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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: OXYGEN-LIMITED METABOLISM IN THE METHANOTROPH METHYLOMICROBIUM BURYATENSE 5GB1C Review round: 1 Reviewer: 1

Basic reporting: This is an interesting study with an appropriate background and rational for the study. Methods are clearly presented and as far as my experience allows appear to be appropriate for the study. In general I found the results to be clearly presented and the discussion and conclusions to be appropriate, however there are a number of areas within the manuscript that need improvement and are confusing. My main concern is with the first three paragraphs of the results. The first on Genomic analysis reads like an introduction and presents no new data and should be in the introduction. The next two on Excretion products are confusing. They contain significant references to past work, are more discussion than results and as such it is very difficult to see what was achieved in this study. In particular references to Gilman et al (lines 209/212) suggest that work presented is from that study and not this. I am sure there are results in these sections but it is not clear to the reader as presented, and needs to be significantly improved. In the results the authors refer to growth rates, but in Table 1 and the methods talk about dilution rates, and then in Table 4 growth rates. There is nothing to say that they are the same thing, this needs to be clarified. In Table 2 one of the columns is headed padj, what is this? 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: OXYGEN-LIMITED METABOLISM IN THE METHANOTROPH METHYLOMICROBIUM BURYATENSE 5GB1C Review round: 1 Reviewer: 2

Basic reporting: This paper examines a type I methanotroph Methylomicrobium buryatense 5GB1 under oxygen-starvation conditions, and used a multifaceted approach to understand its oxygen-limited metabolism. The authors reported their finding in vial experiments, continuous bioreactor experiments, RNAseq, mutant testing and flux balance analysis using a previously developed model, and compared them with previously published results on the strain and a closely related strain. The paper is well-written, well-organized and easy to follow. It provided necessary background information, and conforms to PeerJ standards. Experimental design: This paper addresses an important question, i.e., understanding type I methanotroph’s oxygen limited metabolism, as it is highly relevant not only to understand the methanotrophs’ role in ecosystems, but also to the methanotroph-based biotechnology such as converting natural gas to valuable liquid products. The hypothesis was clearly stated, experiments well designed and carried out, with clear description on different methods. Validity of the findings: By integrating the results from different approaches, it was concluded that M. buryatense 5GB1 maintains a hybrid metabolism of fermentation and respiration, even though a genetic potential for a full fermentative metabolism exists. In addition, it was suggested that the secretion of formate and acetate could be a response to redox imbalance. The conclusion are supported by the findings reported in the paper. Two suggestions: 1. Since the authors compared their results with previously published ones, it would be helpful to list the previously published ones in the table for easy comparison. For example, adding result from Gilman et al to Table 1 to provide a direct comparison. 2. Was produced CO2 measured? I understand it is challenging to obtain an accurate measurement of CO2 in vial experiments. But it should be easier for bioreactor experiments. If the CO2 was measured, then it would provide more information for FBA modeling, and allow a better understanding on the carbon flux shift under oxygen limited conditions. 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: OXYGEN-LIMITED METABOLISM IN THE METHANOTROPH METHYLOMICROBIUM BURYATENSE 5GB1C Review round: 2 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: I am happy with the changes made by the 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: THE NEUROPROTECTIVE EFFECT OF NICOTINE IN PARKINSON’S DISEASE MODELS IS ASSOCIATED WITH INHIBITING PARP-1 AND CASPASE-3 CLEAVAGE Review round: 1 Reviewer: 1

Basic reporting: The study: "The neuroprotective effect of nicotine in Parkin's disease models is associated with inhibiting PARP-1 and caspase-3 cleavage", propose to give evidence that nicotine administration can contrast cell death in the MPP+ cellular model for PD and increase PARP-1 and caspase-3 expression in the 6-OHDA mouse model for PD. They found that nicotinic cholinergic drugs may delete PD progression, in particular through α7 nAChR activation. Besides some typos (Figure 1B in the legend, nicotine; line 231, ih), the article is written using a clear and professional English, and is supported by a sufficient literature. However, there are several concerns for me as follows: 1. the means and the standard errors of the mean of the data obtained are missing throughout the test 2. it is unclear if normality and equal variance tests were run to identify parametric and non-parametric data, please specify how you choose the right statistical test 3. representative pictures of the staining have not been inserted in fig 1, 2, and 3 4. Could you explain why the cell death index value of the treatment MPP+ + nicotine in the figure 3A is different from the one obtained in the figure 1B? and for the treatment MPP+ + choline between figure 2 and 3B? 5. Figure 3: please level out font size 6. Figure 4A and D : molecular weight are missing Experimental design: Regarding the experimental design there are two main concerns for me: • It is known that nicotine concentration in smokers’ body is below 100 µM (Matta et al., 2006; Clunes et al., 2008), so I wonder whether the concentration (2 mM) you used in your paper is appropriate. • MLA want to be used as α7 nAChR antagonist; however at concentrations > 40 nM, the MLA purchased from Tocris (as the one used) could interacts with α4β2 and α6β2 receptors. How do you validate the concentration (20 µM) that you applied? Methods are described with sufficient details, but I ask you to specify the mouse strain used. To further strength Lu et al findings and correlate the in vitro part with the in vivo one, I may suggest three missing experiments: -evaluate PARP-1 and caspase 3 expression in SH-SY5Y cells in control and after MPP, nicotine, or nicotine + MPP treatments. -quantify cell death in the striatal tissue both in the lesioned and non-lesioned hemisphere in mice injected with nicotine or saline. -evaluate PARP-1 and caspase 3 expression in the non-lesioned hemisphere. Validity of the findings: It is almost impossible to evaluate data strength inasmuch means have not been inserted into the text and it is unclear if data have been correctly analyzed. Moreover, some controls are missing (PARP-1 and caspase 3 expression in the non-lesioned hemisphere). 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 NEUROPROTECTIVE EFFECT OF NICOTINE IN PARKINSON’S DISEASE MODELS IS ASSOCIATED WITH INHIBITING PARP-1 AND CASPASE-3 CLEAVAGE Review round: 1 Reviewer: 2

Basic reporting: Clear and concise. Experimental design: Fine Validity of the findings: Findings are novel and likely to be important. Links well with previous studies. Little speculation. Additional comments: Line 301-3: I would suggest removing the following from the penultimate paragraph: “PARP-1 also plays a role in cancers, including those of the prostate and ovary, prompting the development of PARP-1 inhibitors such as olaparib as anti-cancer drugs (Deshmukh & Qiu 2015; Passeri et al. 2016).” This paragraph could be concluded by something on the importance of the sirtuin decrease arising from PARP-1 induced depletion of NAD+, with implications for mitochondrial functioning (e.g. Anderson and Maes, 2014). It should also be noted that a7nAChR agonists are also likely to have significant impacts in PD via the regulation of e.g. the immune system and intestinal permeability (e.g. Anderson et al, 2016). This would not negate the relevance of the current results but would briefly provide the reader with a broader perspective on the role of the a7nAChR in PD. Line 305: In the concluding paragraph, it would be better to refer to PD models and in vitro model, rather than showing “neuroprotective effects of nicotine in PD”. It is widely accepted that these commonly used PD models are poor representations of the complexity of the human condition. Suggested references: Anderson G, Maes M. Neurodegeneration in Parkinson's disease: interactions of oxidative stress, tryptophan catabolites and depression with mitochondria and sirtuins. Mol Neurobiol. 2014 Apr;49(2):771-83. Anderson G, Seo M, Berk M, Carvalho AF, Maes M. Gut Permeability and Microbiota in Parkinson's Disease: Role of Depression, Tryptophan Catabolites, Oxidative and Nitrosative Stress and Melatonergic Pathways. Curr Pharm Des. 2016;22(40):6142-6151.

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: RANGE EXPANSION OF A FOULING SPECIES INDIRECTLY IMPACTS LOCAL SPECIES INTERACTIONS Review round: 1 Reviewer: 1

Basic reporting: The authors presented a well-written manuscript that meets the standards provided for basic reporting. Experimental design: The research question is well defined and relevant, and the manuscript clearly states how this research fills a knowledge gap. There are some details that could be included in the methods, otherwise, the methods are sound. Validity of the findings: The data are statistically sound. The conclusions and discussion are well stated, although the discussion could include more experiment specific comparisons to the literature. Additional comments: The authors present an interesting manuscript examining the role that the boring sponge plays in in oyster predation by crabs. Specifically, they investigate how the presence of absence of the boring sponge affects attach rate and handling time of the stone crab, and important oyster predator. Although the experiment was fairly straightforward, I think this manuscript includes important information on this three species interaction. There are a few grammatical issues, and I think the intro/discussion can be expanded. However, I feel this issues should be easily addressed by the authors, and thus recommend publication after minor revisions. More specific details below: Introduction: In general, the introduction does a good job putting the research in the broader context, however, I feel as though there are some specifics missing. For instance, why use the stone crab? That is, many studies focus on blue crabs and mud crabs as oyster predators. In fact, a number of recent studies have indicated that mud crabs are perhaps the most important oyster predator (Rindone and Eggleston 2011, Johnson et al. 2014, Carroll et al. 2015). Interestingly, the stone crab itself is also expanding its range. I don’t have problems with using the stone crab, but this species should be introduced in a little more detail. Likewise, for the boring sponge, a source showing that fouled individuals are more susceptible to predation would be useful. P 3 LN 35-38: Two small issues. First, it is not entirely clear to me what this sentence is saying, so it probably needs to be reworded. Second, place (1983) after Schmitt et al. on line 36, it does not need to be placed at the end of the sentence. P 4 LN 57-59: Be careful here – this study specifically investigates how the presence of sponges impact the trophic interaction between oysters and stone crabs (Line 60-62), but not how changes in the distributions of sponges are impacting this trophic relationship. This sentence is fine as justification, but this is not being investigated by this study. P 4 LN 60: Here and throughout, make sure this is in past tense – “we investigate” should be “we investigated” P 4 LN 65: Insert “than” between “survival” and “mollusks”. P 5 LN 81-83: Were the crabs all the same sex? P 5 LN 86: “Oysters of similar sizes were used…” What was the size range of oysters used? P 5 LN 88-89: If the 5 hour and 24 hour oyster consumption checks were not used in the overall analysis (which it does not appear to be), this sentence can probably be omitted. Page 6-7 LN 119-129: Make sure the model numbers here are the same as they are described in the results and the table. In the text, model 2 is separate estimates of attack rate, whereas in Table 1 and the results, model 2 is separate estimates of handling time. Results: The authors should delve a little more into the results for model 1, since that was the best fit and estimated different attack rates for the different oyster groups as well. While I agree that the similar support of separate estimates of handling time model (see above comment) supports that handling time is probably most important, the best model also has very different attack rates. This should be mentioned. Discussion: Should start narrow, then expand to broader implications. Summarize the results of the study first, and compare to the literature. Were the rates observed here comparable to other studies for non-fouled oysters? Which other studies of fouled oysters (or other organisms) also showed higher predation on the fouled organisms? The discussion is ok otherwise, but I think needs to include some oyster- and sponge-specific discussion before getting into all the broader implications. Further, I think the authors should also talk about the stone crab as a range expander – it has certainly expanded its range in NC, and since boring sponges are present all along the east coast, expanding stone crabs could be problematic for oysters in Virginia, for example, even if the sponge is not in new areas there.

