL REEF IN THE COLOMBIAN CARIBBEAN Review round: 1 Reviewer: 1

Basic reporting: The ms "Unraveling a resilient reef: structure and composition of Varadero, an imperiled coral reef in the Colombian Caribbean" describes a community of organisms on a newly disovered healthy reef in close vicinity to the outlet of the Magdalena river, Colombia. It is unclear why the term "resilience" is used, which implies that recovery has taken place. Since the reef has just been discovered, it is unlikely that this can be established. Perhaps the authors like to suggest that the reef's good condition indicates that it has been offering "resistance" to local threats? The report is descriptive and mainly of local importance. The discussion could be made more interesting for a wide readership by including comparisons with other areas in the region or elsewhere of reefs in the vicinity of river discharge or in murky water (e.g. Amazon river, Singapore, Jakarta Bay, etc.). Are such reefs in other areas healthy or degraded? Have healthy reefs shown resilience? Experimental design: No comment. Validity of the findings: No comment. Additional comments: Additional remarks are mentioned on the ms pdf.

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.

Title: UNRAVELING THE STRUCTURE AND COMPOSITION OF VARADERO REEF, AN IMPROBABLE AND IMPERILED CORAL REEF IN THE COLOMBIAN CARIBBEAN Review round: 1 Reviewer: 2

Basic reporting: I found the manuscript to be well organized and clearly written, only finding minor grammar errors. The paper reports on the benthic (sponges, coral, and algae) and fish assemblages of a newly discovered, and apparently highly resilient coral reef inhabiting the highly polluted waters within Cartagena Bay. Please consider the following edits: 1) Within lines 30-33, including both the total number of sponge and coral species found at Veradero Reef. 2) Within lines 51-57, authors give examples of reefs found in extreme environmental conditions, yet there is not a single example of a reef being exposed to anthropogenic stresses similar to Veradero Reef. I recommend the authors to consider Appeldoorn et al., 2015 “Mesophotic coral ecosystems under anthropogenic stress: a case study at Ponce, Puerto Rico”. Experimental design: The experimental design as also well organized and clearly written. The survey design and statistical analyses for benthic and fish assemblages were correctly implemented. There are several additions and modifications that I would ask the authors to consider: 1) Line 89-91 Possibly rephrasing the sentence to/similar: Data from the GPS instrument was downloaded and analyzed using the GIS software Garmin BaseCamp, from which a detailed map of the reef was subsequently produced (Figure 1). 2) Line 94 Possibly rephrasing the sentence to/similar: Annotations of coral community composition at multiple depths was analyzed as in Geister (1977). 3) Line 95 – to emphasize and not confuse the reader, please include the name of the reef. “.. a detailed profile of Veradero Reef’s coral …” 4) Line 125 – replace the word “later” with the word “downstream” Validity of the findings: The results of the study are reasonable given the experiments performed. I congratulate the authors for including marine sponges within the surveys and analysis, they are usually left out of major and important survey efforts, even though they are an important component of any coral reef ecosystem. Additional comments: First and foremost, I found this paper extremely interesting, especially since it highlights the discovery of a reef which seems thrive in volatile and toxic environment. As the authors state, Veradero Reef should be protected, I believe that the Colombian government and its citizens should take pride in this newly discovered coral reef, and enforce protection laws against the destruction of this resilient coral reef by the proposed dredging of the Cartagena Bay. I do however, have several comments for the authors to take into account: 1) Line 190 – the genus Helioseris (correct) is misspelled. 2) Line 193 – the genus Undaria is no longer valid and should be replaced by Agaricia 3) Same for lines 194, 202, 207, 210, 211, 227, 232, 233, 4) Same for Figure 2 – Undaria should be changed to Agaricia 5) Figure 2 – genus Helioseris should be corrected 6) Line 225 – replace word “are” with “were” “..were also observed.” 7) Line 226 – start sentence with “In total, 38 coral species…” 8) Line 226 – after the word “identified” add the words “at this depth” 9) Line 235 – I am a confused by this sentence. The authors state that in total, there are 50 sponge species were observed at Varadero Reef, yet, there’s 38 species listed in parentheses. Maybe the authors meant to say that in total, “69” sponge species were observed? 10) Line 237-238 Proposed sentence: Survey transects at upper shallow terraces in Varadero Reef showed higher sponge species richness than that of upper shallow terraces in Baru Reef; 36 and 25 species, respectively. 11) Line 289 – see de Bakker et al. 2016 “40 Years of benthic community change on the Caribbean reefs of Curacao and Bonaire: the rise of slimy cyanobacterial mats” to possibly cite a decline in coral cover. 12) line 291 – a comma is needed within the reference after et al. 13) line 346 – I recommend switching the words “be not” to “not be” 14) Figure 4 – on the X-axis, change the word “Life” with “Live” 15) Within S1 – replace Undaria with Agaricia 16) Within S1 – Porites astreoides is misspelled 17) Within S2 – Amphimedon is misspelled

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.

Title: UNRAVELING THE STRUCTURE AND COMPOSITION OF VARADERO REEF, AN IMPROBABLE AND IMPERILED CORAL REEF IN THE COLOMBIAN CARIBBEAN Review round: 2 Reviewer: 1

Basic reporting: See previous review Experimental design: See previous review Validity of the findings: See previous review Additional comments: The paper has been improved. It reads very well. The illustrations are very nice. I like the artwork in figure 2. Because the ms is almost ready for publication, I congratulate the authors. However, I have found some minor issues for possible improvement. They are indicated in the word document. Based on two remarks in the margin, I have explained the importance of a hyphen. This is not always a matter of personal style. The authors did not take care of the reference list as suggested in my first review. In several references, the spelling of the names of authors is incorrect. In many references, the names of authors still lack initials. I have marked some examples. I have also checked the supplementary material. I suggest that taxon names in tables should be presented in alphabetical order.

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.

Title: MOTOR PERFORMANCE IN PRADER-WILLI SYNDROME PATIENTS AND ITS POTENTIAL INFLUENCE ON CAREGIVER’S QUALITY OF LIFE Review round: 1 Reviewer: 1

Basic reporting: The majority of the manuscript is clear and unambiguous. I have a few suggestions on grammar and language. 1. Please use "patient first" language throughout the manuscript. For example, line 54 should read "the child with Prader-Willi" instead if "the Prader-Willi child". 2. The correct term is "hand grip strength", not "hand gripping strength" (example, line 35). 3. Line 54: The statement that excessive eating begins in infancy is incorrect. Increased interest in food has a median age of onset of 4.5 years and hyperphagia 8 years (Miller et al., American Journal of Medical Genetics, 2011). In infancy most patients with PWS have poor feeding, with or without failure to thrive. 4. line 64 - change to "with an increase in body fat mass" 5. line 73 - change to "devote time and effort to care for them" Experimental design: 1.The researchers do not present an a priori hypothesis. They aimed to investigate the relationship between motor performance and primary caregiver QOL. There were 80 correlations examined in the analysis section. Was there a particular relationship that was of interest? Or was this an hypothesis generating exercise? 2. The question is interesting and attempts to address a knowledge gap. 4. The patient population was adequately characterized. 5. Validated measures were used. Validity of the findings: 1. The researchers analyzed 80 correlation coefficients and did not correct for multiple hypothesis testing or specify and a priori hypothesis/primary outcome. It is not surprising that 5 of 80 results had a p-value <0.05 as this approximates the expected false positive error rate. The conclusions presented in the discussion vastly overstate the meaning of these results. For example, you could perhaps conclude that these results justify a subsequent study to test the relationship between upper limb muscle strength in patients with PWS and their caregiver's bodily pain. It is not appropriate to conclude that better upper limb muscle strength in PWS lowers the intensity of caregiver's pain (lines 215-218). 2. Lines 240-246 -The authors state that a TUG test time <20 seconds indicates that an individual is independent for basic transfers. All PWS patients in this study had a TUG time <20 seconds so how can we draw any conclusions between the TUG test results and caregiver social relationships? 3. I suggest using a composite score from each of the 3 quality of life measures, rather than 16 subscales. If that's not possible, the conclusion should acknowledge the multiple hypothesis testing and need for further research rather than making strong conclusions based on these data. Additional comments: No additional comments

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.

Title: MOTOR PERFORMANCE IN PRADER-WILLI SYNDROME PATIENTS AND ITS POTENTIAL INFLUENCE ON CAREGIVER’S QUALITY OF LIFE Review round: 1 Reviewer: 2

Basic reporting: The authors address an important topic to Prader-Willi, namely, the impact of adult patients’ physical disabilities on their family/caregiver quality of life  There are a number of errors in grammar and syntax noted. As such, the manuscript would benefit from extensive professional and/or peer editing.  The introduction introduces the phenotype and disabilities of PWS, however misstates several particulars. On line 54, the authors state that during infancy the Prader-Willi child starts excessive eating. In reality the hyperphagia of PWS usually starts in school age children, not infancy (Genetics in Medicine, 2012, 14(1):10-26). In addition, PWS is not cause by the deletion of a “specific gene” as the authors state on line 59, but a specific gene region which includes a number of potential genes leading to the phenotype.  I would like to see the authors expand upon the knowledge gap being filled and be more explicit in stating their hypothesis and why this is important to know. Experimental design:  Authors state on line 166 that seven individuals were recruited but on line 167 only describe 2 females and 4 males. Table 1 shows 5 males and 2 females.  The authors state that all patients and their caregivers were recruited through the PWS Association. I am concerned that this may introduce significant recruitment bias as those who participate in such associations may be more engaged and adjusted to the difficulties of caring for family members with PWS. This bias combined with the small numbers in this study severely limit the applicability of these findings to the general PWS population. I would very much like to see additional study participants recruited through other means such as genetics or endocrinology clinic. Minimally, the authors should address this shortcoming in the Discussion.  The authors do a good job of outlining the various physical and QoL tests performed as part of the study.  I would like the authors to comment on the use of handgrip and lateral pinch strength tests in the PWS populations. Have these been validated as tests of upper limb muscle strength in PWS? Given the atypical hand morphology of PWS (brachydactyly with interdigital webbing and joint laxity) could these tests be interrogating hand dexterity more than upper limb strength in these patients? Validity of the findings:  In lines 210-218, the authors discuss the correlation between grip strength and caregivers’ pain as assessed by the SF-26 results. I would like the authors to expand on their discussion regarding these results. Why was there no correlation with the Pinch test? What domain of upper limb strength or manual dexterity is the Grip test assaying that the Pinch test is not? Are these results expected or not? Why or why not?  The authors correctly identify the limitations of this study in lines 255-260. These limitations severely curtail the general applicability of these results. Not only are the study numbers extremely small but there is no control group of any type. Are there similar studies that have been carried out in similar populations that the authors can contrast their results with? Other obese populations? Other disabled populations?  If these results were to be validated, what specific changes in patient care might be made to help mitigate these detrimental effects on caregiver’s QoL? Additional comments: No additional comments

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.

Title: COMBINING OCCURRENCE AND ABUNDANCE DISTRIBUTION MODELS FOR THE CONSERVATION OF THE GREAT BUSTARD Review round: 1 Reviewer: 1

Basic reporting: The study forecasted the spatial occurrence as well as the abundance distribution of Great Bustard based on the priority protection index (PI) by combining occurrence and abundance indices. The results show that the occurrence and abundance models display different spatial distribution patterns, and PI could guide the protection of the areas with high occurrence and high abundance. Generally, the MS did not tell me how to combine the occurrence and abundance indices, and display clearly spatial distribution pattern. Furthermore, only based on the distribution data of Great Bustard in Bohai Bay, the author confirmed that the findings have a wide relevance and applicability, worldwide, and it was difficult to be a global research template. This is strongly non-robust. In Introduction part, the author need to clearly show why many scholars found species occurrence and abundance distribution not to display similar patterns, conservation decisions based on SDMs predictions are insufficient and may even be misleading? And why one time-critical challenge and associated progress will be centered how to combine the useful information that SDMs and SAMs? And one of the objects of the MS was to assess and develop models to predict accurately the patterns of bustard occurrence and abundance. But in Method part, you just used the Random Forest model, no any information about the developing model, if you developed the model, please give detailed information of developing, if not, please describe your method accurately. Experimental design: I don’t know how you choose the eleven environmental variable? Why these variables, not other variable? And why eleven, not ten or eight, or other? This need more details. About the experimental design, you said “we travelled with a small four-wheel-drive tractor along fixed routes”, can you give the detailed information about the route line design context. Actually, this is key for your data robustness. Validity of the findings: Results are not well stated linked to original research question. In Introduction part, you mentioned the second object of the study was to predict accurately the patterns of bustard occurrence and abundance, but in Result part, you did not give clearly the pattern? Why? I suggest you describe your predicted pattern in the Result part. Additional comments: Line 25, best-available presence and count records for an endangered farmland species, Great Bustard (Otis tarda dybowskii) in Bohai Bay, China, as a case study. How did you proved that presence and count records for an endangered farmland species was best-available? And line28, how you know the method was a powerful machine learning method? Can you give some illustration? Line 30-31, You mentioned that the environmental variables influenced bustard occurrence and abundance differently. How the variables influenced? And you found that occurrence and abundance models display different spatial distribution patterns. Please give actually pattern, what pattern would be displayed? Line 36, only by your case study, how to confirmed that your findings have a wide relevance and applicability, worldwide. That means how to extend you scale from the local Bohai bay to worldwide scale? Line 91, The topographical and climate condition varies little in the study area. Can you give information about how varies little? Line 99, According to my knowledge, the bustard distributed widely, why you said Bohai bay is the world’s largest wintering ground ? Is it based on your research or other people’ research? And why is representative of the typical farmland situation in the North China Plain. Can you give actuately base or literature? Line 123-124, please give more detailed information about how to construct a distance layer for these variables using the Euclidean. Line 135-136, Please give detailed information that what range of NDVI representing good condition vegetation, and what range of NDVI represent the bed condition of vegetation, and why? In line 139-155, there are many expression need to be confirmed, such as advanced (line 139), robust (line 141), very good (line 142), performed the best (line 144), robustness (line 146), not so good (line 148), better (line 151), best (line 153). All of these evaluation were based on your own judgment but not citation or proof. I suggested you should rewrite this part. Line 47, you constructed initial abundance model, but you did not tell us how to construct the model? Line 158, you adjusted the simulation abundance, can you give more information about how to adjusted the simulation? Line 171, you extracted the habitat information, in line 172 you created a model file, and in line you adjusted the predicted abundance, can you tell us how to extract, create, and adjust? Line 182, what is better? Line 195, what is the more suitable and scientific? What is not suitable or scientific? Line 258, how do you know the habitat had a fragmented distribution? In Discussion part, some sentence is no cited literature, such as line 264, 366-367, and more. Please check.

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.

Title: COMBINING OCCURRENCE AND ABUNDANCE DISTRIBUTION MODELS FOR THE CONSERVATION OF THE GREAT BUSTARD Review round: 1 Reviewer: 2

Basic reporting: This research looks at an endangered species and proposes a way for better conservation management. In the manuscript, the authors have done a good job to illustrate their design, data collection, methods for modelling, discuss the results and future implications. The references are thorough and figures are informative. Experimental design: The design of the study is generally clear. However, the authors need to address the following questions. First, what is the reason and reference to use a linear regression to convert observation abundance to another? You just want better prediction or other reasons? Why not transform the abundance data before according to its distribution (zero-inflated)? Second, why choose random sampling for pseudo-absence? Do you control for the distance between presence and the random absences? Third, how do you validate your model? Do you have other independent dataset to validate it? Fourth, for this mobile species, for the same survey, how do you avoid counting the same individuals in different sites? Validity of the findings: The findings are useful in conservation planning, especially the way to combine two different kinds of information: presence and abundance. The author could illustrate this findings more, what is the consequences of just using species distribution model or abundance model compared to using the combined result? You can threshold the two maps and compare the differences. Additional comments: It is a good study. I have put other comments for the wording of this paper. Please address this issues and work on the language more carefully. Line 35: spell out the rel. Line 73: Delete “Farmland”. This species habitat contains both grassland and farmland. Line 101: delete “situation” Line 105: delete “winter survey” Line 115: How do you avoid record same individuals during the survey? Line 125: change spacing to pixel size. Line 156: Is there any reference for this method (using linear regression converting observation to simulation abundance)? Line 166: There are various ways to choose pseudo-absence. Why use random sampling? Do you control the distance between absence and presence? Line 210: rephrase the sentence. Not clear what you want to say. Line 215: rephrase this sentence. Line 220: What is multivariate package? Not sure what it refers to Line 263: change “!” to “.” Not common to use “!” in academic paper. Line 276: Please rephrase this sentence. Can’t understand what you mean. Line 309: You jumped around in this section. The author talked about the birds then jumped to plant and crop later. Please make clear transition. Figure2. Change the x axis title for b. Is it observation ID?

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.

Title: COMBINING OCCURRENCE AND ABUNDANCE DISTRIBUTION MODELS FOR THE CONSERVATION OF THE GREAT BUSTARD Review round: 2 Reviewer: 1

Basic reporting: The revised MS solved the issues I have proposed. Experimental design: The authors have given detailed informations about the chose of the eleven environmental variable. Validity of the findings: The conclusions have been stated appropriately. Additional comments: The authors have replied my questiones detailed.

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.

Title: IN SILICO IDENTIFICATION OF OFF-TARGET PESTICIDAL DSRNA BINDING IN HONEY BEES (APIS MELLIFERA) Review round: 1 Reviewer: 1

Basic reporting: The article could be significantly improved by reviewing the use of jargon and revising unclear sentences. A few additional references are recommended. Experimental design: The authors state in the materials section that search terms included “siRNA,” “dsRNA,” “RNAi,” and “RNA interference.” In the Conclusion, they say outcome can matter depending on "whether the organism is exposed to siRNA or dsRNA". This implies that there is a meaningful distinction that is not made clear in either use of terms or methodology. Validity of the findings: With clarification of the issue above, I have no further comment. Additional comments: The paper uses a lot of jargon and that also makes some sentences difficult to understand even for a reader familiar with the jargon. Please reconsider using the word homology or its derivatives (eg, homologies) when referring to quantitative relationships between DNA sequences. Homology is a conclusion of relatedness by descent and not a synonym of similarity nor a quantitative term (Fitch WM. Homology a personal view on some of the problems. Trends Genet. 2000;16:227-31). There is no ‘degree of sequence homologies’ just as there is no ‘degree of pregnancy’ and no information is lost saying ‘degree of sequence similarity’ instead. Sequences may be similar because they are homologous, or similar because they are similar no matter their histories (e.g “random sequence similarities” (line 156-7)). It doesn’t matter why they are similar for this manuscript only that they are. I know that others do it, but such uses are eroding the precision of the term. In fact, given that taxonomy is not a predictor of off-target effects, the use of the word ‘homology’ is particularly troublesome. 33-35 This sentence could be expressed as: “The target gene sequences for these pesticidal RNAs were determined, and the degree of similarity with sequences in the honey bee genome were evaluated statistically.” 37-8 “The likelihood of off-target sequence homology increased with the parent dsRNA length.” Please reconsider this jargon. I suggest “The likelihood that off-target sequences were similar increased with the number of nucleotides in the dsRNA molecule.” What after all is the parent dsRNA (also line 137)? Is it the active pesticidal RNA? 38-9 The dsRNA doesn’t bind to the gene (as in DNA). Instead: “Non-target binding of the dsRNA was unaffected by taxonomic relatedness of the target insect to honey bees…” 42-43 Sequence similarity is not enough to predict biological effect, but likewise there are no bioinformatics ways to ensure that a dsRNA will have no effect, too. E.g. because of secondary dsRNA generation in exposed insects, the primary dsRNA may not be the active molecule in the honey bee. This sentence seems to imply otherwise. 57-59 This sentence describes two methods of delivery but likely readers will be unfamiliar with both, especially since to my knowledge there are no commercially available sprays. Consider introducing appropriate references for both. 59-63 This is true if the dsRNA is longer than the active molecule. It does not have to be. It is possible that plant processed or encapsulated dsRNAs may already be 19-25 nucleotides and not require further processing in the insect. 73-74 This is a grammatically challenged sentence. Gene sequences aren’t silenced, genes are. Please revise to something like: “within a non-target species, this unintentional gene silencing can be due to silencing the intended gene in the unintended organism (non-target binding) or silencing a different gene with sufficient sequence similarity to the dsRNA (off-target binding)” 81 What does “even when sequence homology is identical” mean? Does it simply mean ‘even when sequences are identical’? 79-82 This sentence contradicts many reports in the literature that are more recent than 2004. E.g. Hanning JE, Saini HK, Murray MJ, van Dongen S, Davis MPA, Barker EM, et al. Lack of correlation between predicted and actual off-target effects of short-interfering RNAs targeting the human papillomavirus type 16 E7 oncogene. Br J Cancer. 2013;108:450-60. Unless the authors have other references, this sentence should be removed. The reason bioinformatics analyses have been advocated is not because (82-83) they can avoid false negatives but because they are a starting point to test putative positives. Heinemann JA, Agapito-Tenfen SZ, Carman JA. A comparative evaluation of the regulation of GM crops or products containing dsRNA and suggested improvements to risk assessments. Environment International. 2013;55:43-55. 160-1 Well said. Another good reason to use homology correctly. Moreover, with sprays, non target organisms don’t have to be animals. 162-170 This paragraph is incomplete without discussing secondary dsRNA products. Pak J, Fire A. Distinct populations of primary and secondary effectors during RNAi in C. elegans. Science. 2007;315(5809):241-4. Sijen T, Steiner FA, Thijssen KL, Plasterk RHA. Secondary siRNAs result from unprimed RNA synthesis and form a distinct class. Science. 2007;315(5809):244-7. Please avoid associating predicted binding with actual binding, as in (line 174-5) “67% of all tested dsRNAs had off-target binding with developmental genes in honey bees” This is more accurately “had the potential bind to honey bee developmental genes that were not the target”. In fact, binding is not the key criterion because one base pair is enough to satisfy binding. Some sense of the strength of the binding should also be imparted to the sentence. 178-9 It is not correct to say that a gene ‘performs’ cell proliferation or apoptosis. 180-2 Yes, but in saying that “in silico analysis identified potential gene targets that could present a hazard” it is important to also say that it is not possible to confirm that all or even most potential unintended targets were identified. This is the first line of the conclusion section, but that is too subtle. 189-192 RNAi is not just ‘gene knockdown’. There are 3 manifestations from RNA degradation (involving siRNAs that have 20-21 nt matches usually in the coding region) to translation inhibition (normally when miRNAs are used, ie, those with 2-8nt in the seed region) and RNA directed methylation. The discussion perhaps would benefit from spelling this out. 191-197 Hanning et al (above) would be a good reference for this paragraph. 200-2. Not if it is a spray. 202-3 What is the difference between what you mean by siRNA and what you mean by dsRNA? The latter is usually the generic for si/mi/shRNA etc. Do you mean processed dsRNAs when you say siRNA? Are you talking about the guide strand? 203-7 And secondary dsRNAs.

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.

Title: IN SILICO IDENTIFICATION OF OFF-TARGET PESTICIDAL DSRNA BINDING IN HONEY BEES (APIS MELLIFERA) Review round: 1 Reviewer: 2

Basic reporting: GENERAL The paper fulfills all criteria for professional scientific publication: clear and well written, literature references and structure of paper is professional. This paper offers important information for risk assessment of an emerging new biotechnology with broad application but also risk potential for insects. The authors carried out database research for published sequence information to determine the degree of homology between target gene sequences of pesticidal active RNAs in a nontarget organism, the honey bee. Honey bees served as an iconic case example of nontarget species with critical socio-economic and ecological functions. Off-target effects of any pesticidal substance must be avoided. The authors tested and validated a new methodology for improving the current risk assessment for nontarget effects that are not geared to assess these new biotechnologies. Therefore, I consider this work very important and recommend publication. The research method is well described and carried out, the results are very interesting clearly suggesting the potential for nontarget effects in honey bees to occur if they come into contact with the RNAi. Whether or not that will happen depends on the form of delivery of the RNAi - in-planta or as sprayable formulation - and on timing of the application. But the first step is to determine whether there are any sequence homologies and the authors clearly determined that this is the case. Hence, now exposure assessments must follow and should be carried out prior for assessing the full risk potential. All of this definitely merits publication. My further comments address issues that are not critical to the decision of publication but are for the authors to decide to include if they agree with me that it will improve the paper. Since page numbers are missing I can only indicate line numbers. INTRODUCTION Line 67. The authors mention here 'current risk assessment framework' - personally I would appreciate if the authors would not only focus on the US but also include references to the Cartena Protocol Risk Assessment requirements or EU regulatory risk assessment (mandatory for over 160 nations in the world). The US is a singular case having the most lax standards re risk assessment of biotechnology. This might be used as an excuse to reject the applicability of this work/paper to other regulatory systems of biotechnology as being more stringent. However, none of the other regulatory risk assessment schemes in the world would fully caputure these new risks and, therefore, this work is definitely also of relevance beyond the US. Line 76, last word 'unique' - I suggest the term 'different' instead - and again, also here, reference to other regulatory systems are highly recommendable, so this work has relevance beyond the US. MATERIALS AND METHODS - see below in next section RESULTS AND DISCUSSION Line 153 onward - relating to 'taxonomic relatedness' as an indicator or a criteria for selecting potential nontarget species at risk. This issue drops a bit out of the air, although there is a well-known precedent. This argument for selecting potentially affected nontarget organisms for testing based on their relatedness to the target pest species as indication for susceptibility has long history in the risk assessment discourse of GM Bt plants. And there it has already or also been shown to be a weak indicator for selecting potentially affected nontarget organisms. In general, I find the authors could connect their discussion and paper in general to the on-going discourse about the already existing GM crop plants. Some issues are indeed the same or connect in other ways. And there are several references that could be cited here in support. One issue that is still puzzling to me is on the other hand the fundamental difference in these pest control strategies that have not been properly pointed out - at least to my knowledge which is admittedly limited. It is in my view a fundamental difference if a sprayable pesticidal substance is a low molecular weight chemical or protein (in case of Bt toxins) affecting biochemical metabolic processes or life support systems such as nervous signal transmission or breathing apparatus of insects versus this form of pest control where it targets the genetic machinery. I think we have to think about specificity in completely different way whether it targets biochemical pathways or gene functioning. In particular, in my view, this is argument to broadly reject the above mentioned issue of 'taxonomic relatedness' to be in any way indicative of susceptibility. All organisms share a very similar genetic machinery - hence, all organisms are potential nontargets in a much more broader way than it could be argued with a chemical disrupting certain biochemical processes that are unique to say a kingdom of organism or other fundamental categories of organisms (e.g. photosynthesis in plants, breathing in insects vs mammals, aquatic vs terrestrial organsims etc.). I find that this paper may be a good one to at least raise these issues or point to references where these have been discussed - if at all. The authors are approaching these fundamental questions a little bit in an ad-hoc fashion in the last paragraph of their discussion (before conclusions) within the discussion about what are highly conserved genetic regions in metazoans or not. I think, here a more fundamental discussion along the lines I outlined above would be quite suitable and add relevance of this paper for the general discussion about risk assessment of types of biotechnology applications. Experimental design: MATERIALS AND METHODS All is well described but I think the process could be explained better. Lines 92-94 - the authors list here the search terms for their database search. Did they not include the term 'honey bee' or 'Apis mellifera' or terms refering to target- or nontarget organisms? Later on they list the various species they found published studies for. This means they only looked for anything published on RNAs for pest control and then assessed as part of their analyses which the target species were? And then they set out to compare these (i.e. all RNA applications they found) against the published genome sequences of the honey bees? This process should be explained a bit better, in particular, for readers who are not familiar with this pest control technology. Validity of the findings: Data is robust and well presented and highly relevant to the field of risk assessment of biotechnological applications and products. Conclusions are supported by the data shown and linked to the objectives and research questions. In fact, I suggest that the authors broaden their discussion and connect it to an international discussion of regulatory risk assessment requirements to make it clearly relevant beyond the US. Additional comments: No additional comments

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.