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: RANGE EXPANSION OF A FOULING SPECIES INDIRECTLY IMPACTS LOCAL SPECIES INTERACTIONS Review round: 1 Reviewer: 2

Basic reporting: see below Experimental design: see below Validity of the findings: see below Additional comments: July 31, 2017 PeerJ Editor Re: Manuscript #18935 Review of Speights and McCoy “Range expansion of a fouling species indirectly impacts local species interactions” Speights and McCoy used a series of laboratory trials to assess the effect of boring sponge infestation on predator-prey interactions between eastern oysters and stone crabs. In general, I thought the ms was clearly written and largely straightforward. The results of potentially longer handling times on non-infested oysters are certainly interesting, and could be of wide interest. One overarching comment is that the manuscript is extremely succinct – at time perhaps too much so. For instance, the manuscript notes that there may be changes in the distribution of boring sponge, but only introduces stone crabs as an “important native” (e.g., Ln 61). In fact, stone crabs themselves may be experiencing a change in distribution (i.e., poleward range expansion) that is equally important in the context of this 3-member interaction – particularly in the region in which animals were collected for this study (e.g., Rindone and Eggleston 2011). This point could be expanded on in a revised manuscript (e.g., Ln 147). Similarly, Sara Coleman (UNC-CH) published a thesis in 2014 entitled “The Effects of Boring Sponge on Oyster Soft Tissue, Shell Integrity, and Predator-Related Mortality”. Her results suggested that based on tissue content vs shell integrity, crabs should attack infested oysters over non-infested oysters. However, in feeding trials similar to Speights and McCoy, she found no difference in attack rates on infested vs. non-infested oysters. She concluded that the crushing strength of the crabs she used was so great (based on their size and reported crush force), that no oyster shell was a functional barrier to predation. Perhaps the size of stone crabs used by Speights and McCoy were smaller than those of Coleman – either way, it seems like some broader commentary on how these studies/results fit together could strengthen the discussion. Other comments: Ln 12: Could delete “In this study”, and just start “We investigate…” Ln 37: I wondered how barnacle fouling on their shells increased the proximity of snails to predators. Since this is not intuitive, it would be good to detail the mechanism by which this occurs. Ln 57 (and elsewhere): I understand I’m being perhaps a bit of a stickler, but I encourage the authors to be careful in how they present “recent changes in the distribution” of sponges. To my knowledge, neither Lindquist 2011 or Dunn et al. 2014 actually tested this. Lindquist mapped the present day distribution of sponges, and Dunn et al. examined sponge infestation in various substrates. Both worked across salinity gradients, and both certainly commented on the potential for sponges to be farther up-estuary (indeed, it’s a logical presumption). However, I’m not sure either had the long-term data to rigorously evaluate a shift in distribution. It’s also entirely possible that I’ve missed some piece of evidence those researcher did provide, and I am also unfamiliar with the work of Hong and Shen 2012. I suppose I’m just asking the authors to be certain that they are not overstating what researchers may believe is happening, versus what researchers have actually documented. Ln 86: “Oysters of similar sizes…” is vague. Provide actual sizes (and if sizes changed between trials for an individual crab, or across the four cycles [Ln 103]). Ln 90-104: I thought the authors did a good job of accounting for the issues related to reusing crabs. I have no problem with interpreting their results keeping in mind that reuse occurred. Ln 133-139: The increased handling time for non-infested oysters is the crux of this paper. However, I would like to see more detail on how handling time and attack rate (abort rates, etc.) were documented. Were these factors actually observed/recorded to feed Eqs 1&2? Or are they just estimated from the model fits of mortality. If it’s the latter, I have to wonder what sort of confidence intervals have to accompany those estimates. I think this is another place where the manuscript might benefit from further text/explanation. Ln 151: “inland into estuaries” could be “up-estuary” Good luck moving forward with this ms, yours, [this is a signed review] Joel Fodrie Institute of Marine Sciences & Department of Marine Sciences University of North Carolina at Chapel Hill

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: RANGE EXPANSION OF A FOULING SPECIES INDIRECTLY IMPACTS LOCAL SPECIES INTERACTIONS Review round: 1 Reviewer: 3

Basic reporting: Line 65- please add “than” before survival Experimental design: There are a couple of changes that should be made to ensure this work can be replicated. Please provide the temperature and salinity of experimental tanks. Figure 1- please include n=number of trials for each mean/standard error. Figure 1- Please explain which equation was used for the lines shown here (I believe it was model 1 from your R code). Validity of the findings: No comment. Additional comments: This article is well-written. I was able to replicate all results from the code and data provided- nice job. The use of AIC to interpret the finding of this study was compelling. I am choosing minor revisions just because I think the temperature and salinity are necessary to fully interpret your results and compare them to other systems.

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: DECREASED QUALITY OF LIFE AND TREATMENT SATISFACTION IN PATIENTS WITH LATENT AUTOIMMUNE DIABETES OF THE ADULT Review round: 1 Reviewer: 1

Basic reporting: The article is written in a clear English. The introduction and the background are complete and appropriately referenced to relevant prior literature. All results relevant to the hypothesis are showed in the text and in the tables. Experimental design: The research question is clear, relevant and meaningful and the study is rigorous, well detailed and conducted according to technical standard. Validated tools were used in the language in which the study was conducted. However, it would be appropriate to enclose questionnaires as supplementary materials, in order to facilitate a better understanding of the items. Validity of the findings: The study, showing that LADA patients have a poorer QoL profile than patients with T1DM and T2DM, especially at the expense of DR and insulin treatment, provides new results on a topic that is currently little explored. However, the study included patients with chronic pathology with a view to assessing the impact on quality of life. Notably, quality of life is dependent on mental well-being. Exclusion criteria include mental illness. Therefore, as the mood of these patients was evaluated? Would it not be more appropriate to include this variable in statistical analyses to assess its impact on quality of life and Treatment Satisfaction? Another question. Another exclusion criterion is the presence of dementia. Who and how did patients evaluate to exclude cognitive impairment? 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: DECREASED QUALITY OF LIFE AND TREATMENT SATISFACTION IN PATIENTS WITH LATENT AUTOIMMUNE DIABETES OF THE ADULT Review round: 1 Reviewer: 2

Basic reporting: No comment Experimental design: No comment Validity of the findings: 1) The most important issue is the limited number of LADA patients who agreed to participate in the study: 86 contacted patients and only 48 participants, with a refusal frequency equal to 44% . This high proportion of refusal is an important selection bias that can impact on results validity. Which factors influenced participation in the study? Can authors exclude that participation was influenced by disease status, socio-demographic factors, quality of life or treatment satisfaction? Which are the differences beetween patient who partecipated and those who refused? This point is crucial for results validity! 2) Additionally, given the high number of statistical tests, a Bonferroni correction should be applied. 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: DECREASED QUALITY OF LIFE AND TREATMENT SATISFACTION IN PATIENTS WITH LATENT AUTOIMMUNE DIABETES OF THE ADULT Review round: 1 Reviewer: 3

Basic reporting: 1. Your MATERIALS AND METHODS needs more details about the inclusion criteria for LADA,T1DM,and T2DM, respectively,especially for the inclusion criteria for the LADA. 2. I suggest that the results you describing in Lines 165-177 shoud be moved forward to the part of MATERIALS AND METHODS. 3.It is noted that your manuscript needs careful editing by someone with expertise in technical English editing paying particular attention to English grammar, spelling, and sentence structure. Experimental design: no comment Validity of the findings: Lines 237-239:T2DM patients without insulin treatment showed a significantly lower hypoglycemia frequency perception in comparison with insulin-treated LADA subjects (p=0.006).Please explain why ? Lines 228-229:Insulin-treated LADA patients had a higher hyperglycemia frequency perception than T1DM (p=0.04)...... Please explain why ? Additional comments: The number of subjects with LADA is too small so that the conclusion that "LADA patients showed a poorer QoL profile than patients with T1DM and T2DM" in the current study was not convincing.

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: DECREASED QUALITY OF LIFE AND TREATMENT SATISFACTION IN PATIENTS WITH LATENT AUTOIMMUNE DIABETES OF THE ADULT Review round: 2 Reviewer: 1

Basic reporting: The manuscript is well structured and adequate regarding literature references. It contains relevant results to support the hypothesis. Experimental design: Research question is well defined, relevant and meaningful. Methods are well described. Limitations are adequately described in discussion section. Validity of the findings: Results are significant, but partially inconclusive, suggesting the need for further studies on this issue. Additional comments: It is worth appreciating the attempt to explore what influences the quality of life of patients with LADA, even if the impact that mood alterations may have on the management and perception of the disease is only partially reported. This is a study limitation. However, this point only partially limits the success of the study which is adequate and complete.