Title: EVALUATING THE ROLE OF WILD SONGBIRDS OR RODENTS IN SPREADING AVIAN INFLUENZA VIRUS ACROSS AN AGRICULTURAL LANDSCAPE Review round: 1 Reviewer: 1

Basic reporting: The English is clear and professional, the background information and literature are thorough and appropriate, the structure and figures are appropriate. I could not view the raw data file -- it was all symbols for me. I tried on two computers and try to open it as a text file or import it into Excel. Experimental design: Experimental design: Original primary research is reported, the research question and motivation are clear, the investigation is rigorous and conforms to ethical standards, and the methods are clear and well-described. Validity of the findings: Validity of the findings: Findings are robust, conclusions are well-supported, and speculation is clearly identified and appropriate. Additional comments: The manuscript describes a survey of potential bridge hosts (primarily songbirds and small mammals) to investigate the potential role these species might play in transmitting influenza A viruses from reservoir hosts to poultry. The survey is conducted in Iowa at several poultry and wetland sites during both fall and spring after a large poultry outbreak in the state. Samples were tested for influenza A virus and antibodies and no positives were detected. The manuscript is well-written and very clear throughout. The background information and discussion are very thorough and well documented. I only have a single comment that I think should be addressed (and even it is relatively minor): Line 311 - The authors used a cut-off of 0.6 rather than the manufacturer’s recommended cut-off of 0.5. While the higher threshold is appropriate (and supported by Brown et al. 2009, Clin. Vaccine Immunol. and Shriner et al. 2016, J Vir Methods), the authors use the manufacturer’s sensitivity and specificity values in their analysis of a seroprevalence range. Since the manufacturer’s sensitivity and specificity values are based on a cut-off of 0.5, I recommend that the authors should either apply a cut-off of 0.5 to match the manufacturer’s sensitivity and specificity values (this is the easiest solution since it won’t change results) or they should adopt the sensitivity and specificity values that match the 0.6 cut-off (found in the Brown et al. or Shriner et al. papers). Minor comments Line 55: The authors might consider replacing “epidemic” with “epizootic” throughout the manuscript. Line 85: The authors might be a bit more clear about what they mean by “cyclical” in this sentence. In Ref. 35, I believe the cyclical nature of natural infections is associated with underlying immunity to a particular subtype whereas in Ref. 36, I believe the cyclical nature of infection discussed is the seasonal cycle of infection prevalence. Line 103: I would quibble that this statement may be generally accurate in Iowa, but that natural ponds frequented by waterfowl are not uncommon on or near poultry facilities in other geographic areas (e.g., MN). Also, NPDES ponds attract waterfowl, even in Iowa. Line 111: Recommend deleting the “of.” Line 140: Recommend adding the word “a” before the word “given.” Line 165: The authors might consider changing the “to” to “in.” Line 181: Use consistent capitalization for bird common names. Consider adding scientific names at first occurrence of species in the text. Line 216: I would suggest that variation is dependent on both species and viral strain. Line 221: Did the micro centrifuge tubes contain an anticoagulant (i.e., EDTA or heparin)? Line 367: The authors might consider noting that none of the APHIS/DNR positives for LPAIV occurred in counties where they sampled bridge hosts. Did any of the 3 APHIS/DNR negative counties overlap the counties where bridge host sampling occurred? Line 387: Technically the IDEXX assay tests for influenza A specific antibodies and is not specific for avian viruses. PeerJ does not include funders in the Acknowledgements section.

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.

Title: EVALUATING THE ROLE OF WILD SONGBIRDS OR RODENTS IN SPREADING AVIAN INFLUENZA VIRUS ACROSS AN AGRICULTURAL LANDSCAPE Review round: 1 Reviewer: 2

Basic reporting: The paper is clear and the hypothesis is reasonable. I believe that the negative results are important. The background abstract and background for the study are appropriate. However, the material on surveillance in waterfowl appears as an afterthought. The reader is surprised when the section appears in the methods and the results are barely discussed. Either this section should be removed and the context of influenza prevalence in the areas surveyed are established through published literature. Or, properly describe and analyze the results from waterfolw. What subtypes were present? Were any of the samples isolated? Etc. Experimental design: The design is fine. I think for the results to be truly conclusive longitudinal surveillance is necessary. But this does provide some important data that is relevant to influenza introductions to and spread among poultry populations. Validity of the findings: no Additional comments: After the high path outbreak was detected in farms a number of hypotheses were put forth 1) wild birds were spreading and introducing viruses to each farm experiencing outbreaks. 2) viruses were being spread by fomites. 3) viruses were being spread by other minor hosts such as small mammals and song birds. 4) viruses were being spread by humans passively (tools, workers, trucks etc). It's important that we have data so that we can reject those ideas that are not supported in order to properly identify transmission pathways and effective control measures. I am happy that this manuscript attempt to address some of the spurious or unlikely hypotheses. I recommend that the results about the surveillance in waterfowl be either removed or extended. These results are not treated appropriately, nor is their relevance to the rest of the results clear.

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.

Title: EVALUATING THE ROLE OF WILD SONGBIRDS OR RODENTS IN SPREADING AVIAN INFLUENZA VIRUS ACROSS AN AGRICULTURAL LANDSCAPE Review round: 1 Reviewer: 3

Basic reporting: This study investigated the role of potential avian and rodent bridge hosts for Avian Influenza viruses (AIV) soon after the outbreak in poultry farms of HPAI H5N2 in 2015 in Iowa, United States. The study design focused on sampling these potential bridge hosts at both wetlands where the AIV reservoir occur (i.e. waterfowls) and at poultry farms where HPAI outbreak happened or could have happened. qPCR and serology diagnostics were implemented on both rodent and bird sampled with no positivity detected in the 449 animal sampled. A point to be noted is that mechanical transmission was investigated by swabbing the external body of animals sampled. The authors also compared the community composition of the samples from the wetland and farm sites to identify which species may be more involved in bridging wetland and farms from an epidemiological point of view. In the discussion section, these negative results are discussed in relation to positive results found at the same time in waterfowls in the same state. The authors conclude that the species sampled may not be involved in transmitting AIV from waterfowl to poultry farms and that human induced transmission maybe more involved in farm-to-farm transmission. The article is well written, hypotheses are well stated and tested. The literature references are up to date with the scientific field. I appreciated the approach and the study design and have only minor comments about the study design and some points in the discussion that could be improved and/or further discussed. Experimental design: - L162-170: site selection: the logic behind the site selection is not completely stated. You have 2 “pairs” of sites including a wetland and a poultry farm and other sites (wetlands and farms distant from each other). I guess there must be some constraints or other factors that drove site selection but they must be indicated. For example, why choosing the Malcom site outside any “coloured” cell? And why not in the single orange cell? - L236-247: It is difficult to understand exactly if the waterfowl sampling is part of this study or not. You give a sample size and an AIV prevalence but little other information is available about the sample composition (in terms of species for example). If this information cannot be presented in this manuscript because it was collected by another team, I think this should be clearly stated and only the relevant information should be presented. If the information was collected by the authors, then more information should be given. Did those samples were pooled like the ones for the songbirds? - L331-337: I think you need to state that you assume that the community sampled is representative of the bird/rodent community present. It is necessary because you make this assumption without stating it and without discussing it in the discussion section (see below). Validity of the findings: The findings of the study are relevant for the field, well presented (see below a couple of suggestion for improvement) and will inform further AIV wild bird and rodent surveillance in the United States and beyond. - Figure 1: Maybe use different symbols for wetland and poultry farm sites in order for readers to automatically detect them without referring to the text. - Table 2: I think it is important to present at least the confidence interval of the prevalence estimation for each species (for the total of each species). Despite all your efforts, the sample size is often low for each species and therefore the confidence that AIV was not present in the species’ population is sometimes weak. Additional comments: - L.70: but see also a recent publication: Caron et al. 2016 in Journal of Applied Ecology. This publication could be used elsewhere in the manuscript as well (“songbirds have been found to be capable of carrying AIV” – L105-106). - L.106-108: in case you have not seen them and not to be cited at any cost (and sorry for self-citations but little has been done unfortunately on the topic) but consider also Caron et al. 2009 in Infection, Genetic and Evolution and Caron et al. 2010 in Ecology & Society looking at a conceptual approach and a more risk-based approach respectively to your study design. - L407-417: I agree that you have identified “potential bridge species” within your sample that can connect wetland and poultry farm. But you should discuss at least 2 points. First, the scale of your study is larger than what I would call an ecosystem scale where you sample birds from adjacent wetlands and farms (a few kilometres). This is the case for two pairs of sites but you analyse your results globally across all sites. So maybe that species detected at one wetland were identical to species detected at a distant farm but not at a close farm. In fact, you assume homogenous distribution of species across your study site and should discuss it further. So the risk of AIV transmission from a wetland to a farm is maybe not “real” at all your sites. Second, you only take into consideration the community of your sample and not the “real” community present at site. This should be stated clearly. Capture techniques, time and season do not randomly sample within the bird community. There is a possibility that your potential bridge species (present and abundant at wetland and farm sites) are not the most abundant and shared species between wetland and farm sites within the “real” wild bird community. - Given your approach, you still largely sample blindly within the bird/rodent communities. Estimating rodent community is difficult but bird communities can be characterised through bird counts. You should maybe mention that this could be a possibility to improve your protocol if time allows (which can be tricky in the case of an outbreak). - In the discussion, you should also mention that your species sample size is low for most of the species sampled and therefore that the absence of AIV detection does not prove absence of circulation in those species. L449-452, you mention high sample sizes to reach good estimation of prevalence, but you should also say that at the species level, your sample size is often extremely low and cannot estimate much. You rightly say that further sampling should target (capture techniques should be adapted) identified potential bridge species with the objective to achieve a better sample size. L413-417: you should specify that more targeted sampling could improve our understanding of some of the potential bridge species identified. - Finally, in the last section of the discussion, you qualitatively assess the risk of spread of AIV from wildlife to poultry and vice-versa. Maybe you should also emphasize that an outbreak can be triggered by a rare event and then subsequently spread between poultry farms by farm-to-farm contacts. So AIV prevalence in bridge hosts, even if very low, represents a risk with potential high consequences. - Conclusion: L478-480: can you really say this? I would say that your study did not detect AIV in potential bridge hosts and that it supports the hypothesis that they don’t play a role in AIV epidemiology in Iowa.

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.

Title: EVALUATING THE ROLE OF WILD SONGBIRDS OR RODENTS IN SPREADING AVIAN INFLUENZA VIRUS ACROSS AN AGRICULTURAL LANDSCAPE Review round: 2 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: Great revision; I have no substantive comments or recommended changes; the following comments are meant for discussion only. Line 214: I don’t think a change is needed, but want to point out that the Spackman et al. paper only compared BHI and PBS, neither with antibiotics which can be important, especially for cloacal/anal swabs which are likely to have strong bacterial communities. I know optimal is in the title of the paper, but this seems like an example of making inference beyond the data set, especially since your samples were not stored in an ultracold prior to shipping to the diagnostic lab. Line 249: The “As with samples for small birds and mammals,” is a bit misleading because I think the APHIS surveillance uses 3mLs of BHI whereas you used 2mLs. You might just strike the beginning of the sentence or specify the 3mLs (assuming I’m correct about the 3mLs). Line 277: if you were using an H5N9 isolate, you might mention that the extractions were conducted under enhanced BSL-2 or BSL-3 conditions/in a biosafety cabinet. Line 285: AB? Standardize capitalization of influenza: it is only capitalized in Lines 394, Table 3, and Line 411. Also there is a bit of inconsistency in references (some titles are capitalized, but most are not). Also, in Table 3, remove capitalization of Americana for redhead. Line 415: Although I this is a unreasonable assumption, there are very scant data available on long-term AIV antibody persistence, especially for non-traditional hosts and a single infection. A potential difference between the studies you cite and your study is that geese are long-lived and are likely to have multiple exposures (resulting in an anamnestic response), even within a given year. Multiple exposures increase peak detectable antibody responses and the duration of the response so the 3-6 month estimates may be on the high side for the animals you are sampling. Line 457 – A very minor point, but the 16% might be biased a bit high. I don’t know if you had access to the sample dates for the APHIS data you used, but the APHIS online data (across the US, not just IA, https://www.aphis.usda.gov/animal_health/downloads/animal_diseases/ai/monthlysummary.pdf) show much higher prevalence in August and September than the months during which you sampled so if you didn’t adjust the prevalence for the time period that overlapped your sampling, the 16% might be on the high side. Line 496: seems like there might be a missing “to” in this sentence?

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.

Title: EVALUATING THE ROLE OF WILD SONGBIRDS OR RODENTS IN SPREADING AVIAN INFLUENZA VIRUS ACROSS AN AGRICULTURAL LANDSCAPE Review round: 2 Reviewer: 2

Basic reporting: Introduction - L61-63 : first sentence. The impression left by this sentence is that waterfowl are the culprit. Maybe adding something like “(…) can cross the wildlife-domestic animal interface and find optimal conditions to evolve towards higher pathogenicity in poultry production systems , sometimes (…)”. - L86-87: “most poultry farms now enforce strict biosecurity protocols”: yes… in developed countries. Maybe contextualise here. - L101-102: I don’t think this sentence is necessary there as you repeat it at the end of the introduction. Experimental design: - L326-328: are these tests been developed for waterfowl? Are they species sensitive? You should maybe specify this. Validity of the findings: - The fact that your confidence intervals don’t include zero is a problem for me. You should explain more the reason why, maybe in the M&M section. With only zero results, you cannot have confidence interval above zero, it is impossible within my logic. This could be an artefact of the Bayesian method but at the end I don’t see how all of your results have a non-including zero CI. - The discussion has been improved taking into account the various pointed out in the previous revision. Additional comments: Most of my previous comments/remarks have been addressed. I have only a few minor points to be a considered below. I believe the manuscript has reached the standard of publication.

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.

Title: SHIFT OF SYMBIONT COMMUNITIES IN ACROPORA TENUIS JUVENILES UNDER HEAT STRESS Review round: 1 Reviewer: 1

Basic reporting: 1. The ms is clearly written. Literatures and background are well provided. Figures are acceptable. Experimental design: Research question well defined and it is stated how this study fills an identified knowledge gap. Investigation performed to a high technical and ethical standard. Replicate should be described more clearly (see the general comments for the authors). Validity of the findings: Data is robust, statistically sound, but probably small mistakes in description of the results (see the general comments for the authors). Additional comments: 1. The authors wrote in the Introduction that they studied changes in Symbiodinium communities associated with A. tenuis juveniles for the long-period of 1.5 years (line 97-98). But this is misleading, since actually most experiments finished in 2 or 4 months and only small numbers of juveniles survived up to 1.5 years. 2. The authors described that the range of seawater temperature in tanks (the first paragraph of the Temperature treatment section). Do the values mean daily minimum and mean daily maximum temperatures ± SD for 2 tanks in 2012 and for 3 tanks in 2013? Please explain this more clearly. 3. They compared prevalence of Symbiodinium types among temperature treatments. Is P=0.028 for D1-4 after 4 months not significant (line 251)? What is the level of significance used in this study? Is P=0.006 for A3 (0 day) not significant (line 259-262)? Please check. 4. In 2012 experiment, there was a significant difference in the prevalence of the three Symbiodinium types among temperatures. Still the authors describe the changes of each Symbiodinium type at all temperature conditions together (line 243-248). 5. In 2013 experiments, they found no significant effects of temperature conditions on the prevalence of Symbiodinium types. But they described changes in the prevalence of each type separately at each temperature (line 255-259). It is confusing. The meaning of the latter half of the sentence (line 257-259) is not clear. 6. When they state that type C1 decreased under ambient and 32oC, but increased at 30oC (line 255-257), were there significant differences in the prevalence among different sampling time point? 7. They used two tanks for each temperature condition in 2012 and three tanks in 2013 experiment. Do they use individual juvenile as statistical unit regardless of tanks? They described in the method section that 10-20 juveniles were sampled from each tank at each sampling point. But, if they show the number of replicates in the legend of Figs. 2, 3, and 4, it would be clearer. 8. In the discussion, they state that the observation by Abrego et al. (2009) explains why none of the juveniles in this study harbored C3 Symbiodinium. But it is not clear why? Please make it clear. (line 378-380) 9. They state that similar changes within the symbiont community were observed at higher temperature during later stages (line 366-369). But increase of A3 Symbiodinium as seen under the ambient conditions was not observed in high temperature conditions during later stages (Fig. 3). Please clarify this sentence. 10. The authors stated that colonies harboring only D1-4 increased under ambient (60%) and 32oC (52%) conditions (line 280-282). Under ambient condition, the red bar (D1-4) appears to be about 30% at 2mo (Fig. 4b). Please check whether the sentence is correct. 11. The readers might want to know whether bleaching occurred or not during the high temperature treatment. Small errors 1. Acropora longicyathus instead of longicythus (line 76) 2. Citation of Yamashita et al. (2013) 3. Line 372-375, some words are missing. ‘, which’ can be inserted after ‘Pacific’. 4. Line 477-478. Reference source is not adequate. 5. Change instead of ‘chenge’ (line488) 6. Proceedings. Capitalize the first letter (line 541)

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.

Title: SHIFT OF SYMBIONT COMMUNITIES IN ACROPORA TENUIS JUVENILES UNDER HEAT STRESS Review round: 1 Reviewer: 2

Basic reporting: The manuscript titled “shift of symbiont communities in Acropora tennis juveniles under heat stress” by Yorifuji et al” deals with multiple symbiodinium type association in juvenile coral and how this influences their response to high temperature stress. Authors have carried out the work using the coral A. tenuis, over a period of 2 spawning seasons, effectively repeating the experiment twice. Specific comments 1. Basic reporting A. I recommend the authors to check carefully the English again, I find few mistakes in grammar and usage but this can be easily solved. B. In the introduction, where you introduce Symbiodinium types, please use the scientific names for Symbiodinium that have already been assigned one. For example, type D1a Symbiodinium trenchii C. Background review and references are sufficient D. Figures are OK, raw data has been shared E. I don't see any hypothesis so I cannot comment on that. But the manuscript is self-contained with relevant results Experimental design: 2. Experimental design The research presented is within the Aims and Scope of the journal A. I have some difficulty in understanding the experimental design. it would be nice if authors can illustrate their experiment, B. Why you think 31 ºC is stress temperature? From the value you provided for your ambient seawater temperature range, the highest is already 30.82, and 30.93. How long was this high temperature in the tanks, assuming it to be during summer months? was there any effect on those control juvniles during this time? C. When you say 250-400 juveniles for each temperature conditions, does that mean that all your 2-3 independent tanks for each temperature had 250-400 nubbins? When i see the raw data, it becomes clear that you pick up random number of juveniles for each tile for each treatment to do the analysis. This is not explain in the main text very clearly D. In your raw data, for growth and survivorship, you show for each temperature treatment there are 2 sets of 8 tiles from which you picked up certain number of individuals which amounts to more than 30 individuals for each. However, your Symbiodinium data does not have that many number of juveniles analysed for each treatment. Why so? In the text, line 142, you say 10-20 individuals were collected randomly for the tiles in each tank…..i assume this number is collective sample form 8 tiles, if so, why your raw data shows more than 20 individuals? E. Line 165 is not clear. Please rewrite it 3. Results A. You say in 2012, the observation continued up to 1.5 years, however this does not appear in any of the figures. You do have raw data in the supplementary, why? B. Your results show large difference between 2012 and 2013, why? C. You used the same parental colonies for both the experiment. Response to temperature (ambient and 30 ºC is different between the years. Do you think this might be due to parental effect? I don’t know hoe about the cross between colonies and which colonies sperm fertilised which colonies egg? Paternal and maternal effect also influences the ability of larvae and juvenile to overcome stress. Why do you think that the response you observe in juveniles is solely due to Symbiodinium? why not the host itself? irrespective of what Symbiodinium type is present As you see from your results, there is large variation, especially in 2013, the variation is so big both in prevalence of Symbiodinium types and also D type id always there, so are other types. D. How come there is no adult Symbiodinium type in juveniles? even at 1.5 years? If so, do you have any idea at what age the juveniles have Symbiodinium same as Adults? - in Okinawa, because as you mention in Discussion (line 375), it is by 1.5 years in the GBR. D. D1-4 is outdated nomenclature for Symbiodinium. Please update. Please see the recent publications by LaJeunesse group Validity of the findings: This will be an important contributions in the field of Symbiodinium shuffling in early life stages of corals and how this might be related to the ability of juveniles to overcome stress. However, I do want to say that, the process of resistance in corals to stress is not solely dependent on the type/types of Symbiodinium it associates with. I feel that the authors opinion is biased towards Symbiodinium effect. Unless it is proven for sure that the Symbiodinium play the sole role in coral stress resistance mechanism, it is not fair to go in this direction. I am not forcing authors to think in any direction, but may be try to be neutral and put forth both possibilities? there are many papers to support both aspects. Even from the results of this work, one cannot conclude that Symbidinium shuffling as stress resistance mechanism. Because, the data is from random individuals collected out of hundreds. This might lead to underestimation since the percentage of types in each individual vary. Also the death of juveniles through time could be due to natural process and finally very few survive and then you see certain Symbiodinium type to be dominant and think that the survival is due to the presence of such type/types, but this might not be the case. Again from the data presented in this manuscript (line 275), in 2013 50-70% of juveniles at all temperature associated with A3 or C1 in the beginning and then S. trenchii increased under ambient and 32 ºC. This is good example of random changes. Also when you say that (Line 285) “A greater proportion of coral colonies (this is “Juveniles” and not “colonies” right?) that harboured only S. trenchii survived under 31/32 ºC compared to other treatments”. How do you know this? every time you sampled, you sacrificed the juveniles, there was no way to monitor the type composition in the the juveniles throughout and in the end those survived had S. trenchii, but is it not possible that even those that died also could have been associated with S. trenchii? So, the reason for increase in the proportion of S. trenchii at later stages of the experiment might have been due to increased mortality of juveniles harbouring different types of Symbiodinium including S. trenchii? Additional comments: "no comment"

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.

Title: SHIFT OF SYMBIONT COMMUNITIES IN ACROPORA TENUIS JUVENILES UNDER HEAT STRESS Review round: 1 Reviewer: 3

Basic reporting: This manuscript addresses the changes in symbiont communities in Acropora tenuis juveniles under heat stress, over a period of four months in one year, and over a period of two months in another year. The study found the prevalence of Symbiodinium type D1-4 and the number of juveniles harboring this type to be higher under heat-stress conditions after 2-4 months. The authors did a thorough search of the literature to provide a very detailed introduction and the methods were mostly clear and, again, very thorough in providing all the relevant information of how the experiment was set up and how analyses were conducted. There are some spelling and grammatical errors throughout the manuscript, so careful review of the entire document would be necessary. Experimental design: The experiment was rigorously done, though some continuity between the results in 2012 and 2013 would have been ideal. Validity of the findings: Given that thorough DGGE analysis had been employed for this study, it would be important to show some a figure with DGGE gel pictures depicting the different patterns associated with the different Symbiodinium types, and which were found in which corals, under which temperature treatments and which time points. Even showing a subset of these data would suffice to give readers an example what was analyzed and how different were the types. The authors can then conveniently refer to this figure under 'Symbiont types in coral juveniles and adults' in the Results. Given that clade F was dropped after this section, it would be informative to see what F looks like in a DGGE gel. The terms "prevalence" and "occurrence" require careful explanation in both the text and the y-axis labels in the graphs. At first glance of just the figures, it was unclear what was the difference between the two terms. The y-axis labels would be clearer if changed to "Presence of symbiont type (%)" in Fig. 3 and "Proportion of juveniles (%)" in Fig. 4, or something of the like. The clear distinction should be made in the Methods, Results, and Discussion as well. Fig. 3a shows that type D1-4 is already the most stably prevalent throughout the 4 months at each temperature treatment in 2012 that it would seem difficult to make the case that it is more prevalent at the higher temperatures. The authors can make this case in 2013, however, with the increased prevalence of type D1-4 at 32C in Fig. 3b. But if this conclusion can only be drawn from one set of data but not the other, then there is the question of how repeatable this result is. This would suggest that changes in symbiont communities occur naturally over time, and it is not very conclusive that it is indeed due to temperature alone. The authors also allude to this in lines 331-333, and it is something to carefully consider. Fig. 4 demonstrates a higher proportion of juveniles harboring type D1-4 at higher temperatures in both years, and some arrows indicating the bars of interest (4-month ambient vs. 4-month 31C in 2012, and 2-month ambient vs. 2-month 32C in 2013) would be helpful and easier for the reader to see. Fig. 5 could be enhanced with labels of the Symbiodinium clades/types to the right of each shaded cluster. At present, the reader would have to guess/assume that blue clusters to A, red to D, and green to C. There seems to be some errors in the text where results reported do not match the depiction in the graphs. For example, 63.2% in line 246 is not visible in Fig. 3a, and neither is 60% in line 281 for Fig. 4b. Line 216 should also say Fig. 2a, not 2b. While the study found the prevalence of Symbiodinium type D1-4 and the number of juveniles harboring this type to be higher under heat-stress conditions after 2-4 months, neither of these aspects seem to help the juveniles survive better or grow larger. It is true that the juveniles that did survive after 2-4 months in 2012 seem to be dominated by type D1-4, but overall survivorship is consistently less than 20% at 2 months, 3 months, and 4 months (Fig. S2a). In other words, survivorship did not improve when juveniles were harboring type D1-4. Therefore, this cannot be considered any type of acclimatization, when these juveniles are clearly not doing so well, and survivorship at 31C is lower than at 30C or ambient temperature. And the 2013 results (Fig. S2b) did not show any type of pattern at all. Hence, it is huge leap to make the conclusion that "type D1-4 increases the heat tolerance of the juveniles" (Discussion, lines 350-351). Additionally, the results at 1.5 years in the 30C treatment should be interpreted with caution, as there is no control treatment to compare that to. Additional comments: Examining the potential to shift symbiont communities at the coral juvenile stage is important and currently understudied; hence, this attempt by the authors is appreciated and needed in the field. However, the main concern for this manuscript is that the current results do not support the conclusion that clade D Symbiodinium benefit the survivorship of the juveniles under heat stress. The results even slightly suggest the opposite. Therefore, the authors can conclude that (1) type D1-4 is more prevalent in juveniles at the higher temperature in one year and that (2) it occurs in a larger proportion of juveniles at the higher temperature in both years, but that is it. The question of how these two findings could benefit coral juvenile survivorship, growth, and overall health remains inconclusive.