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: MGST2 AND WNT2 ARE CANDIDATE GENES FOR COMITANT STRABISMUS SUSCEPTIBILITY IN JAPANESE PATIENTS Review round: 1 Reviewer: 1

Basic reporting: All information contained in attached review. Experimental design: All information contained in attached review. Validity of the findings: All information contained in attached review. 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: MGST2 AND WNT2 ARE CANDIDATE GENES FOR COMITANT STRABISMUS SUSCEPTIBILITY IN JAPANESE PATIENTS Review round: 1 Reviewer: 2

Basic reporting: General comments: The language in this manuscript needs to be improved to a more professional and less conversational style. A review by a native English speaker should be sufficient to raise the manuscript up to an appropriate standard and address the overuse of commas throughout the paper. The manuscript seems well referenced and the background in the main body of the paper describes the phenotype well but should expand slightly on the previous work by these authors. The manuscript structure is somewhat inconsistent. The introduction section needs only some language refinement but the method section is very confusing and hard to follow the sequence of work. As a result, interpreting the results and conclusions is impossible. The tables are poorly formatted and contain confusing information which is not always relevant and appropriate. The raw data was available to this reviewer. The manuscript appears self-contained, has clear aims and a relevant result reported which is consistent with the hypothesis. Specific Comments: Abstract: The Section Background/aim should have a more detailed description of the phenotype since this is a general interest journal, not a ophthalmology journal. A sentence or two should suffice to explain the features to the reader. Introduction: Lines 73-80. The language here needs to be improved to a more professional and less conversational style. Figures: The LD figures (1 and 2) are incorrect since no adjustment for kinship has been made and the association analysis they support is entirely inappropriate. Tables: Table 1 should be completely revised. Table 2 contains a column labeled "Other Methods" which is hard to understand. Table 3 contains an eQTL column which adds nothing to the results. Experimental design: General Comments: This is original primary research well within the aims and scope of the journal. The research question of whether SNPs in two previously identified locations by prior work, is well defined, relevant and meaningful. This is a classic fine-mapping approach which seeks to refine a prior signal in two genomic locations and the family-based study design is appropriate in this context. The rigor of the investigation was harder to assess. The methods section is confused and poorly structured and raises several questions in this reviewers mind as to the statistical understanding of the authors. They make several assertions that are incorrect, misleading or outdated that significantly undermine their results. Replicating their analyses with the methods section in its current form would not only be quite difficult but would in fact be inappropriate in some parts. I address these failings in the specific comments below. Specific Comments: The methods section need major revision in order to meet even basic standards of adequate reporting. The way it is currently structured suggests that certain quality control measures were applied after some analyses were performed. Since this seems unlikely, I suggest the authors use a flowchart to clarify the workflow and use this to restructure the methods section. This flowchart could be added to the paper if figure limits allow or added to a supplementary methods section. For the description of the study subjects, the table is poorly formatted and should be revised to reduce redundancy. It is typical to include pedigree drawings unless there is a risk of identification and would greatly assist the reader in understanding the cohort. Samples with available DNA could be indicated on these drawings, rather than detailed in the table. The section describing the genotyping seems straightforward but a table of the primers should be included in the supplementary methods. However, the genotyping rate is rather lower than I would expect and does raise some concerns which the authors should address. Why were there such high missingness rates? This manuscript contains no discussion of power. Power calculations should be performed and included in the methods and results. This manuscript contains no discussion of population stratification. Even within apparently homogeneous populations, significant substructure can still exist and can influence the results of association based tests. A discussion of this in the methods would be appropriate. Although linkage analysis is robust to population stratification, it does require good estimation of allele frequencies in the data. This should be included in the methods. I also have several concerns about how the analyses were performed. The section on association testing uses a completely inappropriate methodology to analyze these data. The authors even seem aware of this themselves, stating that standard chi-square analyses assume independence of the subjects and this standard is not met in this cohort. There is no defending this analysis, it is meaningless and should be removed. A more appropriate association approach would be to use FBAT, EMMAX (with the appropriate caveats about the beta generated) or other methods designed to use or control for kinship between study participants. The next section discusses Mendelian inconsistencies and this section raises several red flags. The authors mention duplicate subjects but do not adequately explain how duplicate subjects were selected for removal. Equally of concern is the large number of reported Mendelian errors and the methods do not sufficiently detail how these errors were dealt with. Typically, one would remove inconsistent genotypes in a SNP just in a family if only one family had an error. If the same SNP has errors in two or more families, the SNP should be deleted across the entire dataset. It is not clear whether the authors did this in their data. Although the authors discuss using TDTae, a method to address genotyping errors without deleting data, this is not used widely in the field as it has been superseded by other, better tests. In addition, the authors describe this method as parametric, which is not exactly correct - the error model is parametric but the test for linkage is not. But no discussion of the other analyses described in the results is included here. These sections should be revised and reordered. Validity of the findings: The association results are the result of an inappropriate methodology and are meaningless. This section should be removed or the analysis repeated with a more appropriate test as I suggested in my review of the methods section. The results also contain considerable discussion of the methods and these portions should be moved to the methods section. For example, there is no mention of the pseudomarker program until the reader reaches the results section. This is not the right way to report methods or results. The exact models used should be presented and the method used for generating allele frequencies. As I've noted, methods are poorly described and as a result, it is hard to assess whether any of the linkage results presented here are meaningful, as I cannot ascertain whether the data were appropriately cleaned before analysis. The use of TDTae is discouraged and it is not clear to me if the authors fully understand the implications of using this test in these data. Finally, the authors make no adjustment for multiple comparisons in these data, despite using several analysis methods and in the parametric linkage analysis, more than one model. It seems likely that such an adjustment would render all results entirely non-significant but should be performed and the manuscript adjusted to reflect the results in the light of this. Additional comments: Although this manuscript presents an interesting fine-mapping study, I have grave concerns about the statistical analyses presented and as a result, cannot recommend this paper for publication without significant revision.

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: MGST2 AND WNT2 ARE CANDIDATE GENES FOR COMITANT STRABISMUS SUSCEPTIBILITY IN JAPANESE PATIENTS Review round: 2 Reviewer: 1

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: BACTERIAL AND FUNGAL COMMUNITIES RESPOND DIFFERENTLY TO VARYING TILLAGE DEPTH IN AGRICULTURAL SOILS Review round: 1 Reviewer: 1

Basic reporting: Most of the for the Basic reporting season is acceptable. However, below are some comments about the English language. Line 50 change Tillage therefore for Tillage, therefore, Experimental design Line 62 This however … The use of "this" brings an unclear antecedent. I suggest that the authors rewrite the sentence making a better connection with the previous statement. Line 83 change Prior to for Before 225 change This for This tillage treatment effect Line 229 and line 231 the use of this brings an unclear antecedent. Line 304 change eachother for each other Experimental design: Some parts of the topic experimental design need to be better explained. Please see the questions and comments below. The authors proposed the nMDS analysis on their material and methods (see line 194). However, during their results and discussion, they used the term MDS, which is a different kind of multivariate analysis. Thus, I think there is a mistake on the use of MDS term and the authors should do a double check on their manuscript. For the lines 227-229 and 231 and 232, I have a doubt about which are the statistical analyses that support the both statements. My suggestion is to rewrite these sentences and citing the analysis which endorses it. The authors used the two-way ANOVA analysis ( e.g. line 233), however, they did not describe in methodology. I suggest that the author use the envif analysis from Vegan package in R (http://cc.oulu.fi/~jarioksa/opetus/metodi/vegantutor.pdf) to evaluate the correlation between the environmental table (table 1) and the nMDS results. This analysis may contribute to a better visualization of which are the chemical and physical attributes that better explain the nMDS results, being helpful to confirm the hypotheses proposed . My suggestion for the lines 296-298 is to include a reference for the ARISA methodology statement. Validity of the findings: no comment Additional comments: I commend the authors for the manuscript, evaluating the effects of tillage practices and depths on soil microbial community. The article is written in a good scientific English and text structure. If there is a weakness, it is in the statistical analysis and some better explanation about some topics during the text ( as I have noted above). In my opinion, after the answer of some question and the inclusion of some analysis that could enrich the discussion, the manuscript will be ready the Acceptance.

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: BACTERIAL AND FUNGAL COMMUNITIES RESPOND DIFFERENTLY TO VARYING TILLAGE DEPTH IN AGRICULTURAL SOILS Review round: 1 Reviewer: 2

Basic reporting: The basic reporting meet the criteria, except for: -The tittles of the figures would benefit from being more descriptive. For example, Figure 2 and three have identical titles. Experimental design: The experimental aspects appear sound. Since different tillage techniques are used, it seems relevant to justify the reason for studying them both in relation to depth. Validity of the findings: I have some comments regarding the validity and discussion of the findings. The authors could discuss the potential for the fungal community results to have been influenced by methodological factors in the molecular approach. It seems the relative error is greater and thus it appears that the sensitivity for the bacterial and fungal analyses may not be comparable? Thus the conclusion of who is more or less responsive to the management, may need to be revised. The discussion in lines 284-293 seems too general and not sufficiently insightful and focused. The conclusion section seems to be too broad, not focused enough of the main findings and their implications. 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: HOW TO NORMALIZE METATRANSCRIPTOMIC COUNT DATA FOR DIFFERENTIAL EXPRESSION ANALYSIS Review round: 1 Reviewer: 1

Basic reporting: Some text descriptions are really confusing. The mathematical formulas are not clear. Overall, the English writing should be improved. Experimental design: The simulation steps are not described clearly. Validity of the findings: Though the simulation results suggest that the proposed taxon-specific scaling outperforms global scaling, the real data results are not enough to support the claim. Additional comments: The authors claim that calculating scaling factors on the level of taxon instead of on the level of global, the performance of differential analysis will be improved. I have some comments on the follows, 1. Some formulas are confusing. For example, formula (3). The estimated scaling factors are only determined by feature i, which is obviously not correct. 2. Some statements are confusing. For example, what does “predictions“ means in line 40? Do the predictions mean DEFs? In line 124, which is “organism-specific abundance”? What is the difference between organism-specific and taxon-specific? 3. The simulation steps are not described clearly. How did the abundance generated, based on what kind of generative model? How did the count matrix of different organism combined together? What is the proportion of each organism? 4. In the real data comparison on page9 13-15, the gold standard for DEF for both global scaling and taxon-specific scaling. The comparisons of the numbers of DEFs identified of both approaches cannot support the claim one outperforms the other, and vice versa. More identified DEFs could be false positive, and few identified DEFs could be lacking of statistical power. The authors should use other criteria to justify taxon-specific scaling is better than global scaling.

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: HOW TO NORMALIZE METATRANSCRIPTOMIC COUNT DATA FOR DIFFERENTIAL EXPRESSION ANALYSIS Review round: 1 Reviewer: 2

Basic reporting: In this manuscript, the authors propose a strategy for normalizing metatranscriptomics data for sequencing depth in the context of differential analysis. They propose the ‘taxon-specific scaling’ to take into account the structure of the metacommunity. This strategy consists in separating the dataset into one dataset per organism, to normalize them independently with standard method such as the method proposed by Anders et Huber in the DESeq2 Bioconductor package and to analyze a combined dataset. They compare this strategy with the ‘global scaling’ one which considers the whole dataset forgetting the notion of species. They study the interest of their strategy, both in simulations and in a real dataset. The introduction will benefit from a more detailed description of the context of normalization for differential analysis. Are standard strategies for transcriptomic directly applicable for metatranscriptomics ? but also are those for differential abundances from metagenomics data applicable for metatranscriptomics (see Weiss et al. 2017). The manuscript is not so easy to read for different reasons : 1/ some essential terms essential such as ‘normalization’ or ‘library size’ are not well-defined and need to be explained in the metatranscriptomics context. 2/ it subsists some redundancy (normalization part of the introduction and taxon-specific scaling and global scaling’ paragraph of the materials and methods section). In order to clarify the manuscript, I suggest to present normalization as between-sample normalization and to define clearly what is the library size. In general, for normalization purpose, the library size is for each sample the total number of sequences which will be analysed. One key point not mentionned here is that normalization depends on the biological question. The main two biases we would like to correct in metatranscriptomics is the composition of metacommunity and the sequencing effort. Both may vary with sample. Depending on the biological question only a correction for sequencing depth or for both is needed. It would be clearer to separate the two and not to speak about ‘organism-specific library size’. I suggest for Simulation II ‘modification in the abundance of species present in the metacommunity’ instead of ‘with library size variation’. The interpretation of the results of differential analysis will depend on the question and on the normalization step and a feature statistically significant can be result of difference in expression or/and in the abundance of the species it comes from. I suggest, for my second remark, to merge the normalization paragraph of the introduction with the paragraph in materials and methods. And in this section to distinguish in different paragraphs i) the taxon-specific scaling, ii)the global scaling, and only in a third part to discuss about the way to compute scaling factor (using DESeq2 for example). Experimental design: The normalization of metatranscriptomics data is a great challenge and the question of when and how to take into account the variations in species abundance is an important question. The authors explore several interesting scenarios. Nevertheless a real metatranscriptomics study contains generally more than 5 or 12 organisms and most of them are uncultivable. Would the authors discuss this point and the application to metagenomic species ? I thank the authors for providing the real raw data and a script with R code for simulation and DESeq2 analysis. Nevertheless, I would appreciate to have a minimal script that downloads the data and either simulated data nor script with simulated parameters in order to reproduce the results of all the analyses. When using the test.main function or the DESeq2.tax.specific function, it results on a matrix of results with 1000*6 samples and not 5000 features as noted l.316. Could the authors explain why? Is it because they sum over all the organisms? L156 the authors wrote ‘There are several reasons why the analysis of the recombined metatranscriptome data can be useful ; first of all, the statistical power of organism-specific tests may be low due to decreased counts.’ I do not agree : if the analysis is done organism by organism, the correction for multiple testing will be less stringent (1000 tests instead of 5000). Validity of the findings: No comment Additional comments: I regret the absence of discussion on 1/ assumptions for normalization which are i) the great majority of the genes are DE and ii) balance between up and down regulated features. Could the author develop this point and the cases other way to compute scaling factors (ie instead of DESeq2 normalization) is necessary ? 2/ validity of the proposed strategy. Could the authors discuss i) on the situations where it is really possible to obtain counts per organisms, and which minimal coverage is needed for each organism, etc. ? ii) when it is reasonable to sum over features? iii) the case of a great difference in metacommunity diversity, e.g. several organisms absent in one condition? In the conclusion, the authors write l. 502-506 « Normalization of metatranscriptomic data must have the goal to eliminate the influence of taxonomic variations from functional analysis. (…) We argue that for a correct normalization the metatranscriptome needs to be decomposed to normalize the organism profiles independently. Then the metatranscriptomic count data may be recombined from the normalized profiles to look for any global trends in the superimposed count data.’ How to superimpose count data is not well explained . Is it always straightforward ? Comments for the authors The reference for DESeq2 is (Love et al. 2014) and in l.186 the authors need to cite Bionconductor and the version they used. One way to obtain references for R packages is to tape « citation(« pkgname ») in the R console. The reference for R is the following one : R Core Team (2017). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/. Minor revisions L50 'transcriptome' is misspelled as 'transciptome'. L133 ‘separated’ is misspelled as ‘seperated’. L211 ‘library’ is mispelled as ‘libray’. l.175 the right name for the normalization method implemented in edgeR is TMM (Trimmed-Mean of M-Values) (Robinson and Oschlack 2010) (l175 , l317,318, 347) The abbreviation used for differentially expressed features is DEF (l.196) but either DE nor DE is used all along the manuscript. Please choose one and be homogeneous. Figure 9. the size of the legend and labels is too small. One way to gain place may be to replace « 13d vs 27d » by « 13 vs 27 » Reference Weiss,S., Xu,Z.Z., Peddada,S., Amir,A., Bittinger,K., Gonzalez,A., Lozupone,C., Zaneveld,J.R., Va ́zquez-Baeza,Y., Birmingham,A. et al. (2017) Normalization and microbial differential abundance strategies depend upon data characteristics. Microbiome, 5, 27.

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: HOW TO NORMALIZE METATRANSCRIPTOMIC COUNT DATA FOR DIFFERENTIAL EXPRESSION ANALYSIS Review round: 2 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: In this manuscript, the authors propose a strategy for normalizing metatranscriptomics data for sequencing depth in the context of differential analysis. They propose the ‘taxon-specific scaling’ to take into account the structure of the metacommunity. This strategy consists in separating the dataset into one dataset per organism, to normalize them independently with standard method such as the method proposed by Anders et Huber in the DESeq2 Bioconductor package and to analyze a combined dataset. They compare this strategy with the ‘global scaling’ one which considers the whole dataset forgetting the notion of species. They study the interest of their strategy, both in simulations and in a real dataset. I thank the authors for the revised version of their manuscript, which considers all my previous comments and suggestions. Just few typos and one minor comment: peerj-17882-Additional_File_4: Fig 4 legend: ‘library’ is mispelled as ‘libray’. Fig 6 legend : the color box with « white », « black » and « blue » is inexact and not useful. I tested the code in the peerj-17882-Additional_File_3.R file on the real dataset and I have got the following error message « Error in `rownames<-`(`*tmp*`, value = colnames(countData)) : duplicate rownames not allowed » I do not use the same versions of DESeq2 and R (R 3.4.0 and DESeq2_1.16.1). It may be the origin of this error. This error appears using the DESeq2.norm.mat function line 384 YMat <- DESeq2.norm.mat(XMat, cond.vec,type.vec) A way to avoid it is to check that colnames of XMat are not duplicated before using the function and rename the columns if necessary.

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: MICROBIAL COMMUNITY DIVERSITY PATTERNS ARE RELATED TO PHYSICAL AND CHEMICAL DIFFERENCES AMONG TEMPERATE LAKES NEAR BEAVER ISLAND, MI Review round: 1 Reviewer: 1

Basic reporting: In this paper Hengy et al. describe the microbial diversity of four lakes with different physical and chemical characteristics. They describe the environmental differences between and within lakes (different depths) at three different times and correlate them with the microbial community structure. Even though the sample sizes are not large I think the results are clear and concise. This study contributes to the knowledge on the potential effect that environmental variables have on the bacterial community dynamics in aquatic habitats. Experimental design: Overall, the experimental design is correct. However, in the general comments section you will find some suggestions and aspect that need to be clarified. Validity of the findings: In the general comments section you will find my main concerns which have to do with rarefying the sequence data and correcting for multiple comparisons when calculating correlations. Additional comments: Below you will find some general or specific suggestions that are mainly aspects associated to the methods. Line 73: Change “reaction” to “reactions” Lines 102-103: I assume that the water sample was passed through the larger filter first and then the smaller one? Please explain with more detail. Line 118: Replace “was measured using and an ion…” by “was measured using an ion…” Lines 130-131: This sentence is not clear. Was the DNA extracted from both filters at the same time? What is the reason to pool both filters? The 2.2 filter excludes all large particles and cells so I would think the 0.22 is the one that should have all the bacterial cells. Please explain. L134: the 27F and 1493R primers amplify the whole 16S rRNA gene, not only the V4 region. Please correct this sentence. Lines 142-151: You need to give more details on the processing of the sequences on this section and some also on the results section: Include how many sequences were obtained initially. Which filtering steps did you do in addition to the analysis of chimeras and how many sequences were analyzed after the filtering steps How many OTUs were obtained? Did you rarefied the samples? At which depth? If the authors didn’t rarefied the samples I strongly suggest to do it. Line 154: Change “Statistical analysis (both chemical and biological) was completed” to “Statistical analyses (both chemical and biological) were completed” Line 153: For this section please clarify why did you retain singletons for alpha diversity and removed them for beta diversity. The same OTU table should be used for both diversity estimates. Otherwise the results may be biased. Also, this section could be organized better. Lines 155-158: This sentence should be included in the “Microbial Taxonomic Analysis” section of the methods. Lines 158-160: Move this sentence to line 155 Lines 169-171: How were the samples normalized? Explain the variance transformation. Lines 185-191. Did the authors correct for multiple comparisons? If not I strongly suggest to do it. Otherwise you might be overestimating the significant correlations. Line 195: Change “was observable” to “were observed” Lines 211-212: Move this sentences to methods. Also it is not clear what the authors mean with relative abundance. Do you mean sequencing depth? Lines 210-224: Is it very important to clarify if the authors rarefied the samples (OTUs) in order to compare diversity values across samples. Otherwise the alpha diversity estimates may be biased. Lines 281-282: This information should be included at the beginning of the results section. You could include how many OTUs were shared among lakes and how many OTUs were unique to each lake or unique to groups of lakes that are more similar in terms of the environmental variables. I suggest including a map of the lakes sampled as an additional figure. Figure 1: You could add a legend for the habitat as is done in the other figures Figure 2: could be improved, so that the legend can be distinguished clearly. The labels on the X axis could be larger and the bars could be thicker. Also I would suggest to change the colors so that it’s easier to distinguish the different bars