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Title: INTROGRESSION BETWEEN ECOLOGICALLY DISTINCT SPECIES FOLLOWING INCREASED SALINITY IN THE COLORADO DELTA- WORLDWIDE IMPLICATIONS FOR IMPACTED ESTUARY DIVERSITY Review round: 1 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: In this paper, Lau and Jacobs report morphological and genetic data that clearly separate the northern Gulf of California sister species, Colpichthys regis and C. hubbsi, and provide evidence for introgressive hybridization between the two distinct species in the Delta region. The paper was very well written and easy to follow. Analyses and presentation of both morphological and genetic data were appropriate and quite thorough, and supported the authors' main conclusions. I only have a couple of comments that I feel the authors might want to address. Although seemingly trivial, the name "Scripps Institute of Oceanography" is not correct; "Institute" should be changed "Institution" in both the text and supplementary material. The second comment regards the bimodal distribution of the mismatch distribution of C. hubbsi (Fig. 4C). Although the SSD and raggedness statistics were not significant, and the sudden expansion model could not be rejected for either species, these metrics are known to have low statistical power. The mismatch plot of C. hubbsi would suggest a stable population. It might be worthwhile emphasizing this a bit more (e.g. line 313), especially since the side-by-side plots of C. hubbsi and C. regis clearly show two distinct patterns, which are consistent with the two distinct haplotype networks.

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Title: INTROGRESSION BETWEEN ECOLOGICALLY DISTINCT SPECIES FOLLOWING INCREASED SALINITY IN THE COLORADO DELTA- WORLDWIDE IMPLICATIONS FOR IMPACTED ESTUARY DIVERSITY Review round: 1 Reviewer: 2

Basic reporting: One relevant reference could be cited: Hastings, P. A. & L. T. Findley. 2006. Marine Fishes of the Biosphere Reserve, Northern Gulf of California. Pp. 364-382. In: Felger, R. & W. Broyles (eds). Dryborders: Great Natural Areas of the Gran Desierto and Upper Gulf of California. Univ. Utah Press, Salt Lake City, Utah. Otherwise, no comment Experimental design: no comment Validity of the findings: no comment Additional comments: This manuscript is well-written and data are thoroughly analyzed and conclusions are for the most part fully justified. It represents an important contribution with relevance not only the system under study but also to similar estuarine systems around the world. The attached pdf has a number of questions, mostly of clarification, suggested rewording, or request for supporting references.

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.

Title: INTROGRESSION BETWEEN ECOLOGICALLY DISTINCT SPECIES FOLLOWING INCREASED SALINITY IN THE COLORADO DELTA- WORLDWIDE IMPLICATIONS FOR IMPACTED ESTUARY DIVERSITY Review round: 1 Reviewer: 3

Basic reporting: Meets all standards. Experimental design: Meets all standards. Validity of the findings: Meets all standards. Impact and novelty is well assessed. Additional comments: This is a well conceived, written, and executed paper on an extremely important topic (the loss of estuarine habitat due to human water usage). It should be published as is (except a minor misspelling of Colpichthys in the abstract)

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Title: SUBSTITUTIONS INTO AMINO ACIDS THAT ARE PATHOGENIC IN HUMAN MITOCHONDRIAL PROTEINS ARE MORE FREQUENT IN LINEAGES CLOSELY RELATED TO HUMAN THAN IN DISTANT LINEAGES Review round: 1 Reviewer: 1

Basic reporting: This manuscript demonstrates that alleles known to be pathogenic in humans arise more frequently in lineages closely related to humans. The work laid out here is interesting, though I have a few concerns that I would like to see addressed. Overall, this manuscript is well written and I have included some minor suggestions to improve clarity in my line by line comments. The introduction and the discussion sections would benefit from further discussion of how this study compares to others. Please see the first of my general comments when addressing these comparisons. Some of the figures can be hard to read due to the large phylogenies being shown. I suggest collapsing clades that do not contain relevant information. The work presented here appears to be self-contained and presents results relevant to addressing the hypotheses. General comments 1. The comparison of the findings reported here to published works demonstrating the occurrence of a mutation in one species is a predictor of its pathogenicity in humans may not be completely valid. From reviewing the cited literature, it appears that this observation is true for very closely related homologs. However, this observation may not be true at larger phylogenetic distances, such as those examined by the authors of this manuscript. It appears that the authors of this manuscript expected to find that human pathogenetic allele would be prevalent in distantly related species. Published results do not seem to suggest this hypothesis. Hence, claiming the results found in this study contradict previous works are not strictly valid because the authors examine much larger phylogenetic distances. I suggest that the authors add a further discussion about how their results differ from that of previous studies addressing differences in phylogenetic distances. 2. Throughout the manuscript, it is suggested that the presence of an allele at a specific site is evidence of the allele’s fitness. The authors should review Kimura’s neutral theory of evolution and address that parallel or convergent evolution is not always indicative of fitness. Zou and Zhang (Mol Biol Evol. , 2015) shows that molecular convergence is often explainable by neutral models. Specific Comments. 3. The figures that include phylogenies are very hard to read, particularly Figure S9. Consider collapsing clades where a change is not observed. 4. Figure legend 1 claims that arrows are on the figure but none are present. The arrows are included in the supplemental figures but why the distance to flies is included is unclear. 5. Include p-values in the text of the manuscript when you are claiming something is significantly different. 6. Figure 7 does not include a y-axis label. Experimental design: The research question is well defined, though a more detailed description the initial hypothesis should be given. Please see my first general comment when addressing this. The technical approaches used here appear to be appropriate. However, the methods section is brief and I would like to see the following questions I had about specific lines addressed. Line 70: How did you constrain the phylogenetic trees? Line 97: How was the binning of phylogenetic distances done? This really needs to be further explained because your results hinge on the size of these bins. Line 102: How were these simulations done? Did you use a piece of software? Line 136: Biased how? Validity of the findings: Overall, I think the results of this project are sound. However, the manuscript itself needs a significant amount of rewriting. The methods section is brief and the results section does not place findings into a larger context. The authors should take care not to oversell their findings because I don’t think the comparisons to existing literature is valid. If the authors feel that their findings are indeed comparable this needs to be carefully described, addressing differences in phylogenetic distances. Further, the authors need to avoid a selectionist view of molecular evolution. The presence of an allele at a site does not necessarily mean that it confers a fitness advantage, rather its presence may be due to neutral process. The conclusion section of the manuscripts claims that Line 240: a variant fixed in humans may be deleterious in non-human species. This was not demonstrated by this study. The last paragraph from the discussion is just pasted into the conclusions section. Additional comments: Minor line by line text comments. Line 38: Include SPFL after you introduce single-position fitness landscape directly after the term. Line 42: Remove “than in distantly” Line 52-55: The connection between two exponential distributions and the number of compensatory changes needs to be better explained. Line 58-61: This is a run-on sentence, consider breaking it up for clarity. Line 65: It is not clear if the 12 mitochondrial are also from Klink & Bazykin, 2007. Line 74: Add a citation for the MITOMAP database. Line 132: What is ‘it’ in the “…these species, it did not share…” sentence? Line 147: Beyond just reporting thing fold change in phylogenetic distance include some context about what this result means. Line 183: Change the word order to read “similar to normal human variations than to non-human variants” Line 207 & 233: Add a sentence about what these differences in phylogenetic distances tells us. Line 255: Equating phylogenetic clustering and fitness is a false claim.

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Title: SUBSTITUTIONS INTO AMINO ACIDS THAT ARE PATHOGENIC IN HUMAN MITOCHONDRIAL PROTEINS ARE MORE FREQUENT IN LINEAGES CLOSELY RELATED TO HUMAN THAN IN DISTANT LINEAGES Review round: 1 Reviewer: 2

Basic reporting: See comments below Experimental design: See comments below Validity of the findings: See comments below Additional comments: This manuscript is a follow-up paper to Klink and Bazykin 2017 GBE. Whereas the original paper used a careful matching scheme to understand how phylogenetic distance influences the relative frequencies of divergent vs. convergent substitutions, this manuscript applies similar methods to understand homoplastic substitutions to variants that are segregating in human populations and to known human pathogenic variants. In particular, the distribution of phylogenetic distances for these homoplastic substitutions are compared with the distribution of distances for divergent substitutions. The authors find that homoplastic substitutions to variants segregating in humans tend to occur at shorter phylogenetic distance than divergent substitutions and observe a similar pattern for human pathogenic alleles. The authors attribute this pattern to changes in site-specific amino acid preferences over time and also consider a handful of specific mutations in more detail. Overall I feel this is a well-conducted study. However, I have some suggestions for clarity and issues of interpretation that I feel should be addressed prior to publication. Major issues: 1. The issue of ascertainment bias in the collection of segregating and pathogenic variants should be addressed in the Discussion. In particular, the database of segregating variants will miss low frequency variants, while the pathogenic variants will miss many mutations that cause lethality during pregnancy (e.g. the pathogenic variants are enriched for mutations whose effects are mild enough to permit viability but severe enough to appear in the medical literature). This means that both types of variants are likely biased in terms of the magnitude of their effects. What impact does this have on the conclusions and interpretation of the study? 2. The current analysis only considers segregating variants and pathogenic alleles that experience at least one divergent and one homoplastic substitution across the phylogeny (line 82). Looking at e.g. Table S2, this can sometimes be a tiny minority of alleles (e.g. for ATP6 only 12 out of 142 polymorphic sites are analyzed). However, this ratio varies greatly between genes and datasets. If a large fraction of segregating (or pathogenic) variants are never substituted in other species that suggests that the preference against these mutations is consistent over time. This contrasts with the authors conclusion that site-specific preferences change. The authors should discuss the extent to which conditioning on at least one homoplastic substitution influences their conclusions. 3. Throughout, there is a lack of clarity in the language used to describe changes observed in amino acid sequences. For instant in the title it says that “Pathogenic amino acids in mitochondrial proteins more frequently ARISE in lineages closely related to human than in distant lineages.” However, this is confusing because the current manuscript deals with both segregating and fixed differences. Does “arising” mean be produced by mutation or being fixed in the population? Similar ambiguity arises throughout the MS, e.g. line 282 where alleles “emerge independently in species more closely related to H. sapiens.” I suggest using a clear and consistent terminology, e.g. always refer to “substitutions along lineages” and “alleles segregating” rather than using alleles “arising” or “emerging” to refer to substitutions. Minor Line 65 — Please clarify for the reader the degree of overlap between the two datasets for both genes and sequences. Should be clear to reader that these are not independent. Line 92-93 More detail on pairing procedure. It is not completely clear from the text how the number of samples is determined or whether the divergent and homoplastic substitutions are at the same site or not. Line 109 “oxydase”->oxidase. Line 255-257 This is a key sentence, but is hard to parse. Please rephrase. Line 281-305 Passages here are highly redundant, as if there was an error in editing.

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.

Title: SUBSTITUTIONS INTO AMINO ACIDS THAT ARE PATHOGENIC IN HUMAN MITOCHONDRIAL PROTEINS ARE MORE FREQUENT IN LINEAGES CLOSELY RELATED TO HUMAN THAN IN DISTANT LINEAGES Review round: 2 Reviewer: 1

Basic reporting: See comments below Experimental design: See comments below Validity of the findings: See comments below Additional comments: I thank the authors for their careful responses and think this version of the manuscript is greatly improved. I feel that all my concerns were proficiently addressed and the changes to the figures significantly improve their clarity. The expanded discussion is especially appreciated and frames this work nicely. My further comments are very minor 1) The first table referred to in the manuscript is S4, so the ordering of materials in the supplemental should be changed. 2) Line 352 includes a typo of the word ‘it’.

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.

Title: SUBSTITUTIONS INTO AMINO ACIDS THAT ARE PATHOGENIC IN HUMAN MITOCHONDRIAL PROTEINS ARE MORE FREQUENT IN LINEAGES CLOSELY RELATED TO HUMAN THAN IN DISTANT LINEAGES Review round: 2 Reviewer: 2

Basic reporting: See General Comments Experimental design: See General Comments Validity of the findings: See General Comments Additional comments: The revised manuscript is substantially improved. The authors have fully addressed my concerns from the previous round of reviews, and I feel the manuscript is now suitable for publication. I also feel that the added background information in the Introduction and Discussion on patterns in the temporal structure of compensated pathogenic deviations will be helpful to the non-specialist reader. The new analysis and discussion of heteroplasmic-only variants (presumed loss of function) is also interesting, and I found the discussion of the relative frequency of loss of function versus quantitative impairment to be quite helpful. In particular, this provides a reasonable hypothesis to account for the qualitatively different patterns of evolution found in previous studies.

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Title: ARTIFICIAL NIGHT LIGHT ALTERS NOCTURNAL PREY INTERCEPTION OUTCOMES FOR MORPHOLOGICALLY VARIABLE SPIDERS Review round: 1 Reviewer: 1

Basic reporting: I enjoyed reading the article of Yuen and Bonebrake. I found it well-written, clear and sound. The authors tested the possibility that artificial light at night could disrupt the hunting success of weaver spiders, which usually hunt by luring flying insects into their webs by means of colour attraction, mimicking with their yellow colour the appearance of flowers. They predicted reduced hunting success under artificial light at night. I found the interpretation of results sound, well-founded and well-referenced. I have however several remarks about the experimental set-up and the description of the methodologies used, which should be addressed clearly for this article to be acceptable for publication. Experimental design: Generally, the description of the light measurements is very poor. In particular: 1) When were the measurements done? Which days of the experiment, and at what time? 2) Was light intensity measured only once per site, or repeatedly? This is important to understand as repeated measurements can reduce the influence of other environmental variables on the results (cloud cover, temperature, etc). 3) The authors mentioned the light type used, but do they know anything about the actual wavelength? Was this ever measured? 4) Similarly, what was the exact light intensity measured at each experimental web? 5) It is unclear what the variable "light" exactly represent in your models. Is this the actual light intensity or is it a categorical variable (light-control)? If the latter, why not incuding the actual light intensity as a predictor? I further think that you should recognise and emphasize in the discussion that the results, although significant in some cases, are based on are relatively low sample size. Also, it would be good to know something more about the species you used in terms of: 1. Is there anything know about the spectral sensitivity of this spider species? 2. You mention (L81) that this species hunt both diurnally and nocturnally. Is there anything know about its actual circadian system? It sounds as if it possesses a very flexible circadian rhythm, which might actually be beneficial in the context of light pollution if they could switch to a more diurnal hunting style. The important of knowing more about circadian rhythms in such a species, and its sensitivity to light, might be stressed more, with some relevant literature added (Gaston et al 2013 Biological Reviews, Dominoni et al 2016 Biol Lett, Davies et al 2013 Global Change Biology). Validity of the findings: As stressed in the previous box, more details on the methodologies and background on the species in terms of light sensitivity and circadian rhythms could be beneficial to the overall quality of the paper. Additional comments: No additional comments

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Title: ARTIFICIAL NIGHT LIGHT ALTERS NOCTURNAL PREY INTERCEPTION OUTCOMES FOR MORPHOLOGICALLY VARIABLE SPIDERS Review round: 1 Reviewer: 2

Basic reporting: The study is very interesting and addresses questions on a hot topic in ecology. I enjoyed how the background knowledge is presented in the introduction and how the authors justify the relevance of the questions they address. Also literature is properly cited and relevant. Description of the figures is good, as well as the labelling, nevertheless I would improve them, but see my comments on the following sections of the review. The manuscripts is written in proper English and well comprehensible and the data are supplied. This said, I have some major issues that, I think, justify my recommendation for the editor. Experimental design: I find the study novel, relevant and interesting and I like the design which is also well described. The investigation has rigorous principles but I see important issues, both conceptual and methodological, that undermine the result interpretation and the main conclusion of the study. Firstly I want to make sure that the authors considered few points that are not mentioned in the manuscripts but are important for the relevance of the study. 1. From the data supplied I see that light intensities in the illuminated orb-webs present are highly variable. If the illumination equipment has been installed in the same manner among webs measurements should be comparable. Of course spiders can build webs in very different fashions making a standardized installation difficult, but I would have tried to standardize the illumination since the sample size is not high enough to make inference on different light conditions. Also, the light intensity detected by the sensor of the measurement device can be very different according to the angle of incidence of the light beam and the distance from the light source, potentially leading to critical bias. Did you control for it? Please provide more explanations on these points. 2. Can you consider the six sites comparable? In the manuscript it is not mentioned whether specific features of the sites are taken into account. For example moon, sky glow, light pollution coming from close by roads, cities, micro conditions around the web (bushes, branches) that could shade those light sources. Being moths highly mobile animals, overall lighting of the site might be relevant. It would be good if these conditions are standardized among sites in order to assume environmental homogeneity. 3. Overall moth abundance and activity and phenology would greatly affect your results. In my experience moth activity can dramatically vary among and within seasons depending on winter and early spring meteorological conditions (larvae development and survival), blooming of some key plant species (within season phenology), temperature, humidity and moon (within night activity). You collected data during one season, each site was sampled four (or three) times simultaneously within one night; this lead me to wonder how did you control for moth abundance and activity in your results. 4. As already mentioned, moths are highly mobile animals and can cover several kilometres in a single night. I therefore ask the authors to justify the independence of the spiders you selected within a site. Albeit I believe that the light treatments do not affect the light conditions of the dark treatments, I am afraid that moth are. For example, you could detect less attracted or intercepted moths in a dark treatment because moths are attracted to the close by light treatment. On the other hand you could detect more moths on a dark treatment because moths are rejected by the close by light treatment. This is probably species dependent and, in order to make proper inference, you need to ensure independence. Validity of the findings: The validity of the findings are undermined by two types of issues: statistical and biological. Concerning the statistical part: 1. the sample size is certainly low and there are no replicates within sites. To make proper inference a higher N and replicates are desirable, for example by sampling the same spiders several nights, looking for more spiders within the same site, increasing the site number and repeating the data collection over two or more years. This point is linked to point 3. in the previous section of this review where I stated the need to control for moth activity among and within season. It is often very difficult to achieve a satisfying sample size in a field study, and if sites can be considered as homogeneous I think that it should not be an insurmountable problem. However, this is a weakness of the study and might seriously undermine the robustness of your results. 2. Spider size is modelled as a quantitative variable but visualized (in figures 1 and 3) and discussed as a categorical. This makes things even harder to understand and interpret. Also, the body size threshold that splits between small and large spiders is arbitrary and this should be stated in the text, or justified otherwise. I am wondering how the results change if the authors model the variable spider size as a categorical with two levels (small and large). 3. When dealing with quantitative variables (size, light intensities, attraction and interception rates, interception efficiency) it is generally desirable to represent data with scatter plots. Consider it in your next version of the manuscript. Beyond the lack of satisfying sample size and of some important predictors (see previous section of this review), there are important conceptual issues that I would like to highlight. 1. I am puzzled by the response variables the authors are considering, especially by the interception rate. What is the biological relevance of it? If only a tiny part of the intercepted moths are eventually captured and consumed, what is the effect of light and painting of the lure on capture rate, moth mortality and spider food intake? In other words, how can attraction and interception affect prey-predator interactions? How could this set up and the results provide clues about the actual pressure of an anthropogenic disturbance such as artificial night light on the ecological interaction the authors are considering? Please, could you specify in which treatments and sites the ten predatory events occurred? 2. Moths flying close to lit webs could avoid them because they can better detect them, resulting in reduced interception events. However, their flying pattern could be greatly disrupted by artificial night light inducing them to aimlessly fly around the lamp avoiding the spider web. On a normal, aimed movement moths might be more likely to hit a spider web. Please, mention the well known fly-to-light behaviour when you discuss your results. 3. What is the time span of the sampling? There must be a typo in line 112. If the fieldwork has been conducted during one month (between Oct 2016 and Nov 2016) I do not see additional problems. If on the other hand several months elapsed from the first to the last sampling, a time variable musts be modelled. Additional comments: The story around this study is beyond any doubts fascinating, the topic is hot and the design cleverly conceived. However, as the authors fairly mention in the discussion, the outcome of the study is difficult to interpret and does not provide conclusive results. It gives clues about the role of the lure on the chephalothorax of Nephila pilipes and the effect of artificial night light on the phases prior to the predatory event. Unfortunately, it does not highlight changes in moths' capture rate and spider food intake making the study of poor biological relevance. Further, the low sample size leads to a poor statistical power, makes the model selection difficult, results unclear and the conclusions unsatisfying. However, I believe that the value of the study can be enhanced. I propose that the authors provide a power test to ensure that the data support the results and that the manuscript will be rewritten in order to better explain the data. Important points that need to be discussed are the definition of prey-predator interaction and the biological relevance for the animals involved in the interaction. If available, additional data could be added: environmental measurements like temperature and humidity, characterization of the light conditions in each site. An important control would be to sample data on lit and unlit orb-web where Nephila pilipes is absent to further disentangle the role of the lure which I believe is important. Light intensities in lit treatments are relatively low if you consider what you would detect within few meters from a street lamp. Increasing light intensity you could get stronger effect of the light. Additional comments and questions on the manuscript: Line 90: why only females? Line 112: typo in the starting and ending date? Line 125: are yellow flowers a common moth food source? According to my knowledge, flowers specialized on nocturnal pollinators do not invest much on striking pigmentation and are therefore mostly whitish/pinkish. However, I am not a specialist of your study area. Line 236: limit the conclusion to the study species or justify the extension of the conclusions to other orb-weaver spider species that might be devoid of lure.

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.

Title: ARTIFICIAL NIGHT LIGHT ALTERS NOCTURNAL PREY INTERCEPTION OUTCOMES FOR MORPHOLOGICALLY VARIABLE SPIDERS Review round: 1 Reviewer: 3

Basic reporting: The paper reports clearly on the methods and results of an interesting experiment. It is well-written, with good reference to the literature and well structured. Experimental design: The experimental design is good. I have two points that should be added to the methods section if possible - first, at the least the manufacturer and the colour temperature (in K) or ideally, the spectral power distribution of the LED lamps used should be included in order to allow the experiment to be replicated. If possible, the sectrum of the lights should be compared to the high pressure sodium lights used in Hong Kong that the authors are trying to replicate. Second, the time of sunset during the study should be stated. Validity of the findings: The data is robust and analysis statistically sound, and the conclusions are well-stated. Additional comments: No additional comments

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.

Title: ARTIFICIAL NIGHT LIGHT ALTERS NOCTURNAL PREY INTERCEPTION OUTCOMES FOR MORPHOLOGICALLY VARIABLE SPIDERS Review round: 2 Reviewer: 1

Basic reporting: ok Experimental design: ok Validity of the findings: ok Additional comments: Overall, I like how authors have changed the manuscript in order to make their conclusions more prudent since they are based on a poor dataset. As they are now, figures better represent the data, also the map is definitely useful.

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.

Title: THE DISTRIBUTION AND NUMBERS OF CHEETAH (ACINONYX JUBATUS) IN SOUTHERN AFRICA Review round: 1 Reviewer: 1

Basic reporting: I am in agreement with the authors that presence data should be made more public as this makes it easier to understand and interpret species distribution. At the same time it is also important to provide enough information so that the study can be replicated and I feel that this needs more attention by the authors. For example, the article could do with some restructuring as some of the methods are presented in the results section. It would also helpful if similar ideas are grouped together and put in the same orders (i.e. results presented in the same order as the methods). At the moment some section jump around a bit making it difficult to follow the authors' train of thought. Experimental design: The authors have collected an incredible amount of data and it is clear that they have put a lot of time and effort into the study. However, there are some concerns regarding the extrapolation of density figures, especially as most figures used violate various assumptions incl. closure and do not account for detection probability. Also, the methods could do with more detail so that the study can be replicated (details are provided in the comments). Validity of the findings: One of the major concerns is that no measure of precision is provided for the overall number of cheetahs and therefore more care should be taken when interpreting results and comparing these to other findings. Additional comments: No additional comments

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.

Title: THE DISTRIBUTION AND NUMBERS OF CHEETAH (ACINONYX JUBATUS) IN SOUTHERN AFRICA Review round: 1 Reviewer: 2

Basic reporting: All my comments are included in the "general comments". Experimental design: All my comments are included in the "general comments". Validity of the findings: All my comments are included in the "general comments". Additional comments: I applaud the authors for this impressive work, including large amount of data and the attempt of introducing a standard methodology for assessing the range and numbers of cheetah in South Africa. As compared to previously available data on the distribution of the species, this paper provides an updated status at high resolution as well as the regional variation in cheetah densities, which is based on a rich and diverse sources of data. It also attempts to determine the possible range of the cheetah based on an assumption of negative relationship between the presence of a large predator and anthropogenic factors. I think, as such, the results presented in this manuscript are generally a valuable contribution to the science as it provides a detailed information on the range and numbers of a rare (difficult to survey) species supported with abundant and statistically elaborated data. However, the manuscript is not devoid of some major and minor problems, which require revision or better explanation to allow the reader understand the reliability of presented results. I see three major issues: 1) It is difficult to follow how the densities were estimated. The information to understand it step by step is available in the manuscript, but it is scattered in different places. For instance, to complete the information presented in the section of methods devoted to “density estimates” one has to look at the results and the Figure 3 to get the general idea. In the “density estimates” section a detailed approach is presented explaining that existing estimates from Zimbabwe and Kruger National Park were used to estimate the total regional cheetah population. This seems to suggest that these data were somehow used for the remaining part of the cheetah range, which is strange as the area of the cheetah presence in both Zimbabwe and Kruger is really minor relative to other populations (especially in Namibia). On the other hand, I learned from results that 14 empirical local cheetah population estimates were used, but it is unclear how all these data were combined with the Zimbabwe and Kruger data and analysed. 2) Although I appreciate the use of the Leslie matrix for estimation of the population growth, I do not understand how was it applied to estimate population densities, and specifically to account for the persecution data. Much information is provided both in the methods, results and the supplementary data referring to this analysis, however, it is not possible to follow the complete procedure and see the results of this analysis in the manuscript. I expect a graph resulting from this analysis showing the population trend based on the relationship between the growth rate and densities, both with and without persecution e.g. to illustrate the scenario mentioned in lines 370-372. 3) I also appreciate the authors’ efforts in trying to statistically support the “possible presence” area of the cheetah and estimating their potential numbers and densities. I understand that human related factors are generally negatively correlated with the cheetah presence. However, I also think that relying on this relationship directly is somewhat misused. There may be both, areas where predator still occurs with relatively high densities of livestock (actually seen on the Fig. 2 and areas where predator does not occur due to other than livestock/human issues. The authors seem to understand this problem as they acknowledge this in lines 445-448. Perhaps, accounting for another factor – wild prey occurrence – would add to the reliability of this estimation, but I assume such data may be equally difficult to get at sufficient resolution. Moreover, may be I missed this info, but I didn’t see the explanation how were these data on virtual cheetah presence farther stratified into densities. Anyway, considering the low reliability of the “possible” part of the cheetah range, I would put yet more emphasis on the untrustworthy character of this part of results. Please note, I have included a number of other comments directly in the manuscript.

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.

Title: THE DISTRIBUTION AND NUMBERS OF CHEETAH (ACINONYX JUBATUS) IN SOUTHERN AFRICA Review round: 1 Reviewer: 3

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: No additional comments

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.