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: MICROBIAL COMMUNITY DIVERSITY PATTERNS ARE RELATED TO PHYSICAL AND CHEMICAL DIFFERENCES AMONG TEMPERATE LAKES NEAR BEAVER ISLAND, MI Review round: 1 Reviewer: 2

Basic reporting: Writing should be improved for clarity. - The emphasis on 'lakes within the Laurentian Great Lakes basin' in the title and abstract is misleading. The Great Lakes basin is massive – 4 lakes is too few lakes to characterize lakes in the Great Lakes basin and it sounds like the study lakes are close together geographically near Beaver Island, MI, making them not especially representative of the extent of the Great Lakes basin. I suggest specifying Beaver Island, MI, not the Great Lakes basin, as the location of the study lakes and being more specific in the abstract concerning the number of lakes sampled. - You claim to show that conditions in the water column ‘significantly impact’ community structure, yet all of your analyses are correlation based. It is more accurate and clear to emphasize the correlations observed in this study than to imply causation. - Additional examples where clarity could be improved: --‘physical, chemical, biological, and temporal processes’ in the first line of the abstract does not make sense to me. Physical, chemical, and biological processes may be temporally variable or dynamic, but I do not think that that there are ‘temporal processes’ that are separate from physical, chemical, and biological processes. --In the next sentence you talk about ‘similar geographic regions’ – do you mean the same geographic region? --It would help to be more explicit about the differences between DOC and DOM (and to define them before using the acronyms) and what information you gain from looking at both instead of one or the other (lines 50-51 of the introduction) Suggestions for improvement on literature references and background/ context: - You need to include a citation for the R statistical environment (type “citation()’” into R to generate the citation – example from the version of R I use on my computer: R Core Team (2016). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/. - The conclusion that ‘despite geographic proximity, each lake harbored a distinct microbial community, suggesting that lake chemistry is a stronger constraint on microbial communities than geographic region’ covers a topic that has previously been explored in freshwater lakes, but it seems like there were a number of relevant studies overlooked from the discussion. I suggest that the authors consider whether previous surveys of lakes provide relevant context to their work, including: Yannarell and Triplett 2005. Geographic and Environmental Sources of Variation in Lake Bacterial Community Composition. http://aem.asm.org/content/71/1/227 Van der Gucht et al. 2007. The power of species sorting: Local factors drive bacterial community composition over a wide range of spatial scales. http://www.pnas.org/content/104/51/20404.short - I additionally recommend that the authors consider whether Newton and colleagues (2011) A guide to the natural history of freshwater lake bacteria may provide additional useful context for their sequence data: https://www.ncbi.nlm.nih.gov/pubmed/21372319. In my opinion, the freshwater database initially curated as part of the Newton review is the best database to use when classifying freshwater sequences (though whether sequence database really matters depends on your question). The database and instructions on how to use it can be found here: https://github.com/mcmahon-uw/FWMFG I commend the authors for including accession numbers for their sequence data. However, I am not sure whether there is sufficient metadata available with your deposited sequences. From what I was able to see when I looked up your accession numbers in MG-RAST, I can tell the location but not the depth or time sampled. A key may be needed to connect name to MG-RAST ID to necessary metadata, perhaps this could be included as a supplemental table. While not necessary for publication, I think it would be useful if the R code, OTU table, and metadata were shared via GitHub. The article structure is professional, though I do have a few suggestions that might improve the presentation of the study sites and results. I think it would be useful to include a table in the main text with the basic limnological characteristics of each lake (e.g., latitude and longitude, ranges of observed water temperature and chemistry parameters described in supplemental table 2). I am not sure what the value is in including an NMDS plot as well as CCA/ pCCA plots. Since constrained ordinations force samples to correspond to measured environmental parameters, I am not sure how useful it is to include these plots in the main text - perhaps they would be more appropriate as supplemental figures. Experimental design: The authors present original primary research that appears to be within the aims and scope of PeerJ. I am unclear on what knowledge gap is specifically being filled. I think the research question could be more clearly defined. The epilimnon/ hypolimnion contrast is interesting; however, it does not appear that the hypolimnion of Lake Michigan was sampled. It is not unusual for the mixed layer depth in Lake Michigan to exceed the 14.5-18.3 m from which bottom samples were collected. Temperature profile data from supplemental table 1 supports the idea that the Lake Michigan hypolimnion was not sampled. The sequencing methods are not described in sufficient detail. First, 27F to 1492R primers span almost the entire 16S rRNA gene, encompassing far more than the V4 region. Does this mean that nested PCR was used? What primers were used for sequencing? If the 27F and 1492R primers were the primers used for sequencing, the MiSeq reads could not have merged. If this was the case, did you only analyze forward or reverse reads? If the number of cycles were varied for each cycle, you should include somewhere, perhaps in a supplemental table how many cycles were used for each sample. How long were your Illumina MiSeq reads (e.g., 2 x 250)? You cite Kozich et al. and say that sequences were processed using Mothur, but if you followed the Mothur MiSeq protocol you should specifically state that you followed the Mothur MiSeq SOP and include the date that you accessed the SOP. Any deviations made from the Mothur MiSeq SOP (if this is what you followed) should be specifically noted. What was the rationale behind removing singletons prior to normalizing samples for beta diversity analyses? I am not aware of this being a standard practice, and it does not seem like a good idea to me. Validity of the findings: Some of the conclusions may be overstated. For example, stating that the data 'show that differences in phyicochemical conditions in the water column can significantly impact aquatic microbial community structure' seems to imply causation when the study was correlational and involved few lakes. It is challenging for me to follow the links between the original research question, supporting results, and conclusions. I find some statements in the results/ discussion to be confusing. For example, the statement starting on line 269 that community composition was indistinguishable between surface water and bottom water communities when oxygen is not stratified seems like a sampling artifact to me. Lake Michigan surface communities from open water stations in the middle of the lake differ significantly from communities sampled from the hypolimnion even though the hypolimnion is oxygenated. A recent paper published in ISME J has also described differences between surface and hypolimnion communities in lakes where the hypolimnion is oxygenated: http://www.nature.com/ismej/journal/vaop/ncurrent/full/ismej201789a.html?WT.feed_name=subjects_microbiology. Additional comments: What does it mean to explore microbial communities within lakes on an 'individual level' - one lake at a time (line 38)? It is not clear to me how relationships between taxa and environmental gradients are being 'deeply' explored (line 84). Is this a reference to sequencing depth? I am not sure that I agree with the interpretation of Jezbera et al. 2012 lines 317-319; Polynucleobacter comprised 1.1-5% of the bacterial assemblage in most lakes with pH between 8.1 and 8.5. I am not sure what it means when Polynucleobacter is described as a 'generalist' genus (line 233). It makes it sound like members within the genus are generalists, when I think the reality is that the members of Polynucleobacter are quite diverse.

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: MICROBIAL COMMUNITY DIVERSITY PATTERNS ARE RELATED TO PHYSICAL AND CHEMICAL DIFFERENCES AMONG TEMPERATE LAKES NEAR BEAVER ISLAND, MI Review round: 1 Reviewer: 3

Basic reporting: This paper is clearly written and relevant, but there are numerous weaknesses that I think preclude it from being published in its current state. With respect to literature cites, the authors should consider referencing other comprehensive 16S characterizations of freshwater lakes, particularly ones in the Great Lakes (e.g., Fujimoto, Masanori, et al. "Spatiotemporal distribution of bacterioplankton functional groups along a freshwater estuary to pelagic gradient in Lake Michigan." Journal of Great Lakes Research 42.5 (2016): 1036-1048) I appreciate the wide array of statistical tests applied, but I don't understand why they are all relevant. Specifically, the NMDS and CCA plots seem redundant and it doesn't seem meaningful for this particular paper to do both, when they are specifically interested in constraining their datasets to the variables they measured. The application of different tests does reads like an after thought, not as something planned with their study. Thus, the data are seemingly made to support their hypothesis, however the experimental design is weak (see below). With respect to the taxa and comparisons to other studies, the authors should use the freshwater 16S database (https://github.com/mcmahon-uw/FWMFG). Experimental design: There are no sample replicates, which is, in my mind, a huge weakness. The authors are making vast generalizations about an entire lake based on 120 ml. They also reference "seasonal" differences (line 95 and elsewhere), but all samples were taken in the summer within a few weeks of each other. The PCR conditions are also problematic; 36-40 cycles is a lot. The Earth Microbiome Project recommends 35 (or fewer) and many papers, past and present, show issues with high cycle numbers (e.g., Qi et al., AEM 2001), especially depending on which Taq was used (which the authors do not report here). Validity of the findings: I only question the findings because of the experimental limitations (lack of replicates, post hoc decisions of statistical tests, and PCR/database issues. I really think they should tone down their findings should this get published. That said, I think the findings fit the data and tests that they have. Additional comments: Line 138: "PCR reactions" is redundant Line 197: So4 is not shown on Fig 1 Fig. 1: "temp" is cut off and abbreviations should be indicated in the figure legend Lines 202-3: Lake Michigan is also stratified in the summer Paragraph beginning on line 257: There are numerous contradictory statements here. I think the authors mean to discuss the lack of an oxycline in Lake Michigan, but instead they say that there was no divergence between LM deep/surface communities, which is stratified, but lake stratification is important in structuring communities. Line 211-12: I don't understand what these numbers mean in terms of relative abundance. Lines 219-222: These two sentences are contradictory. Line 274+ What unmeasured environmental differences? This is very vague.

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: MICROBIAL COMMUNITY DIVERSITY PATTERNS ARE RELATED TO PHYSICAL AND CHEMICAL DIFFERENCES AMONG TEMPERATE LAKES NEAR BEAVER ISLAND, MI Review round: 2 Reviewer: 1

Basic reporting: The new manuscript has greatly improved in clarity and has gained strength by adding additional references and clarifying several aspect of the methods and results. Experimental design: I agree with the experimental design but I have made specific suggestions regarding alpha and beta diversity analyses (see general comments below). Validity of the findings: Data and results are clear. Conclusions are well written. Additional comments: I agree with all the modifications that the authors made to the manuscript based on the reviewers’ comments. I think that manuscript has greatly improved. However, I still have one concern regarding alpha and beta diversity analyses. My self (and other reviewers) had previously asked about why the authors retained singletons for alpha diversity and removed them for beta diversity. The authors gave the following response: “Singletons were retained for alpha diversity as the chao1 index relies on singleton abundances for proper implementation of the chao1. However, as singletons are often removed to calculate beta diversity estimates and visualization, as many singletons could be sequencing artefacts.” Based on this response I still think this is not the best approach for two reasons: (1) Chao1 index is presented on table 2 but it’s not even mentioned in the results and discussion and the authors rely only on the Shannon index results. Therefore, I do not see the need of keeping chao1 in the manuscript and therefore I see no need in keeping singletons. (2) the authors stated that singletons could be artifacts so I do not see the relevance of keeping them at all. The authors should eliminate the singletons and (as I said in my first review) they should use the same OTU table for both diversity estimates.

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: MICROBIAL COMMUNITY DIVERSITY PATTERNS ARE RELATED TO PHYSICAL AND CHEMICAL DIFFERENCES AMONG TEMPERATE LAKES NEAR BEAVER ISLAND, MI Review round: 2 Reviewer: 2

Basic reporting: Basic reporting has been improved. I commend the authors for including additional metadata and access to their analysis scripts through github. There are a few lingering issues needing to be resolved before the manuscript is suitable for publication. There is still remnant language implying causation, for example: 'driven by' in the title line 30 - 'can significantly impact' There are also descriptions obscuring the fact that the hypolimnion of Lake Michigan was not sampled, for example: line 31-32 - hypolimnion of Lake Michigan was not sampled; most accurate representation would be 'surface' and 'deep' line 371 - how deep did Fujimoto et al sample? It is likely that you observed differences because you were not sampling analogous deep samples; I do not think your results are directly comparable due to depth differences and the fact that you did not sample the hypolimnion. line 473-476 - you did not sample the hypolimnion of Lake Michigan and need to be clear about this point Description of study in the final introductory paragraph needs to be edited for clarity (lines 153-156) 1) 4 lakes, 3 holomictic... does this mean #4 is meromictic? Why would you not say something about lake #4? 2) Lake Michigan did not experience "temperature stratification, but did not stratify"; Lake Michigan was stratified; you did not sample a site that exhibited stratification line 430 - need more explanation as to why your results differed from Jones et al 2009. I'm guessing they surveyed lakes covering a much broader range of DOC than you did. Table 1 - There should be separate entries for surface and deep samples; this would improve clarity. Figure 5 - CCA is most useful for the statistics it generates; plots are typically uninformative. I recommend moving CCA plots to supplemental. Minor comments related to language and clarity: Title suggestion: 'near' instead of 'within' Beaver Island, MI line 37 - temperature is a physical parameter, not chemical line 39 - constrained analyses demonstrated that DOC did not constrain? (what does that mean?) lines 115-119 - run-on sentence; break up into more than one sentence line 410 - 'stratified' instead of 'stratifying'? line 412 - what about 'Relationship between environmental conditions and microbial beta diversity'? Current phrasing is not clear to me. line 578 - I don't think 'mitigate' fits here... perhaps 'carried out'? Experimental design: Necessary details are still missing: line 250 - explicitly state the primers used for sequencing line 254 - 'implemented similarly' should be replaced by something to the effect of 'following the Mothur MiSeq SOP with the following modifications' (and then in your brief description, be sure to describe every deviation from the SOP); you need to include the date you accessed the SOP as per the instructions on the website (SOP is not static and has changed over the last few years) line 257 - what version of Silva? line 265 - what version of RDP? Multiple reviewers questioned the removal of singletons and doubletons prior to calculating beta diversity (line 301); rationale and citation provided to justify approach in response to reviewers were not sufficient in my opinion. I recommend re-doing this analysis without removing singletons and doubletons or finding an article that articulates a rationale for this approach, not simply one that went ahead and also removed rarely observed sequences. Validity of the findings: Validity of the findings is tied to some of the issues described above. I think improving clarity of basic reporting will improve the validity of the 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: MICROBIAL COMMUNITY DIVERSITY PATTERNS ARE RELATED TO PHYSICAL AND CHEMICAL DIFFERENCES AMONG TEMPERATE LAKES NEAR BEAVER ISLAND, MI Review round: 2 Reviewer: 3

Basic reporting: The manuscript is improved from the original version. The manuscript is clearly written, uses appropriate citations and background, and the figures and tables are all sufficient. Line 108: This does not make sense: "...experienced temperature stratification but did not stratify." Line 124: How deep was Lake Michigan at the site where you sampled? The depth at which you sampled is not even below the thermocline. Experimental design: I still find some flaw in the design that there is only one sample per lake per depth per time. To extrapolate to whole lake conditions on a couple of liters of water is somewhat problematic, especially since the authors point out that there's lake heterogeneity. Line 176: Methods are still unclear. Aside from the exceptionally high cycle number used to amplify the full length 16S gene, you then amplified the V4 region specifically from these first amplicons? How many cycles did that add? Do you have references that support using this approach, because it is somewhat different than the standard 16S sequencing protocols? I also do not understand why the authors are so reluctant to classify their sequences using the freshwater 16S database; this would provide more information and better taxonomy than RDP. Validity of the findings: Paragraph beginning line 315: the observations/discussion seems to have some logical flaws. The authors claim that these lakes diverge over time and compare that to observations during lake mixing; however, these lakes remained stratified and, in fact, became more (oxygen) stratified over time. In fact, of the variables that were measured, oxygen is the only one to change between the the three timepoints, so it is no surprise that this is what can be linked to the change in community structure. Perhaps the authors should discuss what drives the shift to anoxic bottom waters as well. 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: TOLERANCE: THE FORGOTTEN CHILD OF PLANT RESISTANCE Review round: 1 Reviewer: 1

Basic reporting: The manuscript addresses a fairly major shortcoming in the use of tolerance resistance in plants to arthropods. The material is presented in a straight forward manner and set up in a very readable format. The figures give good supporting back up to the points made in the text, and the references are thorough equally supportive. Some additional detail in the terminology used in the figures would improve their impact and utility in making the point of the review. Experimental design: Well addressed, as to the types of normal exp. designs used in plant resistance research, those unique to the study of tolerance and the limitations posed by lack of development of new methods. Validity of the findings: The logic-based conclusions reached by the authors make use of real-world data to point out the limitations of current knowledge about tolerance plant resistance. Additional comments: A good and timely review. Nice to know others are thinking of management tactics beyond single-gene, high-dose defenses.

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: TOLERANCE: THE FORGOTTEN CHILD OF PLANT RESISTANCE Review round: 1 Reviewer: 2

Basic reporting: All comments are directly on PDF version of ms. Experimental design: NA Validity of the findings: All comments are directly on PDF version of ms. Additional comments: All comments are directly on PDF version of ms.

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: L1 AND L2 READING SKILLS IN DUTCH ADOLESCENTS WITH A FAMILIAL RISK OF DYSLEXIA Review round: 1 Reviewer: 1

Basic reporting: The article is well written, covers most of the relevant literature (except see comment on Siegel’s work) sets out the questions clearly. See also comments on raw data below. Experimental design: I understand that this journal places a premium on technical adequacy rather than conceptual significance, but at the core of this article lies a pointless analysis, the comparison of the HRDys group with the other two groups. Whatever the technical adequacy of the analysis, it is pointless because the groups were selected to be distinct on reading in the first place, so finding a “significant” difference is all but a foregone conclusion. Further, with the well-attested high correlation between reading and spelling, the latter could equally be expected to show a significant difference. It’s less certain but reasonable guess that they will be poorer in commonly related cognitive processes like PA. If the article is to be published, the authors should drop that aspect of their analysis. More worthy of consideration are the questions of how children classified as dyslexic in Dutch fare in English, and how the non-dyslexic family-risk group fare in relation to their non-FR counterparts. The first of these questions was addressed by the subtraction statistic (Dutch WRF – English WRF), with the finding of a greater difference for the HRDys group than the other two groups. The only threat to this counterintuitive finding that I can see would be ceiling effects for the LRnonDys and HRnonDys groups (such as many of these participants reading all the words in the allotted time). Since the authors do not present non-standardized scores, they should address, and hopefully eliminate, this possibility. Supplying raw data would also help readers appreciate is there are any limitations to this finding along the lines that I have speculated. In their discussion of this particular result they might also refer to a finding by Linda Siegel to the effect that RD children can be superior in the “visual route,” to use dual-route terminology. The comparison of the two nonDys groups suffers from low power, I believe, leaving uncertainty about whether there is a population difference between them for the variables that were non-significant. I think the authors would be entitled to use one-tailed tests of significance because they are making directional predictions, which may clarify matters, and in any case their findings are like many others’ and support the continuous nature of dyslexia risk. It’s just a pity that they did not address the question of power and, if possible, design their study accordingly (or use a Bayesian approach based on prior probabilities of difference). I also add that it is going well beyond the data to assume that the results support the polygenic account of dyslexia, that “the HRnonDys group are likely to have inherited some but not all genetic risk factors that contribute to the development of a reading impairment” (lines 431-432). We have no way of knowing this, and it is equally likely that the difference is down to environmental compensation in the nonDYS group, or a range of other possibilities. Validity of the findings: No comment on top of those in previous section. 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: L1 AND L2 READING SKILLS IN DUTCH ADOLESCENTS WITH A FAMILIAL RISK OF DYSLEXIA Review round: 1 Reviewer: 2