Title: A MOLECULAR PHYLOGENETIC APPRAISAL OF THE ACANTHOSTOMINES ACANTHOSTOMUM AND TIMONIELLA AND THEIR POSITION WITHIN CRYPTOGONIMIDAE (TREMATODA: OPISTHORCHIOIDEA) Review round: 1 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: The MS titled “A molecular phylogenetic appraisal of the acanthostomines Acanthostomum and Timoniella and their position within Cryptogonimidae (Trematoda: Opisthorchioidea)” is a well-written, insightful examination of the position of two acanthostomine genera. I found the MS to be clear and well referenced, the data is robust and the images attractive and representative of the data. I have a few comments regarding the MS, and pending consideration of these comments I recommend this article be published in PeerJ Major points 1) The authors do not discuss the position of the acanthostomine genera in the phylogenetic results section, it is almost entirely discussing the associations of those sequences from GenBank. Seeing as this is the basis of the paper I think it needs to be covered in the results section. 2) On line 193 the author mention “Only three high nodal support values (PP ≥ 0.95) from three clades”, yet later in the pargraph they mention that several other clades are support values (PP ≥ 0.95) (line 204 and 208). This needs to be clarified 3) Lines 207-217 and 279-298 discuss clading of species based on host associations or geographical localities. I strongly recommend these be reduced and extensively edited, as these associations are not really evident in the data. No clades can be found by geography (e.g. there are species from the IWP/IP (which is almost the same thing) across the marine crypto clade that do not form any really grouping) or host (e.g. there are species from Lutjanids or Haemulids across the marine crypto clade that do not form any really grouping). It is highly likely that these clades actually represent the selective sampling or Miller et al, from which most of these sequences were generated, in that the studies focused on certain host groups (reef fishes of a few families) in certain locations (GBR/French Polynesia). Minor points Line 15, et al: I think the authors need to make sure they say (at least a few times) that It is partial 28S rDNA, as only 880ish bases (Domains 1-2) of the region were sequenced Line 19: “paraphyly of the genera Acanthostomum and Timoniella acanthostomines” makes it sound the like genera are paraphyletic. I would change to “paraphyly of the Acanthostominae” Line 20: change “by itself” to “alone” Line 46: change to “1) a terminal oral sucker; 2) a body armed with a single row of spines; 3) a preacetabular pit; 4) a genital pore that is not in preacetabular pit; 5) a seminal vesicle that is coiled posteriorly; and 6) a sucker-like gonotyl.” Line 53: comma after Cryptogonimidae; change to “by few, and often trivial, characters” Line 60: remove comma after “astorquii)” Lines 57-74: Some of this paragraph should be moved to the Methods section, it is not an introduction to the subject Line 84: “hydrobiid snails of P. coronatus” to “snails of P. coronatus (Hydrobiidae)” Line 86: “The snails” should be “Snails” Line 90: “As for” should be “For” Line 94: “were” should be “have been” Line 100: change “trematode species from the same fish host, Crassicutis cichlasomae” to “trematode species, Crassicutis cichlasomae, from the same fish host” Lie 105: “The identification to genus level for both Timoniella and Acanthosthomum is certain based on metacercariae morphology” should be changed to “Reliable identification to genus level is possible for both Timoniella and Acanthosthomum based on metacercariae morphology” Line 107: “questioned” should be “questionable” Line 108: “metacercarian” should be “metacercarial” Line 124: insert “fragment” after “ITS1–5.8S–ITS2” Line 148: “with Bayesian” should be “by Bayesian”; add “analyses” after “(ML)” Line 149: change “The ML was” to “ML analyses were” Line 151: change “The BI was” to “BI analyses were” Line 169: delete “those of” Line 203: change “acanthostomines form a paraphyletic group” to “acanthostomines are paraphyletic” Line 204: delete “with Acanthostomum and Timoniella not clustering together”. This is superfluous Line 224: change “…2016). The…”to “…2016), and that the…” Line 227: change “It’s indicate that” to “This indicates that” Line 229: “an appropriate” Line 232: “separate” is implied. Delete. Line 251: I could put the genus names in full here, seeing as the are both C. Line 254: Maybe the authors mean “from freshwater regions”, they just noted that C. urophthalmuscan tolerate brackish water so all records can’t be from “only freshwater fishes” Line 257: change “Acanthostominae was a freshwater group that was sister to the remaining” to “Acanthostominae were sister to the remaining”. As the two genera represented independent lineages, you cannot say “freshwater group” Line 260: Cryptogonimidae is misspelled Line 271: add comma after “infection”; change “e.g.” to “i.e.” as you list all 12 Line 273: is the “?” meant to be in this species name Line 305: “arisen” rather than “arise” Line 311: “suggested” rather than “suggest” Line 314: delete “would” Figure 2: What do the larger black circle inside the nodes indicate? Table 1: the species name in the second line of table needs to be italicized Other trees: why are the species not “cf.” in these trees

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.

Title: A MOLECULAR PHYLOGENETIC APPRAISAL OF THE ACANTHOSTOMINES ACANTHOSTOMUM AND TIMONIELLA AND THEIR POSITION WITHIN CRYPTOGONIMIDAE (TREMATODA: OPISTHORCHIOIDEA) Review round: 1 Reviewer: 2

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: This is a well-written and presented paper describing the relationships of species of Acanthostomum and Timoniella to other previously reported cryptogonimid genera for which comparative molecular data is available. The methods used for molecular characterisation of the cercariae, metacercariae and adults reported here are appropriate and sufficiently detailed. Overall, this is a solid paper and I have no major issues with the work presented. However, I do have some minor comments and edits that the authors should address prior to publication. I have included these as annotations and comments on the pdf file of the manuscript.

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.

Title: MEASUREMENT OF SEDENTARY BEHAVIOUR IN POPULATION HEALTH SURVEYS: A REVIEW AND RECOMMENDATIONS Review round: 1 Reviewer: 1

Basic reporting: I would like to thank the authors of this manuscript for a very interesting study. The topic of sedentary behaviour in population health surveys is very important and it is very suitable for the scope of this journal. Sufficient background and context was provided. However, more information can be added in several paragraphs to make the manuscript more clear for the readers. Line 67) Provide citations Line 189) "Figure 1 provides results of the search strategy used to identify 35 papers that examined the validity"… This is not very clear. Do you mean Figure 1 provides results of the search strategy used to identify [37?] papers that examined the validity? In addition, figure 1 can be used to breakdown the number of included papers undergone psychometric testing in a pediatric population, and in the adult population. For figure 1, it is recommended to provide reasons for excluding articles at full-text screening and the number of articles that are excluded for each reason. Please see “Item 17: Study selection Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram http://www.bmj.com/content/bmj/339/bmj.b2700.full.pdf “ . Since this study is not a systematic review, this is not a serious issue. I just wanted to mention it. In Supplemental_Table_2 Line 184) The reliability and validity of individual questionnaires (n=34 or 37?) has been summarized in Supplemental table 2. Also, I found it to be a bit confusing to know which papers have been discussed in the previous systematic reviews [16, 17] and which ones you are investigating in this study. It would be great if you could clarify. Supplemental table 3 is not reference anywhere in the manuscript. Experimental design: In order to conduct a comprehensive search it is very important to include mesh terms in the search. It is impossible to come up with a comprehensive list of search terms. Mesh searching will assist with retrieving all potential articles relevant to a topic. For example, as a Subjective measurement term, one might use self-rated instead of self report. By searching for "Self Report"[Mesh], you can reduce the risk of missing that specific article. I strongly suggest adding the following mesh terms: "reproducibility of results"/ OR validation studies as topic/ Validation Studies/ Mesh searching becomes even more important if you limit the search to tw field. "The Text Word (TW) index is an alias for all of the fields in a database which contain text words and which are appropriate for a subject search. The Text word index in Ovid MEDLINE (R) includes Title (TI) and Abstract (AB)". (from http://ospguides.ovid.com/OSPguides/medline.htm#TW ) basically tw exclude searching the author's keywords field and mesh field. One of the main limitations of this study is the lack of a comprehensive search. I think it would be great to mention this in the article to increase transparency. Line 123) I do not think searching ‘EBM Reviews - Cochrane Database of Systematic Reviews’ would have been useful as the authors were aiming to find original research articles rather than systematic review articles. Validity of the findings: As mentioned before one of the main limitations of this study is the lack of a comprehensive search. I think it would be great to mention this in the article to increase transparency. Additional comments: No additional comments

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.

Title: MEASUREMENT OF SEDENTARY BEHAVIOUR IN POPULATION HEALTH SURVEYS: A REVIEW AND RECOMMENDATIONS Review round: 1 Reviewer: 2

Basic reporting: Overall a well conducted review. Some stylistic suggestions are made in section 4 General comments. Experimental design: Methods are appropriate. Given there were two recently published systematic reviews that summarized the psychometric properties of sedentary behaviour questionnaires in children and youth, I suggest the authors restrict their paper to adult questionnaires. This would make the tables and text somewhat more manageable. There is little benefit to generating a third systematic review on the same topic (so soon as the first two). Validity of the findings: No meta-analysis or statistical synthesis applied. Narrative description of surveys and their psychometric properties only. The narrative description of results provided in the text is too vague. Statements such as ‘Computer and/or video game time were also frequently assessed’ (line170) should be quantified by including the number of studies and percentage. Another example is ‘Many, but not all, of the surveys’ (line 173). Despite the authors stating that they included questionnaires listed on the Sedentary Behaviour Questionnaire list compiled by the SBRN, there appear to be a number of questionnaires missing, e.g. the Bouchard Physical Activity Questionnaire, the SIT-Q. The Bouchard Physical Activity Questionnaire link from the SBRN site goes to an Am J Clin Nutr article behind a paywall, so this could not be further interrogated easily. The SIT-Q validation paper in BMC Public Health appears to meet the criteria for this review, so it’s not clear why this questionnaire was excluded. Additional comments: Introduction lines 63-65 the authors suggest that evidence supports the idea that different types of sedentary behaviours have different health effects. This is not impossible, but differences in associations with health outcomes are probably a reflection of differential measurement error and/or confounding, rather than different physiological adaptations to sedentary behaviour in different settings. line 75 providing readers with information to support future survey development. Given the plethora of surveys already in existence, do we really want more? Perhaps it would be better to suggest that better psychometric testing and refinement of existing measures be undertaken. Methods line 118 suggest removing the word “Unfortunately” from beginning of sentence. Results Throughout the text the ICCs are reported in a non-uniform fashion. Ideally, the ICC and 95% CIs should be given. In some places the authors simply give the ICC (eg 228), in others it appears to be the CIs only (eg line 242). The first sentence of the paragraph beginning on line 284 is not well worded. I think the authors mean to say something like “validation studies have looked at both single-item estimates of sitting time, or have generated a composite score from a number of items to estimate total sedentary behavior”. Discussion Again, on line 313 the authors note that there seem to be positive health associations for reading (compared to the negative associations seen for other sedentary behaviours). Be very careful with the description and interpretation of this – likely to be confounding, not an actual different physiological adaptation. Lines 325 – 326: this sentence is unfinished. Line 341: actually, a number of questionnaires assessed the validity of screen time, reading and sedentary transportation (SIT-Q; SIT-Q-7d). Do the authors mean “examined the criterion validity”? Line 351: confounding concept again.

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.

Title: MEASUREMENT OF SEDENTARY BEHAVIOUR IN POPULATION HEALTH SURVEYS: A REVIEW AND RECOMMENDATIONS Review round: 2 Reviewer: 1

Basic reporting: I have no further suggestions. Experimental design: I have no further suggestions. Validity of the findings: I have no further suggestions. Additional comments: Thank you for responding to all my comments. I have no further suggestions.

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.

Title: MEASUREMENT OF SEDENTARY BEHAVIOUR IN POPULATION HEALTH SURVEYS: A REVIEW AND RECOMMENDATIONS Review round: 2 Reviewer: 2

Basic reporting: The authors have taken the reviewers' comments on board and done a decent job in revising their work. The manuscript is generally well written. Experimental design: No comment Validity of the findings: Original comment: Despite the authors stating that they included questionnaires listed on the Sedentary Behaviour Questionnaire list compiled by the SBRN, there appear to be a number of questionnaires missing, e.g. the Bouchard Physical Activity Questionnaire, the SIT-Q. The Bouchard Physical Activity Questionnaire link from the SBRN site goes to an Am J Clin Nutr article behind a paywall, so this could not be further interrogated easily. The SIT-Q validation paper in BMC Public Health appears to meet the criteria for this review, so it’s not clear why this questionnaire was excluded. Revised comment: The authors have given a satisfactory response in relation to the Bouchard PAQ, however, they do not seem to appreciate that the SIT-Q and SIT-Q-7d are different questionnaires. The SIT-Q is (the original) domain-specific questionnaire with a past year recall period. It's development and psychometric properties are published here: Lynch BM, Friedenreich CM, Khandwala F et al. BMC Public Health. 2014 Sep 1;14:899. The SIT-Q-7d is a revised version that has changed the recall timeframe to 7 days. This version also removed some of the original items and added others (specifically relating to snacking): Wijndaele K, DE Bourdeaudhuij, Godino JG et al. Med Sci Sports Exerc. 2014 Jun;46(6):1248-60. Both questionnaires should be included in this review. Additional comments: Original comment: Throughout the text the ICCs are reported in a non-uniform fashion. Ideally, the ICC and 95% CIs should be given. In some places the authors simply give the ICC (eg 228), in others it appears to be the CIs only (eg line 242). Revised comment: Restricting the reported findings to ICC only is problematic. The reader cannot draw meaningful conclusions or make an informed evaluation about the psychometric properties of the questionnaires based on ICC alone. I suggest the authors contact the researchers who have published their validation studies without 95% CIs and ask for these data to be supplied for the review paper. After doing this, please include 95% CIs where these are available, and in the case of non-response from original authors please indicated that 95% CIs are not available in your review paper (e.g. by writing something like "for TV viewing time the XXX questionnaire demonstrated reasonable convergent validity ICC=0.68, 95% CI not reported."

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.

Title: MEASUREMENT OF SEDENTARY BEHAVIOUR IN POPULATION HEALTH SURVEYS: A REVIEW AND RECOMMENDATIONS Review round: 3 Reviewer: 1

Basic reporting: N/A Experimental design: N/A Validity of the findings: N/A Additional comments: Thank you for revising the manuscript and including the CIs for ICCs where these were available. This strengthens the reporting and will ensure readers utilising this review correctly cite the reliability and validity outcomes for the measures reported. Thank you also for including the adult questionnaire previously omitted (the SIT-Q) in the Table. However, I note that none of the SIT-Q findings have been incorporated into the text. I think it is important to do so, as this questionnaire demonstrates very good ICC for TV (0.84, 95% CI: 0.75, 0.90) but poor computer ICC (0.31, 95% CI: 0.07, 0.52). It is also one of the few measures that reports sitting during transport.

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.

Title: CRANIAL OSTEOLOGY OF THE PAMPATHERE HOLMESINA FLORIDANUS (XENARTHRA: CINGULATA; BLANCAN NALMA), INCLUDING A DESCRIPTION OF AN ISOLATED PETROSAL BONE Review round: 1 Reviewer: 1

Basic reporting: This is a clearly written and exquisitely illustrated manuscript. I believe that there is no need of additional materials besides the informations provided in the tables and figures. I have only minor suggestions and commentaries on certain parts of the manuscript that I have included in the attached annotated PDF file. Experimental design: This is a well-organized and much needed in depth description of a representative of one of the most interesting groups of fossil cingulates. The results have clear implications for the understanding of the taxonomy and phylogenetic relationships of pampatheres (as well as for other cingulates), and might be useful in future cladistics analysis. The bone-by-bone descriptions are very lengthy at places, but overall there is no excessive details. In fact, I should say that similarly detailed up to date descriptions are lacking for several groups of cingulates. That being said, I suggest that some descriptions may be shortened by reducing or excluding morphological features that are common to all cingulates wherever possible. Validity of the findings: The findings reported in the manuscript are sound and they can be easily checked by any interested reader with the help of the excellent illustrations. Additional comments: I have no additional comments in addition to those stated above

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.

Title: CRANIAL OSTEOLOGY OF THE PAMPATHERE HOLMESINA FLORIDANUS (XENARTHRA: CINGULATA; BLANCAN NALMA), INCLUDING A DESCRIPTION OF AN ISOLATED PETROSAL BONE Review round: 1 Reviewer: 2

Basic reporting: The paper is clear and very well-written; I've noted a few typos in the attached annotated PDF. The literatures references are up to date, and well-cited throughout; I've just made a few suggestions in the annotated PDF, though these additions are not necessary. The article is very well structured and the figures are generally excellent. As this constitutes an extensive description of cranial anatomy, I feel that some more features described in the text should be labelled on the figures (these are specified in the attached PDF). Experimental design: no comment Validity of the findings: no comment Additional comments: Overall, I found this work (a thorough description of the external cranial anatomy of the pampathere Holmesina floridanus) to be very well executed and very valuable. It will certainly count as a major reference concerning cranial anatomy in fossil cingulates and will be much used as a comparative basis. The quality of the anatomical observations and of the figures is very high. I have only minor comments for improvement: 1) additional labeling is needed on several figures 2) comparison of cranial anatomy with a chlamyphorine and/or tolypeutine armadillo is also desirable, given the fact that pampatheres, supposedly close to glyptodonts, may in turn be close to chlamyphorine and tolypeutine subfamilies (according to DNA studies); 3) for some features with high variation (eg, teeth outline), it might be interesting to discuss whether or not ontogeny (eg, tooth wear) may explain the observed differences 4) the discussion on taxonomic ranks attributed within Cingulata (family or subfamily) should be mitigated and existing phylogenetic arguments for having two basal families of cingulates should be given better consideration Please see further details and other minor comments in the attached PDF

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.

Title: CRANIAL OSTEOLOGY OF THE PAMPATHERE HOLMESINA FLORIDANUS (XENARTHRA: CINGULATA; BLANCAN NALMA), INCLUDING A DESCRIPTION OF AN ISOLATED PETROSAL BONE Review round: 1 Reviewer: 3

Basic reporting: Im not an american speaker so I can't check the grammar. The scientific terms used are actualized and in agreement with the modern terminology of mammals anatomy. The list of references is adequate, although suggested to quote a few more for specific topics (comments on the text). The article structure, figs, and tables are adequate. The Figures are fantastic and well planed according to the paper. The submission is Self-contained. Its a solid manuscript. Experimental design: This research fills an important gap in the knowledge of xenarthran anatomy. Research questions are well defined and relevant. The investigation was conducted in a very high quality way. The methods are adequate for this kind of research, although I suggested to explain some of them a little bit more (comments on the text). Validity of the findings: This paper is an absolute novelty for xenarthrans anatomy. The data is robust for this kind of anatomical paper. The discussion and conclusions are well stated. Additional comments: Was a placer to read this paper. Most of my comments tried to make the anatomical part of this paper even more informative for a frequent user of this kind of papers. I made most of the comments on the manuscript. Most of them refers to little details that in such a long description is usual to find. I suggested to take a look of some non quoted references that I think will improve the quality of the paper (comments on the text) I specially recommend to check with other published armadillos phylogenies (see comments on the text) if some of the characters consider here as synapomorphies, were discussed of described by other authors.

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.

Title: SPECIES DIVERSITY AND DRIVERS OF ARBUSCULAR MYCORRHIZAL FUNGAL COMMUNITIES IN A SEMI-ARID MOUNTAIN IN CHINA Review round: 1 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: The authors explored the species diversity and composition of soil arbuscular mycorrhizal fungi communities in a semi-arid mountain – Taihang Mountain. They sampled soils from four habitat types, i.e., agricultural arable land, artificial forest land, natural grassland, and bush/wood land, and used the high-throughput sequencing with MiSeq for arbuscular mycorrhizal fungi. The sequencing depths are sufficient for arbuscular mycorrhizal fungi, and the statistical analyses are robust in detecting the important factors for arbuscular mycorrhizal fungal diversity and community composition. I only have minor comments to make it clearer. Indicate “Taihang Mountain” in the abstract. Did you sample the soil along elevations? As you mentioned the “mountain” in the title, please explicitly consider “elevation” or “temperature” as an explanatory variable in explaining diversity and community composition. Figure 1, 2. Add soil types for replicated samples in figure legend or figure so that readers could understand the figure without referring to the main text. In figure 3, taxonomic information for these 50 OTUs will enrich the information to readers. Table 2. Make clear what kind of “results” you presented. Table 1, 2. What the letters (A, B, C) mean? Jianjun Wang Nanjing Institute of Geography and Limnology, CAS

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.

Title: SPECIES DIVERSITY AND DRIVERS OF ARBUSCULAR MYCORRHIZAL FUNGAL COMMUNITIES IN A SEMI-ARID MOUNTAIN IN CHINA Review round: 1 Reviewer: 2

Basic reporting: The paper is well structured, overall the language is correct but several corrections lust be made. The literature cited is sufficient, recently updated and in accordance with the context of the paper. The pictures, figures and tables are correctly done and there was no overlapping of information. The discussion is mostly based on own experimental data, but further elaboration of own results and literature is needed regarding the effects of water content, and available P and K on AMF communities. Experimental design: No comment Validity of the findings: The discussion is mostly based on own experimental data, but further elaboration of own results and literature is needed regarding the effects of water content, and available P and K on AMF communities. Additional comments: Further elaboration on the effects of most important environment factors on AMF community is needed, and some language corrections are needed. Some language corrections are suggested in the text.

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.

Title: COMPLETE GENOME SEQUENCE AND COMPARATIVE GENOMICS OF THE GOLDEN POMPANO (TRACHINOTUS OVATUS) PATHOGEN, VIBRIO HARVEYI STRAIN QT520 Review round: 1 Reviewer: 1

Basic reporting: The authors describe the genome of a newly isolated strain (QT520) of Vibrio harveyi, and compare it with related Vibrio harveyi strains. The genome sequence of V. harveyi QT520 will be of interest to the scientific community and may lead to a better understanding of its pathogenicity. However, the manuscript is very poorly written; the quality of written English is unacceptable in several occasions and is not suitable for publication unless extensively edited. * Several sections require language edits, including but not limited to: - Line 29: “consists two circular chromosomes” - Lines 34-35: “suggesting strain QT520 were provided with the capacity for a highly virulent phenotype” - Line 41: “and first compared the genome with other V. harveyi strains” - Line 152 “This bacterium was a Gram-negative, aerobic” - Line 158 “Chromosome I was detected of 3211 ORFs” - Line 161 “Chromosome II was contained of 2044 ORFs,” - Line 326 "Strains QT520 were exhibited resistance to tetracycline and ciprofloxacin" * Figure 2: Both A and B are labelled Chromosome I and should be changed * Figure 4B legend missing genome name * Table 1 is missing details of Illumina sequencing technology and related assembly methods * Sequence data is available in public domain but the size of the chromosome and plasmids mentioned in the paper do not match with that in GenBank, examples including but not limited to: - Line 156 - “chromosome I of 3571617 bp”; Chromosome 1 in GenBank (CP018680.2) is 3560044bp - Line 156 - “plasmid p1 of 124998bp”; Plasmid 1 in GenBank (CP018682.2) is 113574bp Experimental design: The Phylogenetic Analysis section lacks credibility and fails to provide an accurate picture with respect to the phylogenetic position of Vibrio harveyi QT520. Which single copy genes were used to generate Fig 4A? While no bootstrap values are provided for Figure 4A, bootstrap values of 24 and 43 in Fig. 4C are way too low to provide any meaningful information. The authors also used different sequence alignment, tree building method and a different set of organisms for the three phylogenetic trees, which are thus not easily comparable. In the single-copy gene tree, QT520 clusters with the other two Vibrio harveyi strains while in the 16S tree QT520 is closer to V. campbelli 1114GL than to other V. harveyi strains. Since this is the first report describing Vibrio harveyi QT520, the authors need to provide more conclusive evidence confirming isolate QT520 is indeed Vibrio harveyi. While the authors mention that 16S rRNA based trees failed to differentiate between the different strains, the statement needs to be substantiated with more robust tree building algorithm and/or complemented with whole genome average nucleotide identity (ANI) values with reference V. harveyi genomes. Validity of the findings: * Provide more specifics on how the genomes were compared using Mummer. Include the run parameters used including cutoffs * Line 208: Define “perfectly matched ORFs” * Lines 210 - 213: Provide literature reference suggesting that the two core genes are responsible for multiple antibiotic resistance to substantiate the claim that QT520 possesses similar antibiotic resistance features as the other two V. harveyi strains * Lines 218-221: Provide literature reference Additional comments: * In the General features section, consider discussing the physiological/biochemical properties separately from the genomic information. * In the LD50 section consider including some information comparing pathogenicity and LD50 values of ATCC 3843 and ATCC 43516 with QT520

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.

Title: COMPLETE GENOME SEQUENCE AND COMPARATIVE GENOMICS OF THE GOLDEN POMPANO (TRACHINOTUS OVATUS) PATHOGEN, VIBRIO HARVEYI STRAIN QT520 Review round: 1 Reviewer: 2

Basic reporting: The work from Tu et al. "Complete genome sequence and comparative genomics of the golden pompano (Trachinotus ovatus) pathogen, Vibrio harveyi strain QT520" is interesting work adding up to our understanding of the pathogenicity and the population genomics of Vibrio sp. Beyond doubt, it is an interesting work worth to be published. Nevertheless, several issues regarding the use of language have to be addressed and corrected prior to publication: e.g. L51-54 change to "... causing mass mortalities in aquaculture species having a major impact on the industry. Species affected throughout the world include the gilthead sea bream ... and prawn (Penaeus monodon)." L55-57 change to "...to infect many aquaculture species in China and is now considered as one of the major pathogens to the fisheries industry". This goes throughout the whole manuscript. I recommend a native speaker or a professional editor to go through the manuscript and correct syntax errors and misuse of English. Experimental design: Line 98-100: Even though I understand what they mean, it is confusing. Please rephrase in a clear way. Validity of the findings: Table 1 and table 3 can be moved to the supplement. Additionally, Table 1 "Sequencing_meth" was combined Illumina Miseq and PacBio; please correct accordingly. Section Discussion Lines 250-256: Please remove completely. Does not adding anything to the scientific impact of the paper and delivers confusing messages. It is well known that different methods/techniques/approaches needed to answer different scientific questions. Moreover, even though it is clear that the QT520 strain is a pathogen isolated in China, the prevalence as a major aquaculture pathogen in China (or anywhere else in the world) is not addressed in this work and not supported by the data. Therefore, a reader should perceive it as another pathogenic strain of Vibrio harveyi, increasing our knowledge of molecular ecophysiology of V. harveyi. Furthermore, it is highly recommended to the authors to submit the strain at least to one culture collection and make it available to the scientific community; I am confident several scientists would be interested to know how they can get access to the strain and this should be mentioned in the manuscript. Additional comments: No additional comments

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.

Title: COMPLETE GENOME SEQUENCE AND COMPARATIVE GENOMICS OF THE GOLDEN POMPANO (TRACHINOTUS OVATUS) PATHOGEN, VIBRIO HARVEYI STRAIN QT520 Review round: 2 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: Line 71: Remove duplicate ‘a significant factor’ Lines 103 - 111 : The authors provide conflicting information about library prep and sequencing method. Library prep was done for Illumina and sequencing was performed by PacBio RS II? Again in the results section the authors mention that sequencing was performed using a combination of PacBio and MiSeq. Please clarify.. Lines 348-49 : Remove last sentence of the paragraph

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.