Basic reporting: Basic reporting, overall, is of good quality. The following, however, are recommended improvements: Ambiguities (i) Lines 150 & 176: As these pertain to reading, "sound-to-letter conversions" (or "phoneme-to grapheme") are not relevant. Letter-to-sound (or grapheme-to-phoneme) conversions are relevant. (ii) 254-257: If the children read aloud, this should be stated. Although labelled as tests of "fluency," they measured the difficulty level of reading accuracy as well as speed of reading. They are tests of "accuracy/fluency." (iii) 416, 426, 439-441: The text should make it explicit that the Spoonerism measures of PA are for LI (Dutch only). (iv) 551, 560: It would be helpful to the reader if the "phonological problem" (or "deficit") were specified as either PA or pseudoword reading, and that neither were obtained for L2 (English). Missing Relevant Background Information An obtained result was that high-risk dyslexics, relative to their L1 (Dutch) word reading levels, had a positive advantage in L2 (English) word reading levels (Lines 480-482). In view of this, inclusion of the ages of the participants are recommended for the two previous studies (Miller-Guron & Lundberg,175-178, and van der Leij & Morfidi, 178-181) that gave results of some similarity. In addition, it is recommended that there be information about the method in the van der Leij & Morfidi study by which their subgroup "was shown to have superior orthographic competence" (178-181). If Miller-Guron & Lundberg obtained research evidence about the influence of irregular conversion rules in their study, then it is also recommended that information about their research method be included. Minor Errors in the Text See lines 296, 298, 459, and 477 Experimental design: The research design fills a knowledge gap and the method has been well executed. The statistical analyses are of a high standard. Lines 521-532 should be included in the Participants section (200- ). Although the instructional and remedial history of the participants was "uncontrollable" (526-527), it should be described in summary form for each of the three main groups. This is of some interest in view of lines 558-560 and 563-565 of the Conclusions. Validity of the findings: (i) The statistical analyses have been appropriately interpreted to support the findings. (ii) However, in 429-432, this "view of" the "polygenetic origin of dyslexia" may be consistent with the results but, without empirical genetic evidence in the study, so also would be a view that there was only familial influence with no significant genetic origin. (iii) The expressed limitations and future directions are pertinent to the obtained results. Additional comments: For testing the "orthographic strategy" speculations (lines 457-461, 492-497, 545-546) the authors should attempt an item analysis of their existing data for the participants' reading of L2 (English) words with only regular grapheme-phoneme correspondences, in comparison with those having one or more non-regular correspondences. If there were sufficient non-regular words in the L1 (Dutch) word reading, the same analysis could be applied to 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: L1 AND L2 READING SKILLS IN DUTCH ADOLESCENTS WITH A FAMILIAL RISK OF DYSLEXIA Review round: 2 Reviewer: 1

Basic reporting: No further comments Experimental design: No further comments Validity of the findings: No further comments Additional comments: The authors have attended well to the worries that I expressed in my original review. I now believe that the article meets the requirements of the journal, and support publication. Congratulations on a nice piece of research.

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: L1 AND L2 READING SKILLS IN DUTCH ADOLESCENTS WITH A FAMILIAL RISK OF DYSLEXIA Review round: 2 Reviewer: 2

Basic reporting: The appropriate improvements have been made in this Revision of July 2017. Experimental design: The changes in this area are also as in 1. Validity of the findings: In their Rebuttal Letter the authors state that their study "cannot determine whether this familial influence is genetic or environmental." Unfortunately this revision, p. 20, lines 18-22, fails to clearly say this. It is obscured by such terms as "multicausal origin" (their text reads "multiclausal"), "may have," and "support." This reviewer approves the authors clear statement of their Rebuttal Letter, which should replace these obscurities. Anything else would not meet the required standard. 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: L1 AND L2 READING SKILLS IN DUTCH ADOLESCENTS WITH A FAMILIAL RISK OF DYSLEXIA Review round: 3 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: In this revision, this area now meets the standard required. 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: INVESTIGATING THE LEVEL OF AGREEMENT OF TWO POSITIONING PROTOCOLS WHEN USING DUAL ENERGY X-RAY ABSORPTIOMETRY IN THE ASSESSMENT OF BODY COMPOSITION Review round: 1 Reviewer: 1

Basic reporting: The article was written well for the most part. However, there were some instances where phrasing and spelling could be improved (see below under General comments). Existing literature was discussed effectively, with limited previous investigations performed in this area. Raw data were mostly made available, however the findings related to participant comfort were not presented effectively and the clarity of graphs contained in Figures could be improved. Experimental design: While the question seems novel it is not well developed in the Introduction section leading from discussion on previous research. Also, there is some confusion over the actual research question at times given “reliability” is referred to consistently and is not actually measured in the submitted article. The methodological approach regarding the scanning procedures and protocols was presented at a high level. Validity of the findings: The findings are mostly presented in sufficient detail; however, some data analyses were not explained in the Methods and surprisingly appeared in the Results section. Further, the conclusion is unclear given a definitive recommendation based on the provided data was not provided and some findings were not discussed regarding differences between approaches. Some adequate explanations for various findings was presented in the Discussion. Additional comments: Introduction Line 86: “on regular” should be “on a regular”. Lines 95-99: This is a one-sentence paragraph that doesn’t really develop an idea fully. I would suggest combining it with the following paragraph or elaborating on the studies presented to present some actual reliability data for supportive evidence. Line 111: “moderate level” of what specifically? Are you referring to “evidence” here? Please specify. Line 113: “is the only study” should be “completed the only study” or similar. Also, why is Shiel et al. not given with a year – is this the unpublished work? If so, probably no need to reference. Lines 120-123: There needs to be a stronger link to this paragraph. You finish the previous paragraph by highlighting that research demonstrates Nana’s protocol is more reliable and comfortable, but then don’t set up the importance of your investigation. Further development to show the need for your study is needed here in 1-2 sentences I feel. Methods Line 131: “the” should be inserted before “scanning bed”. Line 132: Was block randomisation followed whereby the same number of participants completed each scan first? Line 137: “and” should be between males and females without a comma. Line 139: This is confusing as your study doesn’t seem to be a reliability study but rather a comparative study between scanning protocols. Please make sure your terminology and direction match the actual research question throughout. Line 152: “jewelry” is incorrectly spelled. Line 155: Has this device been shown to be reliable and valid? Line 182: “data was” should be “data were” given it is the plural form. Results Line 207: There is no mention of calculating the CV previously in the manuscript? Table 2: Can “Whole body” please be presented horizontally rather than vertically? Figure 3: The numbers of the axes are very hard to see in this figure – can it be reworked to be larger? Overall: There was no detail regarding the participant rating for comfort – this is a key outcome that should be presented in your results somewhere in this section. Discussion Line 238: “This studies primary aim” should be “The primary aim of this study”. Line 254: Please delete “studies regional analysis” and replace with “study” here. Line 264: Please delete the second “as” in this line. Line 268: How does the feet distance apart affect movement artefact? Some elaboration on this suggestion would strengthen your argument here. Line 271-274: This paragraph doesn’t offer anything that is not already known in the manuscript. Further, reliability of the approaches were not assessed but rather the level of agreement between the two was. This needs to be made clear throughout. I would recommend elaborating on these results further to strengthen this paragraph and transmit an important outcome with explanation to the reader. Line 301: “minuet” is misspelled. Lines 300-304: This is not really a measured outcome of the study though and is a little off topic. I would consider removing this paragraph. Line 308: What is meant by “the real difference”? Were your findings not “real” or are you referring to a more valid approach? Line 311: But size was not factored into your approach so I don’t see how it should shape your clinical outcomes. Line 312: So what approach is recommended? From your descriptions here it appears that you are recommending either can be used, however your Bland-Altman data showed the tissue data where consistently higher in the Nana than the NHANES. This is not discussed anywhere in this section. Also, in the Introduction you mention that the Nana has been shown to possess greater reliability, and given reliability was not measured in your study, shouldn’t existing reliability data be factored into your conclusions? References There are inconsistencies throughout the reference list that require addressing – e.g. some journals do not have capitals assigned to words, some article titles are given capitals for each word, & is used in some journal titles, etc.