Title: POPULATION STRUCTURE AND PHENOTYPIC VARIATION OF SCLEROTINIA SCLEROTIORUM FROM DRY BEAN (PHASEOLUS VULGARIS) IN THE UNITED STATES Review round: 1 Reviewer: 1

Basic reporting: -The article is well written. -Literature References are relevant and fits the study and within a broader field. -The structure of the article is of good standard. The format of PeerJ is the opportunity to provide greater detail and more in-depth discussions that may be restricted in other journals. However, there is a delicate balance of introducing as much detail as necessary while retaining the attention of the reader. This article is on the borderline of introducing too much detail. For instance, is the use of Figures 1, 2 and 4 all necessary? The underlying question is, Do the Figures and additional detail in the paper enhance the paper or create a distraction from the primary message of the study? And while there is great detail on different analyses there is lack of depth on the Mexican population? Is this an important population and why? -Complete study without indications of fracturing of the research to increase publication count. Experimental design: -Well defined gap in scientific knowledge, confirms the efficacy of white mold screening nurseries through population genetics analysis and associated aggressiveness of isolates. -Appropriate use of technologies for investigating genetic populations with that are predominantly clonal. Identified and evaluated an appropriate phenotype of interest (aggressiveness). Identified the limitations of the study, in that the pathogen is not host specific and may influence conclusions of the study. -Methods would allow for an investigator to replicate the same or similar study to confirm results or develop new studies. Validity of the findings: -Study provides novel results in the scientific field of S. sclerotiorum on dry beans an important agricultural commodity. Potentially validates the continued use of nurseries for resistance selection in breeding and introduces a potential area of concern/focus for future research with the Mexican population of pathogens. -Data is provided in repository: https://github.com/everhartlab/sclerotinia-366/. Review of data available (includes: year, location, host…) indicates the opportunity for evaluation/use of data to confirm results and/or in future studies. Statistical analysis of data was appropriate. -Conclusions are well stated and appropriate. Additional comments: Overall this paper did a wonderful synthesis of the topic while applying and interpreting the results with good scientific standards. I found only one important area of correction which was in the abstract requiring the clarification of "11 states in the United States of America,...". This is noted in the material and methods but is absent in the abstract. From a scientific perspective, it should be noted that additional phenotypes for aggressiveness should be evaluated in future research. The straw test is only an indicator for one type of pathogen potential and limits interpretation of the populations. The conclusion given in this study is valid but greater resolution may have been possible with more phenotype data (and of course larger populations). I was also interested in more interpretation/analysis as to why the Mexican population did not have MLH shared with any other regions, the absence of clarity on this is done at a loss.

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.

Title: POPULATION STRUCTURE AND PHENOTYPIC VARIATION OF SCLEROTINIA SCLEROTIORUM FROM DRY BEAN (PHASEOLUS VULGARIS) IN THE UNITED STATES Review round: 1 Reviewer: 2

Basic reporting: Manuscript is written clearly, with appropriate references but limited introduction. Please see general comments reagrding that. The authors has clearly stated research questions, hypothesis, detailed M&M, concise results, and extensive discussion. Experimental design: Research questions were clear and addressed appropriately in subsequent analyses. Methodology and data analyses are described with sufficient details to allow reproducibility. Validity of the findings: Data was robust, available for reproducibility and well explained. Additional comments: Population structure and phenotypic variation of Sclerotinia sclerotiorum from dry bean in the United States by Kamvar et al. utilized microsatellite loci to answer number of interesting questions including evaluating phenotypic and genetic diversity of S. sclerotiorum in the nurseries (here referred as natural populations since no control was used to limit disease spread) using regional differences and across different time intervals (span of nine years). In addition, the authors investigated correlation between mycelial compatibility groups and multilocus haplotypes among these populations. Introduction is a bit short and I would suggest expanding few sections (please see specific comments below). Materials and methods are precise and well written and I really appreciated data availability, including all sorts of analyses, which was refreshing. I also like the acknowledgment of shortcomings of the analyses (compound microsats example), which can be challenging to work with. Results were explained well with few exceptions that need some clarification. Overall, well written manuscript with interesting and relevant results for nursery producers/growers. As such, I recommend it for publication with minor revisions.

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.

Title: POPULATION STRUCTURE AND PHENOTYPIC VARIATION OF SCLEROTINIA SCLEROTIORUM FROM DRY BEAN (PHASEOLUS VULGARIS) IN THE UNITED STATES Review round: 1 Reviewer: 3

Basic reporting: The paper focuses on addressing a question of the genetic diversity of Sclerotinia and addressing how this genetic diversity could be ligated to virulence and compatibility between strains. The context provided for the paper is sufficient to state the goal of the research, and the authors show knowledge of the system and the analyses required to achieve the stated goals. The article is sound and data/code for analyses have been made public and easily accessible. In general, the conclusions were supported by the data, there are some points that required some clarification, but overall the research was well developed and the data is nicely presented to follow up the document. Experimental design: The paper is one of the most extensive population studies in plant pathogens addressing questions on the structure of the population and the correlation of genetic diversity with specific traits. The goals and research question stated by the authors were mostly addressed with the data and there are points that despite the difficulty of the question, there are good approximations to the answer. For instance, the limited information provided by SSRs could not potentially lead correlation with phenotypic traits, therefore the authors acknowledge this, and approach the question using different statistical methods. The methods and the analyses conducted are well explained and the authors provided all the code and data for corroborating the results. Validity of the findings: Overall the study provides an extensive view of the genetic diversity of S. sclerotiorum in screening nurseries and commercial fields of dry bean. Despite that the question of how genetic diversity links phenotypic traits like MCGs and virulence has been approached before, the authors analyzed a large number of isolates using multiple markers and different statistical approaches to answer this question. The result is still negative since there are limitations by the markers, and since this pathogen has reduced diversity. Sclerotinia is soilborne pathogen and it is expected that there should be constraint populations at the geographical level, however, there is a reduced diversity suggesting little differentiation among regions. This is addressed by the authors, where soil or contaminated plant material could have played an important role on the transmission of this pathogen. In addition, the goal of establishing a census of the genetic diversity and its relation to aggressiveness is major task but necessary to establish a baseline for breeders to target a representative pool of the pathogen’s population. Nevertheless, the authors go through a good job of addressing the issues and limitations of the study. There are some points or comments that I would recommend to the authors to discuss and/or consider, those were included on the general comments. Additional comments: • The availability of the data and the analyses posted on github was really helpful and it made very enjoyable to read the paper and understand some of the logic of the authors, it has been a great experience. It also provides a good view on the paper and it helps to assess paper and give recommendations on the paper. Kudos! I want to compliment the authors for making these resources available. • Since populations for certain areas were only collected within a single year, variation between years and region should also be looked at with caution. Are year and region still important if samples with more than one year are retained? How much variability is explained if so? It will a good way to corroborate if there is a continuum of genotypes or every year is bottleneck increasing diversity and to determine how much populations sampled once contribute to the analysis. However, the authors are aware of this on line 350-352. • One thing to be addressed is how well these microsatellites represent the whole genome, this was not addressed on Sirjusingh and Kohn (2001) maybe due to the lack of the genome sequence. However, this could explain the lack of power of the existing set to represent the haplotypes in the population. Despite, that most studies are using reduced genome approaches or more powerful techniques, it will be informative for other researchers still using this set of microsatellites to have this information. Njambere et al. (2010; 10.1139/G10-019) did an approximation for S. trifoliorum using linkage groups, but I am not aware of something similar for S. sclerotiorum to corroborate these SSRs. • The relation between MCGs, MLH and aggressiveness is quite interesting, however, it is hard to follow in the text. The graph in github (https://github.com/everhartlab/sclerotinia-366/blob/master/results/mlg-mcg.md), summarizes really well some on this information as well as table 3. Maybe you can consider including this graph either on the article or add a column to table 3 with the average aggressiveness. The graph might be more meaningful since you can see the variability of aggressiveness of the different strains by the different factors. • L384-404 The authors have a discussion on differentiation of the population based on region. Then, the discussion is centered on how regions like WA still had a considerable amount of variation within the US locations sampled. However, one of the points discussed is that one of the locations was inoculated in 2002 and then crop history differed between the two locations sampled. As the authors suggested there is little or minimal differentiation between isolates from different hosts. Nonetheless, the source of the sclerotia used to inoculated the fields is also different. This could be also part of the differences that the authors see in 2008. Despite that other studies have indicated a limited differentiation between hosts, it seems that there is some effect on the genetic. Is the virulence different on these isolates with respect to other isolates? Aldrich-Wolfe et al. (2015) presents information on the allele sizes for the markers used, are these haplotypes present in the current study? It will be interesting to determine if most haplotypes are share or not, and if those present across multiple hosts have a different virulence. However, the major point of the paper is dry bean but the history on crop rotation could explain some of the variability across years. • P6L262 varaibles change to variables

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.

Title: EPIGENETIC CONSIDERATIONS IN AQUACULTURE Review round: 1 Reviewer: 1

Basic reporting: The importance of epigenetics in different fields of biology including fundamental molecular biology, evo-devo problematic and more applied ecological contexts has led to a tremendous increase in the literature, urging for appropriate syntheses of the recent knowledge and perspective in the related fields. Here the authors propose a review about the 'epigenetic considerations in aquaculture' that suits the readership of the journal in my opinion. The work is divided into two parts, with the first dedicated to a short but comprehensive description of epigenetic mechanisms in the different taxa that are considered in the manuscript, which are the most important for aquaculture, fish and shellfish. Then the authors provide a clear perspective of the broad interests of application of epigenetic knowledge for aquaculture present practices and future developments. The manuscript is well written and the quality of the writing was really appreciated. I also congratulate the authors for bringing together data from vertebrate and invertebrate groups. This constitutes to me the strength of the manuscript and its most important contribution especially when compared to a review by Moghadam et al. in 2015 on the same topic but that 'only' considered fish. Nevertheless there are a few substantial comments that should be addressed before acceptance (see other sections below), mostly clarification of apparent contradiction and inclusion of recently published findings. Experimental design: Why did the authors choose to use Google scholar to browse the literature? There are known issues with the quality/relevance/extensiveness of the results provided by this service when compared to 'classical' scientific literature databases (Lewandowsky et al. Online Information Review 2010,Levine & Gil 2009). I believe this might be due to the lack of such a unique database covering the width of the reviewed topic, especially for aquaculture journals. Please provide justification in the text. Validity of the findings: Although the literature reviewed is mostly cited in a relevant, exhaustive and comprehensive manner, there are some places where the authors need to provide additional data, details or explanations of their interpretation of the cited references at the cost of what seemed to me incomplete or contradictive review. There are two places in particular: - Lines 201-203: ' The function of DNA methylation also appears to be similar across vertebrates with the exception of a role in genomic imprinting which is unique to mammals (Potok et al. 2013)'. In this paper, Potok et al. considered 'imprinting ' in the sense of McGowan and Martin, ie. ' imprinting results in an inability to reproduce parthenogenetically because a genetic contribution from both parents is necessary to successfully complete development.' (McGowan and Martin, 1997). However, imprinting is a broader phenomenon which is mostly considered as 'the non-equivalent contribution of the parental genomes to the embryonic genome', or 'the preferential expression of a parental epiallelle over the other'. The existence of such different contribution of parental nuclear genomes, ie imprinting, has been demonstrated in fish and is described by the authors lines 325-332 ' Using genome-wide DNA methylation profiling, authors showed that pseudomales (generated by exposing genetic females to high temperature during a sensitive developmental window) exhibit methylation patterns consistent with genetic males, both of which differ from the methylome of normal females. Excitingly, it was reported that global methylation patterns are inherited by F1 pseudomale offspring generated by crosses between pseudomales and normal females, suggesting transgenerational epigenetic inheritance of environmentally-induced sex reversal in this species (Shao et al 2014).'. Those two claims contradict one another to me. Please clarify. - Lines 225-226 : remove '(and putative promoter regions)', which is debated and not assumable regarding the present knowledge. -Line 336-350: Please include discussion on nutritional influence on phenotype in bivalves (broodstock conditionning....). - The literature about DNA methylation in marine organisms has significantly increased very recently, and brings data that should be included here. importantly, please consider Knecht et al Toxicol Appl Pharmacol 2017, Burgerhout et al PloS one 2017 and Artemov et al Mol Biol Evol 2017 for the fish; Riviere et al PLoS Genetics 2017 and Gonzalez-Romero Aquat Toxicol 2017 for the oyster. An important issue in aquaculture is the control of the reproduction cycle of exploited species. The manuscript deals with sex determination but would gain an additional asset if presenting a short paragraph on this topic on both fish and bivalves. See for example Coveto-Solo et al. Anim Genet 2015, Zhou et al Int J Mol Sci 2016 for fishes, Jiang et al Mar Biotechnol 2015 in the oyster. The reference list needs format editing for homogeneity. Additional comments: No additional comments

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.

Title: EPIGENETIC CONSIDERATIONS IN AQUACULTURE Review round: 1 Reviewer: 2

Basic reporting: The present manuscript represents a very interesting effort in bringing together current epigenetic and marine biology knowledge. It is especially interesting the insight this work provides concerning the potential of epigenetic approaches to improve aquaculture practices. Epigenetics is a relatively young discipline, nonetheless, it constitutes the basis for most molecular biology research done in model organisms nowadays (e.g., cancer research). Still, this approach lacks development in other fields encompassing critical environmental components such as ecology, marine biology, and fisheries, just to mention some. In that sense, the present work has the potential to make a great contribution to bring epigenetics into the latter, helping improve current management, conservation, and restoration strategies. I found the present manuscript well organized in essence, with most of the relevant literature cited as well as many gaps in knowledge addressed. That being said, I believe the work needs substantial improvements to live up to its title. If this work is meant to be a foundational reference (as mentioned by the authors at some point), it requires further elaboration on critical points, as well as additional references and contents completing the information provided. Overall, I think this work is promising, however, I am recommending major revision before further considering it for publication in Peer J. I believe authors can improve this work substantially and I hope the comments below might help them do that in the best way possible. Experimental design: N/A Validity of the findings: N/A Additional comments: Abstract: Current narrative is a bit cloudy with some awkward sentences “… has dramatically increased to where we are now able to characterize …”. Overall, my suggestion is to make abstract more descriptive of the key concepts developed throughout the manuscript. Line 54: refeernces for Wang et al 2009, and for Wu et al. 2011, are duplicated in the references section. Figure 1: Is it correctly represented? Why is there black and grey text? Why is epigenetics box intersecting with genetics? Why not with environment? Where are the references supporting the different markers used? These should be indicated in the figure. Please, provide a more coherent and detailed explanation of this figure. Line 89: Survey methodology is not really appropriate as a section in the manuscript. This looks like some sort of justification in case any relevant references are missing (blame Google!). It seems a bit narrow the fact that this revision can just be based on a google search, rather than on author’s genuine knowledge and passion for the topic through the years. Line 122: what about plants? Since general background is given here, it is relevant to mention that trans-generational transmission of DNA methylation has been observed in plants. Please elaborate. Line 127: Histone modifications are explained here but the epigenetic role of histone variants is completely neglected? What about their role (and the role of their PTM modifications) during epigenetic regulation? This must be addressed here. Line 133: degree in which DNA wrapped around would be probably better explained in terms of electrostatic affinity between DNA and histones? Line 132: core histones are modified at N terminal tails, but linker histones are modified at C terminal tail, it would be better to say that PTMs occur mostly at N terminal tails. Line 136: “These states are dependent on the type (i.e. …) since not all modification types are mentioned here, it is probably better to use “e.g., …”. There are many other less know PTMs in addition to the 4 mentioned (sumoylation, Pro-isomerization, ADP ribosylation, biotinilation …), although this is not a review about PTMs, authors should at least aknowledge that more PTMs exist. Line 137: different chromatin states not only depend on modifications, they also depend on histone variants, extensively! Line 139: Lawrence et al 2016 citation lacks details in the references section. Line 141: the concept of the histone code has been losing momentum during the last few years, not sure if relevant to mention that here. At any rate, it would be interesting to at least dedicate 1 sentence to explain what the histone code hypothesis is about. Line 144: add “among many others” at end of sentence, there are many other histone-modifying enzymes not mentioned here. Lines 145-146: references here (except Lawrence) are a bit outdated, I suggest updating this list, some options could include http://www.readcube.com/articles/10.1038/nrg.2016.59 https://www.ncbi.nlm.nih.gov/pubmed/24614311 Line 147: “modifications IS highly regulated …” Line 157: “code for proteinS …” Line 171: Information about trans-generational inheritance of RNAs is lacking, need to include that, see ref: http://www.readcube.com/articles/10.1038/nn.3695 Line 191: I am missing a reference justifying the affirmation made by that sentence. Line 194: Same, reference at end of sentence will be better. Line 239: There is a significant amount of knowledge about histone variants in shellfish, (see work by Jose Eirin-Lopez’s lab), since everything epigenetic seems so scarce in shellfish, I believe those are worth mentioning. Similarly, this group has recently published a paper summarizing epigenetic methods for the study of shellfish epigenetics, I’m surprised this is not even mentioned here: https://www.ncbi.nlm.nih.gov/pubmed/28848447 Line 254: Recent work has addressed the role of histone H2A.X phosphorylation on oyster responses to toxins: https://www.ncbi.nlm.nih.gov/pubmed/28315825 Line 255: Bivalves replacing histones by protamines in sperm DO NOT EXIST. Bivalves either replace histones by sperm-specific histones or by protamine-like proteins. The only molluscs with protamines in sperm are cephalopods. I suggest that authors check https://www.ncbi.nlm.nih.gov/pubmed/19708021 for additional information. Line 278: This section is where this manuscript starts discussing topics never discussed before. This should be the strength of the present work. I encourage authors to revise and further elaborate this section of the manuscript. Line 285: how does “developmental programming” relates to the concept of hormesis? Line 352: would it be interesting to incorporate the concept of “assisted evolution” in the case of aquaculture? This has been described for corals, raising a lot of controversy, would it make sense in the present context?

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.

Title: IDENTIFICATION AND CHARACTERIZATION OF A NOVEL ZEBRAFISH (DANIO RERIO) PENTRAXIN–CARBONIC ANHYDRASE Review round: 1 Reviewer: 1

Basic reporting: The manuscript is written properly with minor misspellings and other mistakes, like activates (line 94), was taken (?)(127), no comma (135), of (165), contains (lines 514-515), and more. The literature seems correct and well covered. The structure of the manuscript is proper with a few logical flaws, at least from my point of view. For instance, why the "The region after the CA domain...." chapter does not directly follow the "Exon lengths..." chapter? In many instances there are abbreviations used that are not explained, e.g. dpf, MO, hpf that can be difficult for scientists from other fields. Overall the manuscript is interesting, produces a lot of data and partially answers the question of the function of the pentraxin domain in CA VI. Experimental design: Experimentally this manuscript meets all criteria needed to be published in PeerJ. The research question is well defined and it clearly aims at solving the presence of an additional domain in non-mammalian CA VI. Experiments are well performed, however some data is missing: 1. In sequence searches there are no e-values and scores given. We don't know if there are more distant sequences that could be added to the analysis. 2. Why the sequences with only 20 aa insertions were eliminated from the analysis? Was anything else wrong with them? 3. What was the ground on which you decided to edit the longer sequences? What, except for the usual sequence length, lead you to such a decision? 4. Since both DNA and protein sequences seem very similar why did you decide to analyze both types of trees? Usually it is used for more distant cases. 5. In the manuscript you discuss almost the same frequently figures from the main manuscript and the supplemental ones. I think you should decide about that because it is quite inconvenient for the readers. 6. In the "Evaluation of potential..." part of the Methods you state the presence of a hypothetical 18 residues helix. When reading from the beginning the reader has no idea what it is and why you even mention it. 7. At the end of the "Construction of recombinant..." method you mention another PCR. Honestly, I couldn't get what this PCR is for and what is the difference from the previous one. It should be clarified. 8. In "Quantitative real-time PCR" you do not mention how many times you tried this experiment, for statistical reasons. 9. "In the "Non-mammalian CA VI..." Results section you do give details about conservation of the CA VI amino acids (xx out of yyy), but you miss it for the PTX domain. 10. In the phylogeny figures you do not mention if the sequence used is DNA or protein. It should be mentioned in the figure legend. 11. If you want to emphasize specific data from trees you may colour them accordingly. 12. At the end of the first Results chapter you state that PTX is more similar to short pentraxins, CRPs and SAPs. What is the significance of that? I couldn't really find a conclusive answer to that. 13. In the "Platypus is a candidate..." section you do not mention if you tried to fit this contig into the genome. If so where did it fit the best? 14. In my opinion th e"Light scattering analysis..." chapter should be located before the "Recombinant CA VI-PTX..." chapter. 15. Figure 7 is not too well described. Same for Fig. 8. 16. In Fig. 9 there is an inconsistency. What sequence is right: KGP or KQP...? 17. The difference between figures 9 and S6 are not well defined. It needs a better explanation. 18. Your "Conclusions" is a Summary. Validity of the findings: This manuscript describes a novel finding and shows interesting data both in theoretical and experimental parts. The results however do not fully answer the question of the specific function of the PTX domain in the CA VI family. The hypothesis of a replaced attaching function is great and needs future analyses. The results are sound and original and are suitable for publishing after these minor issues are fixed. Additional comments: I lie this manuscript for its broad theoretical and experimental reach. It would be perfect with "the last thrust" that will prove the binding hypothesis. I hope you get to this point soon.

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.

Title: IDENTIFICATION AND CHARACTERIZATION OF A NOVEL ZEBRAFISH (DANIO RERIO) PENTRAXIN–CARBONIC ANHYDRASE Review round: 1 Reviewer: 2

Basic reporting: I enjoyed reading this manuscript. Authors address the interesting case of CA6, which gained a pentraxin domain in the ancestor of vertebrates and then lost it in the ancestor of mammals (eutherians?). This study is very thorough, and puts together high-quality phylogenetics, structural, genomics and functional studies. I recommend its publication. There are a few questions that could be discussed. I would love to understand why this happened as it did (1st gain, 2nd loss, combination of those two domains). I know it is probably impossible to answer this kind of questions with high confidence, but at least authors could attempt to discuss possible hypotheses on why this domain combination, why not in mammals, what impact it may have had on the respective functions of the gene? What about CA6 in mammals? A comparison may help understanding the evolutionary history of this gene. Is the loss of the domain associated to variation in sequence conservation patterns, expression of the gene across tissues, etc? According to GTEx, human CA6 is only expressed in skin. Does this provide any hint on the above questions? It is remarkable that the amphipathic helix has been conserved in mammals (btw, I liked how authors inferred the origin of this helix). This suggests it has an important functional role. Any idea on what this role might be? Regarding the pentraxin domain, it would be interesting to see if the patterns of conservation / divergence differ compared to other pentraxins. This may give insights on functional divergence of this domain. In summary, this is a fascinating case for which further functional studies may benefit from attempts to answer the above questions. Minor comments: -Introduction, lines 87-90: I think that sentence is not needed -Methods, lines 168-169: check grammar Experimental design: Minor comment: the phylogenetic tree of the pentraxin domain would be easier to interpret if paralogous pentraxins from other vertebrates (not only human) were included. This is just a suggestion, it's fine as it is now. In future follow-up studies authors may want to scan for potential signatures of positive selection associated to these domain gain/loss evolutionary events. Validity of the findings: no comment Additional comments: No additional comments

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.

Title: IDENTIFICATION AND CHARACTERIZATION OF A NOVEL ZEBRAFISH (DANIO RERIO) PENTRAXIN–CARBONIC ANHYDRASE Review round: 1 Reviewer: 3

Basic reporting: The manuscript by Patrikainen et al., describes the biophysical characterization and bioinformatic analysis (phylogenetic and 3D modelling) of the carbonic anhydrase VI from Danio rerio. Minor comments: 1- Regarding the 3D modelling of the amphipathic helix. Its 3D localization in the interface between CA and PTX domains is unconvincing (Figure 5). My recommendation would be to remove it. 2- I consider it appropriate to cite the Pfam database, where phylogenetic trees and domain architecture can be found, for example: http://pfam.xfam.org/protein/E9QB97_DANRE Experimental design: no comment Validity of the findings: no comment Additional comments: no comment

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.

Title: RELATIONSHIPS BETWEEN CONSUMPTION OF ULTRA-PROCESSED FOODS, GESTATIONAL WEIGHT GAIN AND NEONATAL OUTCOMES IN A SAMPLE OF US PREGNANT WOMEN Review round: 1 Reviewer: 1

Basic reporting: The present study is a longitudinal study aimed to evaluate the relationship between percent of energy intake from ultra-processed foods during pregnancy and maternal and neonatal outcomes related to weight gain and body composition. The article adheres to PeerJ policies in terms of structure, and is written in a clear language providing eloquent explanations. The introduction section is clear and outlines basic principles about dietary intake during pregnancy that aid in understanding the study design and results sections. However there are certain aspects not discussed in the introduction – for example the outcomes of previously published articles using the same data, or the use of the Diet History questionnaire II. As a result these are discussed in the methods section which becomes over-detailed (see examples at “Experimental design” below). Specific comments regarding introduction: • In lines 33-34 “… has been recommended” – a specific reference should be related to these recommendations. If the authors are referring to recommendations by Imhoff-Kunsch & Martorell 2012 then the location of the reference (now appears as if relates only to previous sentence) should be changed accordingly. • Line 64 – “critical” is a strong word to describe the role of UPF consumption in maternal and neonatal health. Maybe “important” or “of importance” or any other choice of words should be considered. Figures and tables are relevant to the content of the article, and are adequately labeled. However, sometimes the text lacks adequate referral to the tables. The first time that tables 3+4 are presented in the text (line 194) there should be a clear referral as to what information the table holds (like previously reported for table 1+2), so that the even a reader that does not want to approach the table at the same time will understand it’s value. When citing data from table 5 in lines 220-221 it is advisable to include p-values in the text since these are the major outcomes of the study (same as should be mentioned for all outcomes in the text). Specific comments – • The use of an asterisk in the tables to emphesize p-value<0.005 is confusing in tables 3&4 when it is also used in the first column (“for PEI-UPF * Age” etc.). Alternative symbol can be used or significant p-values can be bold text. Experimental design: The study design relies on data collected for previously published studies by the same group. This leads to some undefined issues in the study design. The choice of two groups of patients (lean & obese) and the exclusion of overweight patients is indeed cited as a limitation on the discussion section; however it does require an adequate explanation in the “study design” section. Lines 92-101 describe a comparison between these two groups – if it is a previous one it should be in the introduction and shortened, if this is part of the present study – it should be incorporated into the “results” section, and be accompanied by the p-values and possibly a table (a revision of table 1 to show also subgroup specific data and p-values). Several background explanations should be edited out of the methods study and incorporated either in introduction or in discussion, as appropriate. For example: • Lines 105-114 from “Previous research has shown…” to “such as the questionnaire used in the present study”. Or at least until line 109. • Lines 97-99 – explanation of prior research should be in introduction unless a specific method cited in this article is utilized in current study. Lines 116-124 – a little too lengthy, should consider referring to appendix. The comparison of PEI-UPS to total fat intake and total energy intake is not mentioned in the methods, although is discussed in results section and mentioned in the abstract under “Methods” (“The ability of these dietary indices to predict gestational weight gain was also compared with the predictive abilities of total energy intake and total fat intake”). Specific comments: • Lines 84-87 – the authors do not address the difference in the 2 prenatal visits, and therefore the reader cannot assess the importance of the fact that on average there was no difference in gestational age. If, for example this is relevant for GWG assessment than some data about the average interval between visits could be more appropriate. • Lines 158-161 – exactly repeat lines 87-89 without adding information relevant to the statistical analysis of these outcome measures. Validity of the findings: “Results” section - Even though this is an analysis of pre-published data the results section should be wholesome and represent all information regarding results. Number of patients recruited, how many in each group (lean/obese) etc. prior to presenting table 1. Specific comments – • All data should be presented with p-value in the text, even if p-value appears in table (example line 217-218, 220-221, 223) “Discussion” section – The comparison between HEI-2010 and PEI-UPP discussed in lines 275-284 is unclear and partially inconsistent with data presented in table 5. Lines 277-278 state that PEI-UPF (note spelling mistake since this line says UPP) is a better predictor of neonatal body fat percentage which is consistent with the table (although requires p-values in the text and referral to table). However according to table 5 for skinfold thickness at the subscapularis both tests are significant (and HEI-2010 even with a lower p-value!), and for the thigh both are non-significant. Therefore when stated in live 278 that PEI-UPF is a better predictor in all these categories it is not clear on which statistical parameter the authors rely for this statement (they do not refer to any tale or p-value at this section). Additionally in line 281 the p value stated for association of HEI-2010 with body fat percentage is stated to be 0.334 whereas in table 5 it is 0.3 and the value of 0.334 cannot be found elsewhere in the data. Line 303 – not only pregnant women may underreport food intake, it is a human tendency. The words “subjects” or “participants” is probably more appropriate. “Conclusions” section – The conclusion sections should briefly outline the main findings of the study without repeating general observations already presented in the discussion. Therefore should consider omitting lines 315-317 (from “excessive gestational weight gain”…”to “associated comorbidities”) and lines 319-320 (from “thus, from a clinical standpoint…” to “that energy”). Additional comments: This study is addressing important issues regarding maternal nutrition during pregnancy and its effect on maternal gain weight and neonatal obesity measures. This field is of growing interest in many countries.

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.

Title: RELATIONSHIPS BETWEEN CONSUMPTION OF ULTRA-PROCESSED FOODS, GESTATIONAL WEIGHT GAIN AND NEONATAL OUTCOMES IN A SAMPLE OF US PREGNANT WOMEN Review round: 1 Reviewer: 2

Basic reporting: Manuscript : 18220v1 Title” Relationships between consumption of ultra-processed foods, gestational weight gain and neonatal outcomes in a sample of US pregnant women” 1. Title: It is clear. 2. Abstract : Background, Methods, Results are Ok . In "Discussion": 1) Spell out "PEI-UPF". 2) The first sentence should be more specific. 3) Instead of 'several maternal outcomes", the authors should say "increase gestational weight gain" . 4) Instead of " several neonatal clinical outcomes", the authors should say" increase neonatal body fatness" 3. Introduction: It is clear and to the point. 4. Method : It is very well written and clear. 5. Results and Discussion are very well written 6. Conclusions: It should shortened. I suggest to delete the two sentences on line 315 and 319 that start with “Excessive gestational weight….” and ends with “ food consumed.”. 7. References are pertinent and updated. 8. Tables and Appendix are OK. Experimental design: "No comments" Validity of the findings: The results are novel and important. Additional comments: Important article that demonstrates that diet quality seems to be more important than the amount of food consumed in pregnancy.

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.

Title: RELATIONSHIPS BETWEEN CONSUMPTION OF ULTRA-PROCESSED FOODS, GESTATIONAL WEIGHT GAIN AND NEONATAL OUTCOMES IN A SAMPLE OF US PREGNANT WOMEN Review round: 2 Reviewer: 1

Basic reporting: NA Experimental design: NA Validity of the findings: NA Additional comments: The authors have revised the manuscript in accordance to our previous comments, and is now vastly improved.

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.

Title: THE IMPACT OF CHOOSING WORDS CAREFULLY: AN ONLINE INVESTIGATION INTO IMAGING REPORTING STRATEGIES AND BEST PRACTICE CARE FOR LOW BACK PAIN Review round: 1 Reviewer: 1

Basic reporting: This article is generally well written, appropriately referenced, conforms to PeerJ standards for structure, and has clear tables. There are three areas where I believe the basic reporting could be improved: 1) The way in which the intervention groups are described is frequently confusing, both within the abstract and the paper itself, for example “to compare the effect of receiving imaging results with best practice care (without imaging)”. I suggest rewording this with something along the lines of ‘to compare the effect of receiving imaging results with the effect of receiving best practice care that does not include imaging’. It may be helpful to provide a numbered list of the primary aims in the same way the secondary aims are presented. 2) The last sentence of paragraph four in the introduction “Also of interest is evidence that providing patients with information about normal results - prior to their receipt of their own results - may optimise the reassuring potential of normal findings” is discordant with the previous paragraph that suggests imaging is not reassuring. I suggest rewriting the sentence. 3) It would be useful to have exemplars of the pre-information, the normal scan report, the epidemiological information, and the enhanced report that were provided. These could be added to supplement 4 or 7. Experimental design: I have two key queries in relation to study design that it may be helpful for the authors to address in the discussion: 1) Why was a best practice care plus imaging group was not included? Although I am not a proponent of scanning for reassurance, I could imagine that if I was I might also consider that I do this in addition to providing best practice care (excluding the explanation that scans are not helpful). This means that I could look at these results and see that best practice care was well received, but also that scores in the scan groups appeared to improve rather than deteriorate after receiving scan reports, so there is a possibility that receiving both of these interventions could be helpful. This dichotomy is carried over to the discussion where it is inferred that GPs choose between either scanning or providing explanation, rather than being able to provide both. 2) Why was the TSK-11 was not completed at baseline? With no baseline scores it cannot be assumed that the small differences between group found post-intervention are related to the intervention. It is also uncertain whether these between group differences are meaningful (given a four-point change in TSK-11 score is needed to identify an important decrease in fear of movement (Woby et al. 2005) ). I have some additional queries and suggestions for the authors’ consideration. 3) In terms of participants, were health professionals and other academics included in the authors’ networks and eligible to participate? This may have biased the results as these people may share similar views about imaging to the authors. The risks and benefits of using these networks could be discussed in the strengths and limitations section. 4) Was there any assessment of the psychometric properties of the BRP (which I assume was developed for this study). These could strengthen interpretation based on changes in this measure. 5) When and why was the Back Pain Attitudes Questionnaire completed and what were these data used for? These data are not currently presented. Validity of the findings: The data generally appear to be robust, however, I am not qualified to comment on the statistical analysis. 1) It would be useful to have mean change scores and 95% CIs presented for the ANCOVA analyses. 2) The sentence “This study provides evidence that simply offering best practice care is indeed effective management for recent-onset LBP” considerably overstates the results from this study as no one with recent onset LBP was included in the study and no clinical outcomes were measured. Additional comments: Thank you for asking me to review this paper. It is an interesting study design and makes innovative use of technology to explore a hypothesis in a large sample of people. The paper is generally well written and related to current literature and thinking. Given the design, my impression is that the results can only be exploratory and the strength of these is overstated at times in the discussion. Overall, I think that this paper makes a useful contribution to the knowledge base, and I am supportive of its publication, however, there are a number of places where I think it may be challenged and these may benefit from being addressed.

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.

Title: THE IMPACT OF CHOOSING WORDS CAREFULLY: AN ONLINE INVESTIGATION INTO IMAGING REPORTING STRATEGIES AND BEST PRACTICE CARE FOR LOW BACK PAIN Review round: 1 Reviewer: 2

Basic reporting: Overall the article demonstrated a clear, professional and unambiguous English language.In order to improve the clarity I suggest that the author rewords line 5 to 8 in the background section to make the aim less ambiguous. The sentence line i282 is rather awkward and should be restructured,. Appropriate literature reviews, references and context of this novel work in order to address gaps in the literature, Good links to the high prevalence of degenerative disease in asymptomatic individuals and potential for harmful misinterpretation of imaging. In order to provide more context on the role of imaging I would suggest referring to current guidelines for the use of imaging in LBP to identify cases where imaging is indicated and when it is not. Also add a line to clarify that most cases of LBP are self-limiting and subside within a particular timeframe and does noit require investigations.. Professional article structure, tables and raw data appears to be accurate and clear. Relevant results were given to confirm the hypothesis Experimental design: This study is an original primary research within the scope of the journal. Research aims and secondary aims were well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap. You did not define the research question but identified the knowledge gap being investigated and the study contributes to filling that gap. Appropriate and novel investigation performed to a high ethical standard. simulated patient, 3-armed, randomised online experiment. However, how valid and reliable is his simulated patient approach compared to real life patients this needs to be justified. Are there any reviews or studies looking at this? If so I would add to the methodology section. You mentioned that many adult volunteers suffered with LBP but did not indicate the severity or disability associated with this.. You could have included information on participant levels of anxiety or depression, work status and hobbies since these factors could have affected the results. Perhaps add more detail regarding the back pain questionairre and how that impacted upon the results. Methods described with sufficient detail to replicate the study. Validity of the findings: Data is robust overall and clearly presented, a sample size calculation was performed the dtests utilised were statistically sound. The participants were randomised, and blinded to the other interventions.. The Primary outcome measure used was the BPS Line 149. Has the validity and reliability of this measure been established? If so it needs to be stated. What was the authors rationale for combining the 3 numerical rating scores. This needs to be stated. The data on which the conclusions are based are provided and is statistically sound.. Appropriate discussions made where rationale & benefit to the literature and patient management in primary care is clearly stated. Conclusion are well stated, linked to original research aims & limited to the authors supporting results and the limitations of this study using simulated patients has been addressed. Additional comments: This is an interesting and novel study with acknowledged limitations but has a sound conclusion and clear implications for primary care practice in the management of low back disorders.

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.

Title: THE IMPACT OF CHOOSING WORDS CAREFULLY: AN ONLINE INVESTIGATION INTO IMAGING REPORTING STRATEGIES AND BEST PRACTICE CARE FOR LOW BACK PAIN Review round: 2 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: The authors have appropriately addressed the comments and suggestions.

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.

Title: THE TETRAPOD FAUNA OF THE UPPER PERMIAN NAOBAOGOU FORMATION OF CHINA: 1. SHIGUAIGNATHUS WANGI GEN. ET SP. NOV., THE FIRST AKIDNOGNATHID THEROCEPHALIAN FROM CHINA Review round: 1 Reviewer: 1

Basic reporting: The manuscript has been improved since last I reviewed it, and my comments are fewer, see below. I remain unconvinced that this is not just a Chinese specimen of Annatherapsidus, but at this point the authors and I will just have to agree to disagree on this point. In particular, though, I have doubts as to the presence of eight upper postcanines in this specimen. On the right maxilla there are only five alveoli, and that large of a discrepancy in tooth count between sides would be highly unusual in a therocephalian. It is difficult to see the alveoli in the figure provided, and it appears the alveoli in the left maxilla may not be fully prepared. A better figure of the specimen in palatal view would greatly improve this situation and make the identification as a new taxon much more convincing. Line 12: “well know” should be “well-known” Line 18: “akidnognathid” is misspelled Line 19: “one species is known from Russia”—incorrect. There are two species of Annatherapsidus from Russia (A. petri and A. postum). (Furthermore, there are other Russian therocephalians that may be akidnognathids based on some phylogenetic analyses, e.g., Scylacosuchus.) Line 30: “component” should be “components” Line 35: “represented by a few species”—well, there are 55 named species of these therocephalians, none of which have been formally synonymized outside of an unpublished PhD thesis, so it is hard to say. I agree that once all the revisionary work is done there will be few left, however. Line 68: As noted above, multiple species of Annatherapsidus from Russia are known. See Ivakhnenko (2011). Line 87: “von Nopsca” should just be “Nopcsa” (note spelling) Line 170: “is much robust” should be “is much more robust” Lines 203–204: “Middle Permian” and “Late Permian” are no longer formal chronostratigraphic subdivisions, please use the informal “middle Permian” and “late Permian” instead. Lines 209–211: Change this sentence to “Although middle and late Permian deposits in China have revealed a diverse, species-rich vertebrate fauna, therocephalian records have proven elusive.” Line 264: Throughout this paragraph the family names Akidnognathidae and Perplexisauridae need to be capitalized. Also, why is it called Perplexisauridae when Figure 5 (and existing therocephalian literature) calls this clade Chthonosauridae? Line 288: “akidnognathid” should be “akidnognathids” Figure 1: It would be easier to interpret this figure if you note the type locality of Shiguaignathus by listing the genus next to the dot, like you do for Gansurhinus and Daqingshanodon. Figure 5: “Euchambersia” and “Lycideopidae” are misspelled. Also, the genera “Ophidostoma” and “Microwhaitsia” are currently unpublished. Unless you are absolutely certain that these names will be published before the Shiguaignathus manuscript, please replace these names with the respective specimen numbers so as not to create nomina nuda. Experimental design: See above. Validity of the findings: See above. Additional comments: No additional comments

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.

Title: THE TETRAPOD FAUNA OF THE UPPER PERMIAN NAOBAOGOU FORMATION OF CHINA: 1. SHIGUAIGNATHUS WANGI GEN. ET SP. NOV., THE FIRST AKIDNOGNATHID THEROCEPHALIAN FROM CHINA Review round: 1 Reviewer: 2

Basic reporting: Liu & Abdala present a well-preserved palate and snout of a putative new taxon of akidnognathid therocephalian. The overall presentation and illustrations are good, although they would benefit greatly from comparative photos of other akidnognathids and close-up illustrations of some of the purported anatomy, especially the dentition (or what little of the dentition is preserved). Experimental design: The experimental design is good and follows standard geological, paleontological, and phylogenetic procedures. Validity of the findings: The findings are important and convincing with regards to the presence of an akidnognathid therocephalian in this fauna. However, I have some questions about the generic diagnosis and justification of the new taxon (see detailed comments below). Additional comments: I congratulate the authors on their discovery of a very nice centerpiece specimen for this study, and on the excellent presentation of their paper. Regarding my specific concerns, I want to make clear to the authors & editors that I have reviewed a previous version of this manuscript that was submitted to another journal. At the time, I did not feel that it merited publication in its current form because the major claim that the authors were erecting a new taxon was not justified by the available information in the fossil as illustrated. Since then, there's been some effort to address my concerns, although the authors continue to treat the specimen with a new genus name. I take issue with most of the characters in their diagnosis, but recognize that one or two subtle proportional traits could be useful in erecting a new taxon, and I am open-minded about these--namely the possibility of a high postcanine count and the obviously small size of the suborbital vacuity. Taking on the the 'Diagnosis' point-by-point, the shape of the choana looks identical to the Russian Annatherapsidus and other early akidnognathids (it is not autapomorphic as their Diagnosis suggests), the extra palatal ridges are also present in Olivierosuchus and Promoschrhynchus (and perhaps others), and the postcanine tooth number appears at first consistent with the primitive number (6-7) based on the photographs, although they dashed-in extra putative alveoli in their drawing that suggest as many as eight postcanine positions--note that a previous version of the manuscript presented only 6 on one side and 7 on the other, which would be consistent with Akidnognathus. Given the ambiguity, I'd like to see this better illustrated with a close-up photo before I am convinced of this, but I suppose it's possible there may be up to eight. The most convincing evidence of a new taxon is in the extremely small suborbital vacuities. I agree with the authors in that I have not seen vacuities this small in another akidnognathid specimen, so perhaps the authors are on to something here. Maybe the importance of this structure should be emphasized for now as evidence of a new taxon until more complete specimens are found in the future? Overall, the new record adds important information about the biogeography and biochronology of Permian therocephalians, and I think it should be published in some form. I will make the recommendation to publish with minor revisions, pending that the authors can (1) add one or two useful comparative photos from the palates of other specimens, (2) better illustrate the dentition to support their claim about the high number of postcanines, which is not super convincing right now, and (3) revise the Diagnosis accordingly. There are also a number of minor spelling/grammar errors in the manuscript and figures (e.g., Lycideopidae is misspelled and placed at the wrong node in the cladogram) that the authors/editors will need to address at some point, as I am only commenting on the manuscript's scientific value at this point. Thank you and good luck! Adam Huttenlocker University of Southern California

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.

Title: MASS MARKING OF JUVENILE SCHIZOTHORAX WANGCHIACHII (FANG) WITH ALIZARIN RED S AND EVALUATION OF STOCK ENHANCEMENT IN THE JINPING AREA OF THE YALONG RIVER Review round: 1 Reviewer: 1

Basic reporting: The paper presents a detailed study on stock enhancement of S. wangchiachii and is written in a clear and concise manner. I would have liked to see a more precise definition of stock enhancement as well as a reference that shows such marking techniques are still clearly visible after several years in some species, e.g. in the otoliths of Acanthopagrus butcheri alizarin complexone was still visible after 15 years (Cottingham et al., 2015). I would also like to have more information on the species, such as maximum size and age and its importance as a targeted species. The figures and tables are clear, however, I suggest smaller font on Table 2 would enable the table to be on one page instead of three. There are also some inconsistencies in spacing. Experimental design: The experimental design was well thought out and executed and within the scope of the journal. The research question was well defined and fills an important knowledge gap. However, the description of the statistical analyses was not sufficient. This section needs to be expanded. Also, in the material and methods it needs to be reiterated that the means of the instantaneous growth and mass were calculated for different batches, otherwise it is not clear to the reader how statistical analyses can be undertaken. Validity of the findings: The study demonstrates that restocking can be used as an effective measure to enhance the population of this species. The data was robust in that enough replicates were employed to undertake statistical comparisons. It was concluded that this study offered a cost-efficient method for mass marking, but the costs are not discussed in the text. Also, there is nothing in the introduction about total length, mass and condition factor and what these measurements represent, their meaning and why such measurements should differ between marked and unmarked fish. Additional comments: I outline some general points below. Use mm throughout instead of cm. No dash required between numbers and units. Line 29. It is not clear what slightly increase means. Line 43. otolith marking should be mentioned first as it is the most important in your paper. The same goes for fluorescent marking in line 46. Line 137 Of the 600,000 marked fish, why were only 400,000 released. Line 153/154 Consider rewording sentence. Line 174 Three pairs, do you mean Sagitta, Asteriscus and Lapillus? If so, which ones did you mainly use. Line 196/197 different font and double spacing Line 229 The slight difference was statistically different, which one was less. Line 234-242 What about condition factor between marked and unmarked fish. Line 253 are the instantaneous rates unitless? Line 323 What is the relevance of this? Table 1 caption- treatments spelt wrong. Figure 4 caption. Consider rewording

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.

Title: MASS MARKING OF JUVENILE SCHIZOTHORAX WANGCHIACHII (FANG) WITH ALIZARIN RED S AND EVALUATION OF STOCK ENHANCEMENT IN THE JINPING AREA OF THE YALONG RIVER Review round: 1 Reviewer: 2

Basic reporting: This ms fulfills the criteria given. General comments: An interesting and most clear paper on an important problem, i.e. how to evaluate the outcome of massive restocking activities using young/small fish individuals. My main concern, besides some details below, is that the paper is unnecessary long and complex. The main reason is that so much statistics is presented not only in the tables, but also in the running text. Please consider to omit most of those details in the text, but comment on them using a reasonable amount of details and “digits”. I am not that familiar with this journal and it rules, so the following are most as questions and suggestions; • As the same words and terms appear in both the title and as listed key-words, the key-words may be changed to increase the chance of being found in a search. • Seems the references seem to come in the wrong order when several are given within brackets in the running text, i.e. the come in alphabetic order and not in time order that is the most? common way in scientific papers. Experimental design: Seems OK with me. In a way there are even two control groups, one is the unmarked fish stocked in the river and the other one are the groups held in ponds, where I assume life and growth is more "comfortable". Validity of the findings: OK, but as statistics is not my expertise I cannot judge that part in any details, but looks convincing to me. Seems the results are so obvious. Additional comments: See above (1. Basic reporting)

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.

Title: STEM-LOOP STRUCTURE PREFERENCE FOR SITE-SPECIFIC RNA EDITING BY APOBEC3A AND APOBEC3G Review round: 1 Reviewer: 1

Basic reporting: In this manuscript, Sharma et al. have examined the role of stemloop structure and sequence in A3A/G activity. The work follows up on previously published findings by the group that A3A can edit RNA in monocytes and macrophages. In this manuscript, the impact of RNA shape and sequence is examined. Experimental design: I found experimental design to be quite standard and sound. A3A can be purified from eukaryotic or prokaryotic expression systems, and in the present work the authors have used a C-terminally His-tagged version purified form a bacterial expression system. This protein is incubated with the RNA substrates, mutations are measured by sequencing. Overall, the protocol follows established assays and data analysis was sufficiently detailed and rigorous. Validity of the findings: The study identifies important structure and sequence aspects of the RNA substrates. The data is in line with the well-established sensitivity of all AID/APOBEC family member enzymes to ssDNA shapes and sequence motifs. The functional data correlated nicely with predicted RNA secondary structures in a manner expected from the known biochemical properties of AID/APOBEC family member enzymes. I did not find grounds to doubt the validity of the presented findings. Additional comments: In general, I found the manuscript was very well put together, clear and easy to follow. The conclusions are supported by the data and the work was rigorously carried out. My suggestion to the authors are to include additional more comprehensive references: -For instance, in regards to the role of A3s in viral restriction, there is an emerging body of evidence that A3s can also do the opposite, by helping retroviruses escape from drugs and adaptive immune recognition (suggested reference Monajemi et al. 2012, Retrovirology) -In regards to the structure/function of A3 catalytic motifs as discussed in the introduction, there are some nice current structural studies focusing on the catalytic pockets of AID/APOBECs which can be referenced (e.g. King et al. 2017 Frontiers Imm, King et al. 2015 Structure, Shi et al. 2017 NSMB) -The discussion will be improved if the authors also discuss their work in the context of previous works on the relative importance of secondary structure vs. primary sequence for determining AID/APOBECs. For instance, Larijani et al. 2007 MCB (amongst others) did similar work on AID and found that secondary structure (shape) is a more important determinant of AID targeting to stemloops and bubbles, as compared to its trinucleotide WRC sequence specificity.

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.

Title: STEM-LOOP STRUCTURE PREFERENCE FOR SITE-SPECIFIC RNA EDITING BY APOBEC3A AND APOBEC3G Review round: 1 Reviewer: 2

Basic reporting: Very minor comment on the references: it is a bit strange that introduction starts by citing reviews, which is perfectly fine considering the time passed from the initial characterisation of the APOBEC3s, yet only one review of the many labs involved in the initial characterisation (with a few still working on the APOBEC3 genes) is used. Then the introduction moves to describe the APOBEC3 locus, going back to an original paper (Jarmuz 2002 - incidentally the organisation A3 locus described in that paper is different from the current understanding), but Jarmuz 2002 is not the proper reference for the Zn-coordinating residues in the deaminase domain (it should be Betts 1994). Experimental design: no comments Validity of the findings: The findings support the conclusions, pending the clarification of the problems highlighted below: a) lack of negative controls - With regards to the experiments with patients' MEPs, it would be useful to also have a sample in which APOBEC3A is silenced (the same authors have used siRNAs in the past). In Figure 1 there are background peaks in the chromatograms from Donor 2 and 3 HI that are the same height as the edited peak. It seems that even the edited peak could be considered background (which does not interfere with the conclusions) - With regards to the in vitro experiments and the A3G ones, it would be necessary to use a catalytically inactive mutant of the enzyme to show the background levels of editing. All this is necessary as it is not clear what is the dynamic range and the linearity of the editing assays. b) It is not specified how many times the experiments were repeated, and there is no sign of error bars (or statistics) in any of the experiments. c) in parts of Fig.1 the editing of the mutants is quantified through different methods (Sanger sequencing, AS-RT-qPCR). How do these assays compare with each other? Could it be better to keep the experiments separated? Additional comments: The topic is fascinating and the findings have the potential to help us understand the dynamics of APOBEC-mediated RNA editing. The striking observation that specificity and strength of RNA editing seems to be deriving from a segment of 10-16 nucleotides is somehow at odds with the specificity of editing previously described. How can such specificity be conferred by a few bases in the loop? What other elements are necessary for other the editing to be targeted to those transcripts? Considering the data the Authors have accumulated in these years, it might be worth reanalysing the RNA-seq data and seek whether other transcribed sequences bearing identical stem-loop segments lack some other feature present in the edited transcripts.

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.

Title: STEM-LOOP STRUCTURE PREFERENCE FOR SITE-SPECIFIC RNA EDITING BY APOBEC3A AND APOBEC3G Review round: 2 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: The rebuttal of the Authors answers most of my criticism. Yet I wonder why none of their argumentations end up in the manuscript. I would have thought some of those answers would provide context to the presented work and deserve a place in the study. Specific comments: - as I have already written, the background peaks in Donor 2 and 3 do not change the interpretation of the phenomenon, but they do cast doubts on the background levels of the assay and therefore on its dynamic range. In short, if you want to use a live cell assay to study biochemistry, you have to define the dynamic range of your assay, otherwise all your observations are just qualitative (which might be fine, but it should be noted). This is something the Authors should deal with. - I apologize for missing the number of times the experiments were repeated (though it is not the usual notation) and the standard deviation. Would it be meaningful to add some statistics to describe the various efficiencies? - Do the authors realize that maybe not every reviewer/reader is familiar with Supplementary Fig. 8 of Sharma 2015? It might be helpful a citation in the text to support the correlation between the two techniques. Moreover the slope in Suppl. Fig 8 is 0.7. Does this mean that the values were normalized between experiments in order to compare the two techniques? Would it be useful to label each substrate to highlight which experiments the data came from? - Actually (aside from correcting the Betts reference), the references were just fine. It was just odd that the only original work cited at the beginning was the Jarmuz paper, whose importance with regards to the research presented is marginal if compared to other original research (e.g Sheehy 2002, Harris 2003, Mangeat 2003, etc.). It was odd, but a legitimate choice. On the other hand, if Jarmuz is there because it is the first reference for A3A, then it would be fair to also cite the original Madsen 1999 [phorbolin] and Sheehy 2002 [identification of A3G] papers.

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.

Title: COMPARISON BETWEEN RADIAL ARTERY TONOMETRY PULSE ANALYZER AND PULSED-DOPPLER ECHOCARDIOGRAPHY DERIVED HEMODYNAMIC PARAMETERS IN CARDIAC SURGERY PATIENTS: A PILOT STUDY Review round: 1 Reviewer: 1

Basic reporting: no comment. Experimental design: no comment Validity of the findings: In figure 4, besides Bland-Altman plots, linear regression plot with Pearson’s r2 coefficient should be shown to address the CO and SV measured by the two different methods. Additional comments: This paper is well designed to address the priority of the DMP-life vs. TTE and showed convincing data for the accuracy of DMP-life, which could be a proper candidate for the hemodynamic detector for the patients. I recommend the author add more statistical graphs like the linear regression plot with Pearson’s r2 coefficient in Fig.4.

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.

Title: COMPARISON BETWEEN RADIAL ARTERY TONOMETRY PULSE ANALYZER AND PULSED-DOPPLER ECHOCARDIOGRAPHY DERIVED HEMODYNAMIC PARAMETERS IN CARDIAC SURGERY PATIENTS: A PILOT STUDY Review round: 1 Reviewer: 2

Basic reporting: Minor comment: In the exclusion criteria, please confirm the information of LVEF: 40% or 45%? Experimental design: No comment. Validity of the findings: The authors of current study well described their work and got the conclusion. Additional comments: As an alternative choice of pulmonary artery catheter or transthoracic / transesophageal echocardiography measurement, non-invasive radial artery tonometry test has been introduced and functionally well described to measure the aortic pressure, artery stiffness or heart function during the past 20 years. However, less than 20% (107/572) total operated patients could use this method shows that it still need to be well evaluated before widely usage.

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.

Title: COMPARISON BETWEEN RADIAL ARTERY TONOMETRY PULSE ANALYZER AND PULSED-DOPPLER ECHOCARDIOGRAPHY DERIVED HEMODYNAMIC PARAMETERS IN CARDIAC SURGERY PATIENTS: A PILOT STUDY Review round: 1 Reviewer: 3

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: In this manuscript, Zayat et.al first time compared the DMP-life tonometry device measured SV and CO with TTE in preoperative cardiothoracic surgery patients. Their data indicate reasonable accuracy and precision between two technologies. Manuscript is well written and experiments are straightforwardly designed, but the conclusion needs more data to support. Following is my concerns: 1. Authors only used the Bland-Altman analysis to compare the differences between TTE and DMP-life measurement (both SV and CO). Linear regression analysis to illustrate the correlation efficiency can be considered to compare these two methods. 2. In the discussion section, authors discussed the advantages and disadvantages of TTE and DMP-life. But to better explain the feasibility of DMP-life, authors can compare their data to other reported SV/CO monitoring methods. For example, Castro et al (Castro V De, Lhotel L, Mabrouk N, Perel A, Coriat P. Comparison of stroke volume (SV) and stroke volume respiratory variation (SVV) measured by the axillary artery pulse-contour method and by aortic Doppler echocardiography in patients undergoing aortic surgery. 2006;97: 605–610.) compared SVs measured by the axillary calibrated artery pulse-contour method with TTE, and their percentage error of both minimum and maximum SV was 26%. 3. According to a lot literature, many SV/CO monitoring devices have not yet replaced PACs due to poor trending ability. Authors better to have serial measurements at different hemodynamic states (instead of single measurement) to assess trending ability, and analyze ΔSV values using four-quadrant plot or a polar plot analysis.

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.

Title: COMPARISON BETWEEN RADIAL ARTERY TONOMETRY PULSE ANALYZER AND PULSED-DOPPLER ECHOCARDIOGRAPHY DERIVED HEMODYNAMIC PARAMETERS IN CARDIAC SURGERY PATIENTS: A PILOT STUDY Review round: 1 Reviewer: 4

Basic reporting: The aim of this study is clear that the authors tried to use the non-invasive BPPA to replace PWD method in clinics especially in cardiac surgery patients. Experimental design: There were some data showed the mean CO and SV were comparable between BPPA and TTE in coronary artery bypass surgery patients. However, there are no data to show the sensitivity and accurate/reliable dynamic range of BPPA. Validity of the findings: The concept and data in the manuscript are original and useful for clinics. It is much easier to get cardiac and hemodynamic information using this non-invasive way than conventional TTE. Thus, patients will benifit much if this method will be widely used in hospitals. Additional comments: The authors tried to compare the accuracy and reliability of BPPA used in cardiac surgery patients. Over all, this manuscript is well organized and the data are clearly presented. However, there are some places need to be improved: 1) Figure 1 can be removed from the manuscript. Although it shows some standards that exlude the patients from BPPA test, it does not have any information about the 107 patients enrolled in the test, which should be the key information other than the excluded ones. 2) Figure 2 shows the principle of DMP-life system, however, there were not any explanations in the text. Can the authors add some information to explain the key principle of this system and how it is used in patients. 3) Figure 3 shows the results were got at different applied pressure for each tested patients. What do red rectangles and arrow mean in the figure? The shape are different at different pressure for different persons, so is the shape related to the severity of the heart disease? 5) In Figure 4, the description of the figure legend and the titles in the figure are not consistent. Please check and make the figure in the right order and label. 5) The authors only tested the applicability of DMP-life system in patients with cardiac diseases. Why did not you test it on normal people? so that then we can better know how reliable/accurat it is for this system. I encourage the authors add these data in to make the conclusion more solid. 6) Check the writing and format, and keep them consistent. For example, line 39 and line 193 "5.2±0.85" could be "5.2 ± 0.85".

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.

Title: COMPARISON BETWEEN RADIAL ARTERY TONOMETRY PULSE ANALYZER AND PULSED-DOPPLER ECHOCARDIOGRAPHY DERIVED HEMODYNAMIC PARAMETERS IN CARDIAC SURGERY PATIENTS: A PILOT STUDY Review round: 2 Reviewer: 1

Basic reporting: No comment. Experimental design: No comment. Validity of the findings: No comment. Additional comments: The article is ready for publish.

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.

Title: COMPARISON BETWEEN RADIAL ARTERY TONOMETRY PULSE ANALYZER AND PULSED-DOPPLER ECHOCARDIOGRAPHY DERIVED HEMODYNAMIC PARAMETERS IN CARDIAC SURGERY PATIENTS: A PILOT STUDY Review round: 2 Reviewer: 2

Basic reporting: No comment. Experimental design: No comment. Validity of the findings: No comment. Additional comments: The authors of current study well described their work and got the conclusion.

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.

Title: COMPARISON BETWEEN RADIAL ARTERY TONOMETRY PULSE ANALYZER AND PULSED-DOPPLER ECHOCARDIOGRAPHY DERIVED HEMODYNAMIC PARAMETERS IN CARDIAC SURGERY PATIENTS: A PILOT STUDY Review round: 2 Reviewer: 3

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: Thanks for the authors performed the pearson correlation efficiency analysis. But the correlation of CO measurements between DMP-life and TTE (r = 0.501) is not a good correlation, which means the CO measurement might be not accurate as TTE. Moreover, authors claimed that this study is to ensure the accuracy of DMP-life device without reproducibility analysis. Then it is very important for authors to include direct comparisons between DMP-life and standard PAC measurement (and can also compared with TTE), especially at this case, the CO measurements didn’t have a significant positive correlation.

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.

Title: COMPARISON BETWEEN RADIAL ARTERY TONOMETRY PULSE ANALYZER AND PULSED-DOPPLER ECHOCARDIOGRAPHY DERIVED HEMODYNAMIC PARAMETERS IN CARDIAC SURGERY PATIENTS: A PILOT STUDY Review round: 2 Reviewer: 4

Basic reporting: The revised manuscript is well improved. The methods, results and discussion are more clearer. Experimental design: n/a Validity of the findings: n/a Additional comments: The authors addressed my concerns and the manuscript was well improved.

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.

Title: GENOME-WIDE IDENTIFICATION OF ABA RECEPTOR PYL FAMILY AND EXPRESSION ANALYSIS OF PYLS IN RESPONSE TO ABA AND OSMOTIC STRESS IN GOSSYPIUM Review round: 1 Reviewer: 1

Basic reporting: No comment. Experimental design: No comment. Validity of the findings: No comment. Additional comments: The manuscript submit by Zhang et al. regarding genome wide identification of ABA receptor protein family PYLs in four different species of cotton is an extensive report. Though the PYL family members are reported from many other plants, but the current report is the first from Gossypium spp. In spite of no novelty in the entire research the information generated will be of interest for researchers working with Gossypium spp. Apart from identifying the entire family members from progenitor G. arboretum, G. raimondii they also identified them in the tetraploid G. hirsutum and G. barbadense species. The authors also performed the chromosomal location of each gene, synteny among all four species and phylogeny with PYLs of other known plants. The members of G. hirsutum was further investigated for its tissue specific expression, regulation under ABA treatment and osmotic stress. They also analysed GhPYLs interaction with two members PP2C proteins, GhABI1A and GsABI1D known to be involved in ABA signalling. Even though, the later part of the work is fragmented and ends abruptly, the information in the manuscript is interesting. I have following minor concerns for the submitted work: The nomenclature of the Gossypium PYLs are solely based on that of Arabidopsis PYLs. It seems this nomenclature (giving a specific number at the end) does not hold true when PYLs of rice is also considered. Can author take this opportunity to propose a uniform nomenclature of PYLs across different plant species. Tissue specific expression of GhPYLs are compared with the expression of PYLs in root (Fig. 6). As such the modulation of these genes could not be assessed in root. It will be better if an house keeping gene is taken as internal control. The Y2H analysis result may also be presented in tabular form, that will be easy to see in one glance which PYLs are interacting with the two selected proteins.

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.

Title: GENOME-WIDE IDENTIFICATION OF ABA RECEPTOR PYL FAMILY AND EXPRESSION ANALYSIS OF PYLS IN RESPONSE TO ABA AND OSMOTIC STRESS IN GOSSYPIUM Review round: 1 Reviewer: 2

Basic reporting: The authors have identified the PYL genes in Gossypium species. They have identified 21 genes in G.arboreum, 20 in G.raimondii, 40 in G.hirsutum and 39 in G barbadense. They have done thorough bioinformatics analysis of these genes, and have identified conserved secondary structure that are essential for the function for ABA receptors in arab. They have analysed the G.hirsutum genes for the relative gene expression in osmotic stress and in response to ABA. Using two hybrid system they have also shown the interaction of GhPYLs and GhABI1A and GhABI1D. Experimental design: The material and methods are clearly written. Validity of the findings: The authors have done several experiments to validate the claims made in the manuscript. The relative gene expression shows the role of PYL in ABA response and osmotic stress. Similarly the yeast two hybrid analysis shows the interaction of PYL with Gh ABi1A and Gh ABI1D. Additional comments: There was one paper by Chen et al published in PPB on the Overexpression of cotton PYL genes in Arabidopsis enhances the transgenic plant tolerance to drought stress. The authors have not mentioned this in their manuscript and since this paper is on PYL in G. hirsutum, the authors need to revise the manuscript accordingly.

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.

Title: GENOME-WIDE IDENTIFICATION OF ABA RECEPTOR PYL FAMILY AND EXPRESSION ANALYSIS OF PYLS IN RESPONSE TO ABA AND OSMOTIC STRESS IN GOSSYPIUM Review round: 1 Reviewer: 3

Basic reporting: No comment Experimental design: Details were missed in some experiments. For example: Line 148：Which parts of the plants were sprayed with ABA? How to treat with 10% PEG6000? The paragraph of Line 144: You have both treated plants (after Line 145 which is clear to readers) and untreated plants (before line 145). Please make it clearer that the plants were not treated. The roots of the treated plants were sampled and frozen. How were the samples of the untreated plants treated after sampled? Line 150: How many grams of the samples were used for RNA extraction? How were the samples homogenized before the extraction? Line 151 and line 152: The reaction conditions of cDNA synthesis of RT-PCR? Please rewrite this part. Line 155: “Experiments were repeated at least three biological replicates.” Do you mean the experiments were repeated at least three times? How many replicates did you have each time? Line 154 primer: please provide primer sequences in Materials and Methods or in the supplementary Validity of the findings: No comment Additional comments: Generally, the manuscript is written in professional English with minor errors. The study is novel and meaningful. The introduction has sufficient background information.The hypotheses and experiment design are appropriate. But, details were missed in some experiments. The result section including figures and tables are generally OK. But, 1) the explanation of some results are not clear or sufficient; 2) the figure may not be a very good format to present the results shown in Figure 6, 7, and 8. The discussion is OK. Please read the detailed comments and suggestions in the annotated PDF manuscript.

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.

Title: GENETIC DIFFERENTIATION AND PHYLOGEOGRAPHY OF MEDITERRANEAN-NORTH EASTERN ATLANTIC BLUE SHARK (PRIONACE GLAUCA, L. 1758) USING MITOCHONDRIAL DNA: PANMIXIA OR COMPLEX STOCK STRUCTURE? Review round: 1 Reviewer: 1

Basic reporting: No comment Experimental design: No Comment Validity of the findings: No comment Additional comments: The authors use mtDNA sequencing to examine population structure between Mediterranean and Northeast Atlantic blue shark populations. The paper is interesting and will make a nice contribution to the literature. I have made comments and suggestions below. General comments Given the proximity of the populations, I was surprised the authors found genetic differentiation in their dataset. This is a very interesting result. In some areas, the authors are a bit too verbose (examples: last sentence of Abstract; lines 76-80; lines 215-219; lines 237-242; lines 254-258; lines 316-330) I commend the authors for the interactive map of their sampling—this is very cool! Why were the sequences not combined to a concatenated sequence and the same set of analyses run? Also, why the discrepancy between sample sizes for the two genes? Specific comments: Abstract: I suggest changing “failed in finding evidences of constant migrations” to “failed to find evidence of migration” I suggest changing “The network results revealed apparently no genetic structure through the Mediterranean-Atlantic seaway, quite the opposite, the Phist AMOVA and pairwise Phist analysis found a significant genetic structure among four geographical groups” to “Although no obvious genetic structure was apparent from the haplotype network, Phist analyses indicated significant genetic structure among four geographical groups.” Line 3: make “shark” plural and change “spreads” to “is found” Lines 17-18: change to “on the western side” and “on the eastern side” (change “in” to “on” in each case). Line 19: delete “and” Line 55: delete period after Mediterranean Lines 65-66: authors cite a study stating that almost all BS caught in the Mediterranean were immature, yet in their dataset (Table S1), this is not the case. The authors do not speculate as to why this is. It would be interesting to touch on this in the Discussion if the authors have any insight. Lines 74-75: In the abstract, the authors state 8 populations—please clarify. Line 80: change “Expected” to “These” Lines 116-119: Normally, final concentrations are reported (this is a minor point). Line 154: change “females outnumbered significantly males” to “females significantly outnumbered males” Line 178: Which second one? It looks like only the haplotype with 61 individuals is shared by all populations except NNEATL (which is mentioned in lines 176-178). Lines 231-236: which markers were used in these other studies and how do they relate to data here? Lines 242-245: Do you mean female philopatry when you say “reproductive movements?” The mtDNA structure found could very well indicate this, although it would help to have corresponding nuclear data to verify. Also, the authors don’t really stress whether or not these sampling locales were nurseries (although they do mention in passing)—I would suggest explicitly stating which sites are known (or suspected) nursery sites—the authors do this for the Mediterranean but not the Atlantic sites. Also, I would suggest analyzing sites without adults—if female philopatry could explain these results, I would expect an even stronger signal when solely analyzing juveniles/immatures from each site. Lines 258-260: This isn’t necessarily true—they can be panmictic (with males mating across populations) with female philopatry. Based on other studies, I’d be surprised if nuclear data exhibited structure. Following, (lines 266-268)—nuclear data may not necessarily exhibit structure. Lines 269-283: This paragraph seems out of place. Most of it belongs in the INTRO. Lines 316-330: Also seems a bit out of place, with a lot of this belonging in the INTRO. Ending with a comment on the fin trade doesn’t fit with this study. Instead, I would suggest stating the novelty of the Mediterranean population based on your results and how specific conservation measures should be implemented for the population (for example, leaving lines 321-325). Fig 1: This is minor, but I suggest changing “sampling design” to “sampling sites” as some of these sites weren’t designed but were instead from commercial fisheries. Fig 1: In both the figure (either on the figure itself or in the legend) and also in the text, please include sample sizes of the four regions. Fig 2: I understand why the authors have multiple colors within certain haplotypes (for example, three green in haplotype 42), but without knowing the sample locales, these are meaningless. Since the authors break this down in the supplemental figure, I would suggest combining the colors for this figure.

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.

Title: GENETIC DIFFERENTIATION AND PHYLOGEOGRAPHY OF MEDITERRANEAN-NORTH EASTERN ATLANTIC BLUE SHARK (PRIONACE GLAUCA, L. 1758) USING MITOCHONDRIAL DNA: PANMIXIA OR COMPLEX STOCK STRUCTURE? Review round: 1 Reviewer: 2

Basic reporting: The paper is nicely presented, has an adequate number of references and has a sufficient number of tables and figures. I have added minor suggestions for the authors. Background “but genetic structure has not been confirmed even at interoceanic distances.” Remove the word “even”. There are plenty of recent papers on BS describing genetic structure on an intraoceanic level. Results Remove “apparently” and replace “quite the opposite” with whereas. Introduction Line 3: Please use a reference for the previous statement. Line 40: The authors use the term mating clubs. It might be better to include them into quotation marks. Line 55: remove the first “.” Line 71: Add a space before “On the other hand” Material and Methods Lines 87-90: Please add the number of individuals per sampling area in case readers will not have to check supplementary material. Lines 110-111: Why did the authors required to design new primers for the species, since they could follow previous attempts? See Verissimo et al., 2017 for CR primers. Can they justify their decision? Line 120: replace “annealing at 60ᵒC for 30s” with “annealing at 60ᵒC for 30 s”. Line 139: Describe the four groupings for the AMOVA analysis. Results Line 179: What does the N≥10 mean? Number of individuals? Number of haplotypes? Please clarify. Discussion and Conclusion Line 244: Replace “Φsts” with “Φst values”. Lines 252-253: I am wondering about the role of the Adriatic as a nursery for BS. The authors claim to confirm Megalofonou et al., 2009 suggestions about the matter. I also assume that the authors have probably used samples provided from the same dataset used in that manuscript. Considering the lack of information on sampling dates (I do not remember seeing a table with the actual dates that the samples were caught), have the authors compared the sizes or sex ratios based on time of capture? Do they have other individuals caught in the same period for direct comparison and subsequently prove their argument? Figures and Tables Figure 2: What is the point of the additional small figure displaying the size+number of individuals per haplotype, when you have the number of individual in each haplotype? Please remove it, as it does not add anything to the Figure. Figure 3: You need to add the details for the sampling locations. NNEATL: North North-eastern Atlantic; SNEATL: South North-eastern Atlantic; WMED: Western Mediterranean; EMED: Eastern Mediterranean. Experimental design: The importance of the sampling area is highlighted throughout the manuscript. The Mediterranean is a very important mating and nursery ground for chondricthyans. The methods are sufficiently described. However, I was wondering about the sampling dates. Sampling took place from 2003 to 2016. Have the authors checked for temporal differences? Validity of the findings: Despite the complexity of the results, the authors do a very good job describing and justifying their results. Their findings are robust. and statistically sound. There are a couple of things that need to be clarified. There is a lack of discussion regarding the sex ratio differences found between the two seas despite the results they reported in Lines 154-164. Do the authors have any suggestions? Moreover, can you justify the differences in male/female sex ration found in the Mediterranean between your results and the ones reported by Megalofonou et al., 2009? Lines 254-268: You are trying to justify the complexity in structure between the Mediterranean and adjacent North-eastern Atlantic BS. You are doing an excellent job by justifying your data with the results from tagging data (previous studies). Considering the lack of nuclear data, I believe that you can also use studies with similar distribution might partially help you justify this weak structure between the Med and the Atlantic. There are at least three papers (2 on Scyliorhinus canicula and one on Etmopterus spinax) that might be able to help the authors add to their discussion, particularly in the absence of nuclear data. Additional comments: This is a nice study with sufficient mtDNA data that is well analyzed and well presented. The figures are clear. I feel that the study will be of broad interest to readers of the journal because of the interesting region being sampled. Our understanding of elasmobranch genetic structure is greatly improved by studies such as this. This is especially true in light of recent papers that have presented genetic structure in the Mediterranean. I think there is the basis for a nice publication on the population structure of Prionace glauca, and I believe that the manuscript could be improved as few things are not presented adequately.

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.

Title: GENETIC DIFFERENTIATION AND PHYLOGEOGRAPHY OF MEDITERRANEAN-NORTH EASTERN ATLANTIC BLUE SHARK (PRIONACE GLAUCA, L. 1758) USING MITOCHONDRIAL DNA: PANMIXIA OR COMPLEX STOCK STRUCTURE? Review round: 2 Reviewer: 1

Basic reporting: The paper is nicely presented and questions/concerns raised by both reviewers have been dealt with by the authors. Experimental design: The importance of the sampling area is highlighted throughout the manuscript. The methods are sufficiently described. I would like to thank the authors for the extra analyses they performed and replying to all questions raised. Validity of the findings: My concerns from my previous review have been addressed. Additional comments: This paper is a nice contribution to the literature, particularly for Mediterranean species.

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.

Title: ECOMORPHOLOGICAL INFERENCES IN EARLY VERTEBRATES: RECONSTRUCTING DUNKLEOSTEUS TERRELLI (ARTHRODIRA, PLACODERMI) CAUDAL FIN FROM PALAEOECOLOGICAL DATA Review round: 1 Reviewer: 1

Basic reporting: refer to general comments for the author Experimental design: no comment Validity of the findings: refer to general comments Additional comments: I recommend publication with minor revisions as listed below. I am not familiar with the software programs used so I will not comment on their efficacy. I will list below comments and errors by their line number. Ln 45 hypocercal instead of hipo- Ln 49 founded instead of funded Ln 72 pteraspidomorphs instead of pteraspidomorfs Ln 103 Add Carr (2010) to the references about Dunkleosteus inhabiting the seas of Euramerica. Ln 110 Add Carr (2010) to the references, since it directly address the pattern of disarticulation in Dunkleosteus. Ln 112 In line 333 you refer to Coccosteus as a “close related placoderm.” In this line you refer to Coccosteus as a ”smaller basal arthrodire.” Neither case is accurate. Coccosteus is a member of the coccosteomorph arthrodires while Dunkleosteus is a member of the pachyosteomorph arthrodires. These two groups are sister groups, thus neither is more basal to the other. Terminology more appropriate might be what you used to describe sharks as “non-closely related” since the two arthrodires are from distinct orders. Ln 124 In this line you refer to type 1 and type 2 and direct the reader to Fig. 1A where you show the 8 landmarks. Is Fig. 1A the type 1 and Fig. 1B the type 2? You may want to note then that type 2 has two additional landmarks as noted in Fig. 1B. You could add this within the parentheses. Also, in the figure caption you should identify which figure represents type 1 and type 2 so that the text and caption parallel each other. Ln 142 Add “v.1.4.1” to the software (tpsRegr v.1.4.1) to be consistent with your references to other software packages (e.g., tpsDig1 software v.1.4). Ln 149 Elsewhere in the text you only refer to TBL, not BL (e.g., in line 188 and Supplemental Data S2). Ln 153-154 The plate names will not be familiar to non-placoderm workers so it would help if you labeled those plates on your Fig. 1C. As in Carr & Jackson, 2010: fig. 5-7, you can add SO, PN, and R to your Fig. 1C. For the site of the quadratomandibular articulation you can place an asterisk on the PSO plate. Be sure to define your abbreviations in the figure caption. Ln 158 Since you use the abbreviation “JM” later (e.g., line 183) you should introduce it in this line, e.g., “five infergnathal metric variables (JM1-5).” Ln 164-164 This sentence contains a double negative (No, with neither, nor). Either use “no, either , or” or possibly “Significant phylogenetic signals have been detected neither…nor….” Ln 165 Add a comma after sharks (“sharks, respectively). Ln 166-167 Drop the article “the” before the percentages, i.e., “PC1 explains 41.7% of the total variance whereas PC2 explains 32.7%.” Ln 193 In this line you refer to “deep peduncles” while in line 341 you call it a “narrow peduncle.” Deep (to me) implies a wide peduncle so change line 193 to match line 341. Also in this line change hipocercal to hypocercal. Ln 202 In the references, you list this as Thomson & Simanek, 1977. Ln 212 Thunniform instead of tuniform (the same change elsewhere). Ln 217 lobe instead of love Ln 237 Change phrasing: I suggest “our work has allowed the establishment of a comparative framework” Ln 246 bouyancy instead of bouayancy Ln 258 Change phrasing: “As in sharks” Ln 307 founded instead of funded Ln 280 thunniforn instead of tuniform Ln 286 mechanisms instead of mechanism Ln 294 Is your reference to the entire book by Boucot or more specifically to Williams, 1990, Feeding behavior in Cleveland Shale fishes, p. 273-287. In Boucot, Evolutionary Paleobiology of Behavior and Coevolution. I think the specific reference would aid the reader in finding the reference. Ln 307 founded instead of funded Ln 325 Spell out the first use of the genus name in C. carcharias. Ln 333 refer to line 112 comment. Does membership in a separate but sister order make it “close related”? Ln 341 refer to line 193 comment Ln 343 Instead of “involves” would “implies” be more appropriate? Ln 358 Usually references to a figure in another document use a lower case “f” while references to figures in the manuscript use an upper case “F”. What are the instructions to authors guidelines for PeerJ? Ln 369-370 In line 513 you refer to Paul, Chase & Hodges, 1989 as Paul & Chase and note that it is in book edited by Hodges. Which is correct? Ln 374 Use either “This kind of approach can be” or “These kinds of approaches can be” Ln 384 Phrasing: “how” versus “what”? Ln 384-388 This is a fragmented sentence. Can you break it up into two or more sentences? It is hard to follow as it is now. I checked that all the references are listed in the text and visa versa. Figure 1 As noted above, it would help the non-placoderm reader to add plate abbreviations for the SO, PN, and R as well as a possible asterisk for the site of the jaw articulation on the PSO (refer to Carr & Jackson, 2010:fig. 5-7). Again, my question of adding type 1 and 2 in the figure caption to match their mention in the text. Tables 1&2 (JM1-5) instead of simply (JM)

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.

Title: ECOMORPHOLOGICAL INFERENCES IN EARLY VERTEBRATES: RECONSTRUCTING DUNKLEOSTEUS TERRELLI (ARTHRODIRA, PLACODERMI) CAUDAL FIN FROM PALAEOECOLOGICAL DATA Review round: 1 Reviewer: 2

Basic reporting: In the interest of open and honest dialogue in science I do not wish to remain anonymous, and encourage the authors to contact me if they have any queries. Regards and best, Tom Fletcher, University of Leicester • Clear, unambiguous, professional English language used throughout. Suggested improvements: L28 - “However, body design of fishes is determined in a large extent …” should be “determined to a large extent”. L29 - “… being possible to clearly recognize different morphological traits that have evolved several times in non-closely related groups with similar lifestyles” I would suggest should be “making it possible to recognise”. I feel clearly recognising is too general in that instance, as it is only very extreme adaptations that are obviously for a specific purpose. L45 - “fin span or the hipocercal” should be hypocercal L46 - “The application of this ecomorphological approach to the concrete case of Dunkleosteus terrelli has led to a new reconstruction of this emblematic placoderm.” It is unclear what you mean here, what is a concrete case? L49 - “in contrast to classical reconstructions funded on the phylogenetic proximity with much smaller placoderms known from complete specimens” Funded is an inappropriate word here. I would suggest “… reconstructions based on the…” L52 - “which entails that the caudal fins of placoderms could have displayed a greater range of morphological variability than thought up to date”. I would suggest “which suggests a greater morphological variability in placoderm caudal fins than previously thought.” L55 – “The Fossil Record…” should not be capitalised, suggest “The fossil record…” L55 – “…glimpse into the extinct creatures” is unclear, suggest “glimpse in to the lives of extinct creatures”. L70 – Sentence a little long “However, large dermal plates are typically restricted to the cephalo-thoracic region in early vertebrate groups (e.g., placoderms and most agnathan ostracoderms including pteraspidomorphs, galeaspids, osteostracans and pituriaspids) and scales, that normally cover whole body surface, easily disarticulate being found as isolated elements in the vast majority of cases (Janvier, 1996).” I’d suggest “However, large dermal plates are typically restricted to the cephalo-thoracic region in early vertebrate groups (e.g., placoderms and most agnathan ostracoderms), and in the vast majority of cases scales disarticulate to isolated elements during preservation (Janvier, 1996).” L76 – Some repetition here, and a little unclear suggest deleting “ leading in conjunction to a very vague 79 idea of the general body shape and size of a big number of early vertebrate representatives, 80 especially in regard to their post-thoracic region.” Instead explain briefly (7-8 words) that endochondral bone has a greater preservation potential (with reference). L85 – ‘Fabricational’ is not an appropriate word here, do you mean ‘developmental’? Nevertheless I would suggest “including not only the phylogenetic legacy of the species but also environmental and functional aspects (Seilacher, 1991).” I would use ‘species’ instead of ‘group’ here, since you are arguing (and I agree) that every species is uniquely shaped by the factors you list. L86 – I suggest “… aquatic vertebrates is determined to a large extent by their swimming mode” L88 – Suggest “… the functional constraints of different types of locomotion are so strong that…” L93 – Suggest “…between the mode of life and body pattern in living aquatic vertebrates, we propose that the identification of ecomorphological relationships…”. L96 – This is a very long sentence. Suggest “With this aim, we have assessed the relationship between the locomotory patterns and the morphological variability of the caudal region in extant sharks by means of geometric morphometrics and allometric regression analysis. We present a comparative framework for predicting some anatomical aspects in extinct species from palaeoecological data, and vice versa” L103 – ‘Paradigmatic’ is not an appropriate word here. Suggest “Dunkleosteus terrelli is an exceptionally large and iconic carnivorous placoderm that inhabited the seas of Euramerica during the Late Devonian (Carr & Jackson, 2010).” L106 – I suggest “Given its large size* and fearsome appearance, D. terrelli has been a focus of interest for the general public for decades.“. *I would also include a length in metres here, with reference. L108 – Suggest “However, despite popular interest in the species D. terrelli is only known from disarticulated plates of the head shield, and a few articulated remains of incomplete pectoral fins (Carr, Lelièvre & Jackson, 2010).” L111 – Suggest “ … remain unknown, and to date reconstructions of D. terrelli have relied on the morphology of smaller basal …” L113 – I think the paragraph starting “As a consequence …” repeats the previous paragraph and I’d delete it L128 – Suggest “This procedure allows the removal of variations in….” L131 – Suggest “The degree of homplasy was checked by plotting … “ L163 – Should be “… of a Pinocchio effect …t” L164 – Double negative, suggest “… detected either for the … “ L166 – Should be “…PC1 explains 41.7% of the total variance whereas PC2 explains 32.7%.” L168 – Should be “… caudal fins of sharks …” L184 – Should be “ … both variables shows a good fit to a linear model…” L208 – This whole sentence is unclear and I think repeats what follows. If you want to keep this I’d suggest “The shark body patterns proposed by Thomson & Simanek (1997) are recognizable after our morphometric analysis. Separate groups, as extremes of a morphological continuum, are probably due to different locomotary styles (Fig. 2B).” L214 – Should be “… adapted to strong continuous swimmimg… “ L217 – Should be “well-developed ventral lobe and wide …” L224 – Suggest “There is evidence that increased size results in lower muscle contraction frequency (Altringham & Johnston, 1990; Altringham & Young, 1991), which results in lower tail beat frequency (Wardle, 1975). Consequently, a loss in swimming speed and hydrodynamic lift is compensated for with proportionally wider caudal fin spans (Motani, 2002; Lingham-Soliar, 2005).” L233 – Suggest “where hydrodynamic lift is not as important, with decreased need to control …” L237 – Confusing sentence, suggest “Our work is the first to establish a comparative framework with geometric morphometrics of body-caudal fin propulsion on which to base ecomorphological inferences of extinct early vertebrates.” L240 – Suggest “… approach is applicable for …” L245 – Suggest “… placoderms and ‘acanthodians’)…” L247 – Should be “… that contribute to buoyancy … ” L253 – Suggest “ … remains of large pelagic arthrodires… “ L254 – Suggest “… make it difficult to base…” L258 – Unclear, I suggest “As in modern sharks, arthrodire…” L260 – Suggest “ … as the main propulsor organ, are also be subject to strong selection pressures driven by ecology, locomotion and body size” L266 – ‘In this sense’ is not the best way to phrase this, and th term cruising describes the contuous swimming. Suggest “Recently, Carr (2010) has provided strong statistical evidence for active pelagic cruising in this species, based on taphonomical and sedimentological data.” L270 – Autocorrect has resulted in typos here “… Basin (EEUU) and the Tafilalt Basin (Morocco), suggesting a…” L276 – Suggest “… modes could also be present …” L287 – Suggest “… armoured animals, for example arthropods… “ L293 – Suggest “ … thus constituting direct evidence of the …” L307 – Misuse of the word funded again, suggest “…placoderms have been based on the proportions of …” L314 – To clarify I suggest “ …, including C. cuspidatus itself (Miles & …” L318 – Typo, should be “ variables in placoderms (see Carr….” L323 – I would try and reduce the length of this sentence. L343 – This sentence is unclear, what outlines? I think this needs to be more specific. L346 – This sentence does not provide additional information and repeats previous points. I would suggest deleting it. I am also unclear as to what is being suggested in the line 348 sentence starting “In this sense…”. Again not a common phrase in scientific writing. L352 – Need to explain the significance of the single dorsal fin and Coccosteus here. Sentence is unclear. L357 – Need to define macruriform L374 – Should be “ … kind of approach can be …” L376 – Should be “ … in order to make inferences about …” L379 – Should be “…sensitive to convergence…” L384 – Should be “ … us to know what some of the first vertebrates…”. However, placoderms are far far from the first vertebrates, so I would reconsider this sentence. L388 – The closing remarks are a little long and repetitive, I would consider rewriting these. • Intro & background to show context. Literature well referenced & relevant. L66 – would be beneficial to include a date with reference here L76 – again, include a date and reference here L291 – explain what Orodus is, a smaller fish? Aside from these minor points, I think more emphasis needs to be placed on the controls on morphology. Function is mentioned but not discussed in detail. The relationship between jaw size and ecology again is not discussed. So I feel that there needs to be a more comprehensive introduction to the theory. E.g. why do fast cruising sharks have the tails they do? • Structure conforms to PeerJ standards, discipline norm, or improved for clarity. Yes. But please see notes above. • Figures are relevant, high quality, well labelled & described. Yes. Experimental design: • Original primary research within Scope of the journal. This article provides a good framework for the estimation of Dunkleosteus size; a large and iconic fish. However, the sole comparison with the great white jaws is not justified adequately in text. • Research question well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap. The research question is well-defined and relevant for the estimation of fossil fish body size. However ecological correlates are neglected, which are an important part of the study. • Rigorous investigation performed to a high technical & ethical standard. In essence this is a statistical approach to reconstructing Dunkleosteus postcrania. While competent, there is little ecological justification presented to support the findings, and I feel a more the problem deserves a more thorough approach. • Methods described with sufficient detail & information to replicate. L145 – Might be beneficial to explain what the pinnochio effect is here or in the introduction. L149 – unclear as to why you are using 33 white sharks, and not all shark species here. Is there evidence for a direct comparison with a single species? L205 – Should be “… structure involved in thrust generation …” • Raw data supplied (see PeerJ policy). Yes. Plots in Supplementary data 3 may need axis labels or for clarity. Crosses and circles need to be explained. Validity of the findings: Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated. • Data is robust, statistically sound, & controlled. While an impressive array of statistical tests have been utilised, I do not believe enough justification has been provided for the comparison of Dunkleosteus with the great white shark. This justification is a very important part of the analysis, since the authors themselves state the variability of morphology based on ecological and functional factors. For the comparison to be valid, I would need to see more evidence that the feeding mode and locomotion style of the two were very similar. The alternative is to run a similar analysis of other sharks of similar size (e.g. whale sharks, basking sharks, Greenland shark etc) to show a universal trend separated from ecology. Secondly, the predictive power of this technique is stated on numerous occasions (e.g. L323); however the authors have not applied it to modern sharks to test this. E.g. Does the regression predict the size of a whale shark based on jaws? • Conclusions are well stated, linked to original research question & limited to supporting results. L268 – Needs a reference Otherwise the links to original research are good. Conclusion is a little repetitive, which reflects the limited scope of the investigation. This is not necessarily a bad thing. • Speculation is welcome, but should be identified as such. Speculation is separated from fact well. Additional comments: This is a competent statistical comparison of Dunkleosteus with modern sharks. The question is not novel, but the approach used here is for this species. I like this paper a lot, and it deserving of publication, but not in its current form. The comparison of jaws with only the great white shark is not justified or explained in text. The regression used to predict body size or tail type in an extinct animal is not tested here on modern sharks. Were the authors to choose a large pelagic plankton-feeding shark and predict tail span, this may go some way to testing the validity of their 'comparative framework'.

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.

Title: ECOMORPHOLOGICAL INFERENCES IN EARLY VERTEBRATES: RECONSTRUCTING DUNKLEOSTEUS TERRELLI (ARTHRODIRA, PLACODERMI) CAUDAL FIN FROM PALAEOECOLOGICAL DATA Review round: 1 Reviewer: 3

Basic reporting: This is a really clever study which seeks to reconstruct the unknown body shape and size of a large, extinction charismatic megafauna (Dunkleosteus) using inference from living analogues among the sharks through the inference of general rules for fish body form. However, this worthy effort has a number of important issues which reduce the strength of the arguments and the ability to infer the morphology of extinct fishes with high probability; These need to be resolved before publication. It requires better organization, stronger justification of specific assumptions and arguments, and reconsideration of the methods. Experimental design: Comparison of sharks and placoderms: I can see the basic logic for comparing Dunkleosteus exclusively with living sharks. Dunkleosteus is a large, generalist predator with sharp-edged jaws found in an open water setting, so modern great whites and other sharks are likely the best modern analogues. However, this argument is not explicit in the paper, nor is the case for selecting actively pelagic sharks as the group with the right body shape and proportions made clear. There needs to be a real comparison of the head and jaws and head size of Dunkleosteus and sharks to determine which shark ecological group is the ideal analogue. Reliance on past speculation is not enough. Shark morphospace analysis and results: The results of the morphospace analysis are a bit muddled - there's quite a lot of overlap between sharks with different ecologies near the center of PC1 and along most of PC2. How do we know Dunkleosteus exhibited a morphology matching that of active pelagic sharks at their most distinct (e.g. the 4 with the most negative scores on PC1) and not at their most generalist (the other 6 points)? It is possible the restricted set of landmarks artificially increased the degree of overlap between different shark ecological groups. Why was body depth/shape and first dorsal fin position/form not captured by the landmark set? These aspects also vary in sharks, are important aspects of fish form related to locomotion and ecology, and are unknown in Dunkleosteus. Indeed, the body depth of the Dunkleosteus reconstruction in Fig. 4B is much greater than that of the sharks used as as analogues for inferring its body size and caudal shape, and the dorsal fin is unlike that of known large, pelagic fishes. It would be better to base all aspects of the body on quantitative inference. It is also not clear whether caudal fin edge shape, another ecologically relevant trait, was recorded, as the landmark sets in Fig. 1A and 1B disagree. Dunkleosteus life-mode: This paper also needs more explicit justification for why Dunkleosteus was likely to be an active pelagic, "body and caudal fin"-type continuous swimmer than just citing Denison and Carr, and more shark-like than teleost-like. Some of the required evidence is referenced throughout the discussion (e.g. sedimentological evidence; the paragraph from 283-302 which should be moved forward) but not elucidated up front. A large range might be evidence, but body and caudal fin swimming, and continuous swimming, are not required for such (see the spread of Indo-Pacific reef fishes, for example). Inference of body length: It is not clear why body length was estimated only from great white sharks. Are body length/jaw length ratios already known to be more dependent on ecomorphology across sharks, or fishes, than ancestry? While an argument is made for not estimating size based on smaller arthrodires, would it not be more appropriate to apply the shark scaling formula to placoderm relationships than assume the same relationship as in one species of shark? Validity of the findings: Dunkleosteus reconstruction: While it is likely that Dunkleosteus did have a caudal form resembling that of modern pelagic sharks given what we know about fish ecomorphology, the arguments in the paper are not yet strong enough to support this assumption as laid out above. In addition, the new reconstruction of Dunkleosteus may have a correct tail, but the body depth, and other fin forms are still based on Coccosteus, which the authors clearly argue is not a good analogue. A full attempt to infer the form of early vertebrates requires consideration of these ecologically important aspects as well. An improved landmark set for sharks would go a long way to resolving this. Additional comments: line 72: "pteraspidimorphs" 193: "hypocercal" 269: Appalachian Basin, Tafilalt Basin 280: thunniform 285: Lamsdell and Braddy 2009 did not explicitly test the trophic position of Dunkleosteus. 360: This section seems unnecessary. Paleoart should be based on the most up to date reconstructions as a matter of course and good paleoartists know this. Fig. 1 The landmark sets in Fig. 1A and 1B disagree. Fig. 4A It is not clear from the paper where these outlines come from. Fig. 4B The reconstruction Dunkleosteus has much greater body depth than the sharks which provided the inferred caudal fin shape. Why is this assumed? Also, why are the median and paired fins assumed to be more like Coccosteus than pelagic sharks, since the shapes of these are also related to locomotion?

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.

Title: A CDK1 PHOSPHOMIMIC MUTANT OF MCAK IMPAIRS MICROTUBULE END RECOGNITION Review round: 1 Reviewer: 1

Basic reporting: The microtubule depolymerizing kinesin-13, MCAK, plays critical roles in spindle assembly and in correcting improper kinetochore attachments. Because MCAK is a potent depolymerase, its activity is spatially and temporally regulated, but the mechanism of this regulation is not well understood. In the current paper the authors perform a thorough characterization of a T537 phosphomimic mutation of MCAK that lies in the catalytic domain of MCAK and which has been shown to be phosphorylated by Cdk1. They report that the T537E mutation results in decreased microtubule depolymerization activity due to its inability to remain at microtubule ends. This finding is a logical extension of their previous work in which they showed that mutation of highly conserved residues in alpha-helix 4 of MCAK also resulted in reduced microtubule depolymerization activity and a reduced binding to microtubule ends. Because T537 lies is positioned adjacent to alpha-helix 4, the current work provides strong corroborating evidence that this region is critical for recognition of microtubule ends and is an ideal way to modulate MCAK activity. • The data presented is short, sweet, and to the point. The text is clearly written, and the presentation of the data makes it easy for the reader. • The data is put in the context of the field, but the authors may have missed an opportunity to discuss the broader mechanisms of regulation of MCAK activity, discussed in more detail in point 3 below. • It would be helpful to the reader if the authors reported what is plotted. For example, 1B represents a box and whiskers plot and 1C represents mean +/-???. The box and whisker definitions nor the error bar values (SD or SEM) are presented to the reader. • For the original data supporting Fig. 1C, there is only one set of data for the +tubulin and +microtubule samples, and yet the Table says that n=3. Please clarify. • In Fig. 2A, please provide the colors on the kymographs to the reader either on the figure or in the legend. • For Fig. 2C and 2D, it would be helpful to the reader if the authors reported the fit of the data in the graph so that the reader does not have to go back and to the table. This is also true for Fig. S2. In addition, for each of these data sets, the excel sheet only has one set of data, and yet the tables say n=3. Since the data appear to be normalized, is it not possible to plot the average +/- SD of the normalized data in the graph? This would present all of the data to the reader. Experimental design: • This is a well-done characterization of a mutant form of MCAK. The data provide a potential mechanistic understanding of how phosphorylation may regulate the catalytic function of MCAK. • This is the first detailed biochemical characterization of a regulatory phosphorylation that occurs in the catalytic domain. • All experiments appear technically sound with sufficient replicates. • The methods are well-described. Validity of the findings: • The results are clear, and the differences between wild type and mutant are clear and robust. • The conclusions of the study are valid and well-justified. Additional comments: • In the models discussed at the end of the paper, the authors are not taking into account the multitude of observations showing that the other domains of MCAK target it to the kinetochore, inner centromere, spindle poles, or plus-tips of microtubules. Therefore, MCAK may be positioned near the microtubule end, so the key event is likely the residence time with the curved tubulin not just being at the end. • It has also been well-established that MCAK’s microtubule depolymerization activity requires the positively charged neck region in addition to the catalytic domain. The neck is also the site of a critical Aurora A/B phosphorylation site, which has been shown to regulate microtubule depolymerization activity. Both the Wordeman Lab and the Walczak Lab have done extensive mechanistic analysis of how the neck and the regulatory phosphorylations affect MCAK activity. It would be helpful to the reader if the authors included some of this work in the discussion. For example, Ovechkina et al, 2002; Moore et al, 2004; Wagenbach et al, 2008; Cooper et al, 2010; Ems-McClung et al, 2013.

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.

Title: A CDK1 PHOSPHOMIMIC MUTANT OF MCAK IMPAIRS MICROTUBULE END RECOGNITION Review round: 1 Reviewer: 2

Basic reporting: The authors describe the role of CDK1 mediated phosphorylation on the activity of MCAK. The background information provided in the text are well described and give a full picture of the biology of the kinesin MCAK. The main conclusions of the work are that the T537E-MCAK phosphomimetic mutant has i) an attenuated microtubule depolymerisation activity; ii) a reduced ATPase rate in the presence of polymerised microtubules; iii) a shorter residence time at the microtubule end than the WT and iv) it cannot promote ADP dissociation. Experimental design: The experiments are very well presented and designed. However, although the raw data support some of the conclusions, the general interpretation of the results need major revisions. Please find below the list of comments that the authors can consider during the revision of their work. Validity of the findings: 1. First set of results (described from line 99 to line 121). According to the data, the T537E-MCAK can turn over ATP in solution and bind to unpolymerised tubulin. How do the authors explain the differences in the ATPase rate of T537E-MCAK in the presence of unpolymerised vs. polymerized tubulin? Could the T537E mutation reduce the lattice-stimulated ATP cleavage due to a conformational change of T537E-MCAK which does not occur upon binding of unpolymerised tubulin? The authors could measure the binding rate of T537E-MCAK to unpolymerised and polymerised tubulin and suggest some hypothesis on the mechanism of action of the MCAK mutant. 2. In the second paragraph of the results (lines 132-133) is written that “Wild type MCAK makes short diffusive interaction with the microtubule lattice”. It can be assumed that this is due to the initial high microtubule binding affinity of ATP.MCAK and, subsequently, to the lattice promoted ATP cleavage which weaken the MCAK interaction with the microtubules (Friel and Howard 2011, The EMBO Journal). Given the reduced ATPase rate of the MCAK phosphomimetic mutant in the presence of microtubules, it is very likely that T537E-MCAK is predominantly in the ATP-bound isoform. Thus, it is surprising that the affinity for the microtubule lattice is not significantly changed for MCAK mutant (line 138-139). Since ATP.MCAK tightly binds microtubule filaments, should not the authors expect a longer residency time for T537E-MCAK along the microtubule lattice and a reduced diffusion rate than the WT? For this reason, the authors might consider that the attenuation of the depolymerisation activity of the phosphomimetic mutant might be also due to the loss of ability of T537E-MCAK to reach the microtubules end (see also ‘General comments’ below). 3. In the third set of results (from line 148 to line 176) is shown that the T537E-MCAK mutant has a slow ADP dissociation rate at microtubules end. However, the rationale and the conclusions of this experiment (see also ‘General comments’ below) do not seem very clear and robust. Concerning the rationale of the experiment, I am wondering whether the number of T537E-MCAK molecules bound to ADP at the microtubule end is sufficient to test the ADP dissociation rate. The data presented in paragraph 1 and 2 of the results show that it is very unlikely that T537E-ADP.MCAK can reach the microtubule end because of i) the reduced ATPase rate of T537E-MCAK bound to microtubules (0.335±0.081s-1 compared to 4.75±0.057s-1 in WT) and ii) the short residence time of T537E-MCAK at the microtubule end (0.64±0.02s compared to 2.03±0.13s for the WT). For these reasons, I suggest moving these experiments in the supplementary data section because they are supporting previously results but do not significantly add novel information. Additional comments: a. At lines 213-215 the authors write that “This mutant still displays the characteristic diffusive interaction of MCAK with the microtubule lattice and can still reach the microtubule end”. However, this statement is not very well supported in the text/figures in which, instead, is shown that T537E-MCAK has a reduced residence time at the microtubules end. In addition, this sentence does not sustain the conclusion that T537E-MCAK does not distinguish between the microtubule lattice and the microtubule end. Although this conclusion might be very intriguing to explain the attenuated T537E-MCAK depolymerisation activity, there is no direct proof for this and sounds too speculative at this stage of the work. b. The conclusion that the reduced depolymerisation rate of T537E-MCAK is dependent on the attenuated ADP dissociation from T537E-MCAK at the microtubules end, might not be fully appropriate (see also point 3 above). Indeed, the authors do not consider the hypothesis that, given the reduced residence time at the ends, the mutant MCAK that does not get to the microtubule tip because is not able to diffuse along the lattice (see also point 2 above). This hypothesis is also in accordance with another work showing that T537E-MCAK predominantly localizes along the microtubule lattice of the mitotic spindle (but not at the ends) and display a very weak staining at centromeres in HeLa cells (Sanhaji et al., 2010, Mol Cell Biol). In other words, the authors might discuss and consider the hypothesis that the CDK1 mediated phosphorylation of MCAK does not directly affect the catalytic activity of the kinesins at microtubule end but regulates the ability to diffuse along the microtubule lattice by modulating the ATP-ADP cycle on MCAK.

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.

Title: A CDK1 PHOSPHOMIMIC MUTANT OF MCAK IMPAIRS MICROTUBULE END RECOGNITION Review round: 2 Reviewer: 1

Basic reporting: The authors have addressed all points listed during the first revision of the paper. The results are very well explained and the conclusions are robust. Experimental design: I appreciate the authors have now performed the experiment to investigate the ability of the T537E-MCAK mutant to get to the microtubule end. This additional experiment clearly show the T537E-MCAK phosphomimetic mutant can reach the microtubule end although its residence time is much shorter than the wild type. Overall, this experiment greatly contributes to support the conclusion that the T537E-MCAK is not able to distinguish between microtubule lattice and microtubule end. Validity of the findings: The findings about the role of the CDK1-mediated phosphorylation of MCAK are strongly supported by the data and I recommend this work to be published in PeerJ. Additional comments: No additional comments

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.

Title: A MICROBIAL SURVEY OF THE INTERNATIONAL SPACE STATION (ISS) Review round: 1 Reviewer: 1

Basic reporting: The submitted manuscript reports the microbial assemblage of the International Space Station (ISS), and compares the ISS assemblage to microbiomes of homes and humans. The manuscript is generally well-written with somewhat sufficient and relevant references to support their claims. However, given the very low number of samples (which is in itself understandable because of the sampling site and circumstances), one may expect more rigorous analyses to be done to further describe the ISS assemblage based on the limited number of samples. For example, the manuscript can add the following analyses/discussions to make up for the limited number of samples included: 1.1 The authors have compared their findings with the previous work by Venkateswaran et al. (2014), but do not compare their findings with that of Ichiro et al. (2016). 1.2 Given that the crew vent sample appears to differ from the majority of other samples, would be interesting to investigate this sample further at the sub-genus/strain/species level. 1.3 Greater efforts will need to be addressed to the figures. For example, in Figure 2, the title of the legend groups appear to lack consistency in capitalization ("Surface_Type" but "node"). Also, there should not be underline in the figure legend "Surface_Type." In Figure 4, it is extremely hard to visualize the different sample groups, using the current colour labelling scheme (which is very gradient-like). The authors may want to manually fix the colour for each sample group. Also, the list of sample groups currently are in alphabetical order, but this order does not help in the overall understanding of the ecology. Alternatively, sample groups can be listed by their built environment group (e.g. all sample groups within Homes listed first, then sample groups within ISS, or vice versa). Figure 5 Panels B and D, and Figure 6 suffer from the same weakness. Experimental design: 2.1 The Methods section is described in detail. 2.2 It is implied that the authors have filled the knowledge gap by using the Illumina technology to increase the sequencing depth to explore the ISS microbiome in greater detail. Perhaps strengthening that point in the introduction and conclusion will add impact to the manuscript. 2.3 The analyses described appear to have done well, however additional analyses should be performed to strengthen the impact of the work given the very low sample number. 2.4 The lack of controls in assessing for potentially contaminating taxa is a great weakness of this work, but the authors have explained the reason for not having them. However, it would still be beneficial to include controls that the lab group has subsequently produced after the start of this work, to show readers that the authors made an effort to account for potential contamination during sample prep and sequencing. If possible, this is highly recommended. If not, please explain why this is not possible. Validity of the findings: 3.1 Given the low number of samples, it is necessarily to indicate in discussion that the lack of replicates may affect the validity of the results. 3.2 There is no indication where the sequences of the ISS samples can be accessed. Please make that available in a public repository. 3.3 While the authors indicate that the findings will have in space exploration in the future, as most readers will not be heading to space in the immediate future, can readers learn anything about the built environment on Earth from this ISS study? Additional comments: Line 39: change "Earth homes" to "homes on Earth" Line 93: change from "example" to either "exemplify" or "assess" Line 185: twelve base change to "twelve-base" Section "Comparison of ISS surfaces in homes on Earth and to the Human Microbiome Project:" besides the point that surfaces on the ISS were selected to mirror that selected for a previous home study, please indicate more succinctly the rationale for comparing the microbiomes of the ISS to homes. Is this because humans will potentially living in the space in the future? If anything, it is my humble opinion that the ISS environment may more resemble that of cleanrooms. Unfortunately, cleanroom environments are not described in this study, and a simple taxonomic or community analysis should have been described. Also, as any differences in methodology can potentially affect sequence-based microbial community surveying results, please indicate whether these different studies adopted identical sample collection, preparation, and sequencing methods. If not, add a disclaimer that method differences may have affected the comparison of the studies. Line 272: outside air change to "outdoor air" Line 311: suggest to remove double negative in sentence. Line 326-330. Is there an ecological/rational reason for the discrepant community found in the crew vent sample, and its resemblance to human gut communities? Please also fix the in-text citation and references throughout. Additional proof-reading of the manuscript will be beneficial to minimize typos (random hash-tags appearing in manuscript, mathematical power symbol represented as ^ instead of a superscripted 2, etc.). Refer to https://peerj.com/about/author-instructions/ for more information.