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: INVESTIGATING THE LEVEL OF AGREEMENT OF TWO POSITIONING PROTOCOLS WHEN USING DUAL ENERGY X-RAY ABSORPTIOMETRY IN THE ASSESSMENT OF BODY COMPOSITION Review round: 1 Reviewer: 2

Basic reporting: The manuscript is generally well written and clearly describes the study (although see comments below). Fig 3 is unclear, the Y scale should be compressed and the axis titles and legends are uninformative (what does Mmeanlean actually mean?). The 2 SD limits should be stated. What are the units? Please redraw. Tabulate LOA data for all body regions. How widely is the Nana protocol used? The paper has been cited 52 times on Web of Science but it appears that citing papers are concerned with the main aim of the Nana pape, the effects of daily activities on DXA" rather than the use of the protocol. If it is not widely used then this point should be made. This should not be seen, however, to question the worth of the present paper. Experimental design: The authors have generally described the methods adequately. However a few points require attention. Was the survey of participant's protocol preference undertaken in a systematic manner? was a specific survey form used or simply anecdotal comment? Provide more details. The authors used data automatically provided by the equipment software. This is OK but does need clarification, for example, what is exactly defined as "tissue". This is a generic term that is presumably being used in specific way by Lunar. Please explain. The statistical analysis is generally acceptable but I question the correctness of the use of ICC for method comparison. I believe that Lin's concordance correlation is more appropriate. I suspect that you will find that Lin's r and ICC are very similar but statistical rigour should be applied. The authors also refer to Bland Altman plots. This is colloquial, it would be more appropriate to refer to limits of agreement analysis. Paired t tests can also be used to assess the significance of position differences (or combined for all body regions in an ANOVA to see if there is segment-position interaction). Single scans for each position only were performed. This begs the question as to whether the inter-position differences are similar to intra-position differences and hence may not be "true" inter-position differences. This needs to be addressed. Validity of the findings: The authors present limits of agreement analysis but fail to discuss adequately these findings. For example, are the positioning interchangeable? The mean difference in lean is small (Fig 3) but the LOA are about =/- 2 (kg?) for a mean lean mass of around 50 kg. This equates to a 4% difference. Is this clinically acceptable? Can you justify the final sentence of the Discussion in the Abstract? This implies exchangeability of methods. The LOA plots are for whole body only. I suspect that the LOA may be wider for body segment data. This should be provided. Rather than additional plots a table of bias and LOA for all body regions could be supplied. Discussion requires a strengths and weakness paragraph. Additional comments: Minor points to note Line 77 It would be more correct to refer to different degrees of attenuation rather than "rates". Line 109. It would be better to phrase as " Shiel et al (unpublished data) have systematically...." Line 181. The website has a number of spreadsheets - which one was used. Why was this used rather than SPSS? Line 262,. It is unclear how the studies have accounted for biological error? Please explain. Line 182 and elsewhere. "Data" is a plural, i.e. data were...

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: INVESTIGATING THE LEVEL OF AGREEMENT OF TWO POSITIONING PROTOCOLS WHEN USING DUAL ENERGY X-RAY ABSORPTIOMETRY IN THE ASSESSMENT OF BODY COMPOSITION Review round: 2 Reviewer: 1

Basic reporting: Elements of basic reporting in the submitted manuscript meet the journal standards. Experimental design: Elements of experimental in the submitted manuscript meet the journal standards. Validity of the findings: Elements of validity in the submitted manuscript meet the journal standards. Additional comments: The authors should be commended on addressing the author comments thoroughly, and as a result producing an improved manuscript. I do however have some minor points that require consideration: INTRODUCTION Lines 107-109: This seems like a bit of a stretch regarding development of a need for your study. I think speculating that bias existed in previous work should not be included and that an alternative reason/explanation should be presented here to show more work is needed in this area. Perhaps some of the positioning issues raised in this study might be useful. DISCUSSION/CONCLUSION Can you make a recommendation using the collective evidence from past research and your findings, considering aspects such as reliability, validity, comfort, etc.? You conclude that the protocols should not be used interchangeably, but which is ultimately recommended?

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: INVESTIGATING THE LEVEL OF AGREEMENT OF TWO POSITIONING PROTOCOLS WHEN USING DUAL ENERGY X-RAY ABSORPTIOMETRY IN THE ASSESSMENT OF BODY COMPOSITION Review round: 2 Reviewer: 2

Basic reporting: The authors have satisfactorily amended the manuscript in response to queries raised in my original review. Experimental design: The authors have satisfactorily amended the manuscript in response to queries raised in my original review. I accept the author's arguments for the use of ICC although I still believe that concordance correlation rather than ICC is preferable for the type of analysis described here. Validity of the findings: The authors have satisfactorily amended the manuscript in response to queries raised in my original review. Additional comments: The authors have satisfactorily amended the manuscript in response to queries raised in my original review.

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: INVESTIGATING THE LEVEL OF AGREEMENT OF TWO POSITIONING PROTOCOLS WHEN USING DUAL ENERGY X-RAY ABSORPTIOMETRY IN THE ASSESSMENT OF BODY COMPOSITION Review round: 3 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: The authors have sufficiently addressed all suggested revisions. Thanks for the opportunity to review this revised version.

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: TUMOR-INFILTRATING CD8+ LYMPHOCYTES PREDICT DIFFERENT CLINICAL OUTCOMES IN ORGAN- AND NON-ORGAN-CONFINED UROTHELIAL CARCINOMA OF THE BLADDER FOLLOWING RADICAL CYSTECTOMY Review round: 1 Reviewer: 1

Basic reporting: Language used: The language should be edited for clarity. Sentences that are unclear: Lines 64-65: please edit for clarity/make more concise Lines 152-154: Unclear if “the authors” defining high CD8 density are the authors of the present study or if this sentence refers to the most recently referenced papers; please edit for clarity and insert reference(s) as appropriate. Lines 163-165: This sentence invokes possible (and likely very important) immunosuppressive effects of the local tumor microenvironment (TME), but is unclear; please edit and insert reference(s) as appropriate. Lines 174: As is, this is a long sentence; suggest refining to minimize redundant statements. Sentences that could be improved for style and readability: Line 148-150: awkward construction of final clauses Lines 158-160: awkward construction Line 175-176: wordy Literature references, etc: No comments. Article Structure: Lines 132-135: The statement describing the results of the multivariate Cox proportional hazards regression model is closer to a conclusion than a simple statement of findings; please focus on describing the correlation identified without straying into interpretation (value as prognostic/predictive factor) in the results section. Self-contained with relevant results to hypothesis: See comments below (Experimental Design section) for recommended additional analyses to include in the manuscript; several may have already been performed but should be explicitly stated. Experimental design: Aim & scope: Appropriate. Research Question: The study adds to the literature by demonstrating differential correlation between overall survival (OS) and infiltrating CD8+ T-cells in organ-confined and non-organ confined disease. OS is a strong, important end-point while the semi-quantitative method for scoring CD8 positive cells infiltrating into neoplasm is likely reproducible in the clinical setting. Rigorous Investigation: It would be interesting if the authors examined correlation of CD8+ TILs and OS for pT2-4 lesions as a comparison to Sharma et al (PMID: 17360461); this would also test the hypothesis that the negative correlation between CD8+ TILs and OS in organ confined disease is driven primarily by pT1a lesions. Method description: 1) The authors should clarify their scoring methods – is the percentage CD8+ cells out of total cells or total lymphocytes? Was this an estimate or was this counted? An estimate is acceptable, but should be stated explicitly. 2) The authors should explicitly discuss in the manuscript why/how they selected bins for CD8+ TILs in their analysis (I.e., CD8+ vs CD8-negative, as opposed to other score combinations such as CD8+ score = 0 & 1 vs CD+ score = 3 & 4) 3) If known/recorded, it would be nice to include how many cases required a consensus discussion between the reviewing pathologists for scoring. Validity of the findings: Data Robust; details provided. Concerns are expressed above about method description and other possible analyses that might make the results more impactful and demonstrate both consistency with prior literature and highlight new findings (i.e., differential correlation of CD8 TILs with OS in low-stage vs high-stage disease). Conclusions Conclusions would benefit from increased clarity in writing and more concise statements. A minor point: the authors have not demonstrated activity (pro or anti-tumor) of the TILs. They have demonstrated presence of CD8+ cells alone; activity would require markers of T-cell activation. In general this distinction is clear, but grows slightly fuzzy in lines 162-5. Afterall, as the authors note in Lines 164-5, the local tumor microenvironment very likely has an important role. Speculation 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: TUMOR-INFILTRATING CD8+ LYMPHOCYTES PREDICT DIFFERENT CLINICAL OUTCOMES IN ORGAN- AND NON-ORGAN-CONFINED UROTHELIAL CARCINOMA OF THE BLADDER FOLLOWING RADICAL CYSTECTOMY Review round: 1 Reviewer: 2

Basic reporting: The manuscript meets standards. Minor points and comments are as follows: 1. Row 48: These 2017 numbers are projected and not actual numbers. Please revise. 2. Did any of the patients receive any immunotherapy. A comment on the existing knowledge on immunotherapy of TCCs could be useful. 3. Rows 142-143: The authors write “…however, which was a favorable…”. Please rephrase for syntax. Experimental design: The manuscript meets standards. Validity of the findings: The authors investigate the presence of CD8-positive tumor infiltrating lymphocytes (TILs) in organ-confined and non-organ-confined excision specimens of bladder transitional cell carcinomas. Based on their findings, the authors conclude that the presence of TILs is an independent prognostic factor in both organ-confined (unfavorable) and non-organ-confined (favorable) disease. No other clinicopathological correlations are identified based on the findings of the present study. While technically statistically significant in both cohorts (p<0.005), the statistical correlation is not compelling, as the reported p values are approaching the 0.005 cutoff. To further weaken the findings, the results are contrasting between the two cohorts. The authors should state this as a limitation of this study and should also try to offer possible explanations to the contrasting results between organ-confined and non-organ-confined disease. Finally, some information should be offered, with regard to the current state of immunotherapy in bladder transitional cell carcinoma, as this closely relates to the presence of TILs. Although unlikely, information (or lack thereof) should be included on whether patients have had immunotherapy as part of their treatment, and report any correlations with presence of CD8+ TILs. 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: TUMOR-INFILTRATING CD8+ LYMPHOCYTES PREDICT DIFFERENT CLINICAL OUTCOMES IN ORGAN- AND NON-ORGAN-CONFINED UROTHELIAL CARCINOMA OF THE BLADDER FOLLOWING RADICAL CYSTECTOMY Review round: 2 Reviewer: 1

Basic reporting: This is a re-review of the manuscript submitting following minor requested revisions. I have four truly minor, albeit important, requested modifications. 1) The authors have now twice addressed concerns expressed from multiple reviewers about the selection of bins of CD8+ scores and explained their rationale for using CD8+ TIL negative vs positive (grouping 0 & 1 scores vs 3 & 4 scores). They have adequately explained in the rebuttal letters that there was no statistically significant association for other combinations of scores. However, unless I missed something, the authors have still not included this explanation in the primary text. I strongly believe this rationale should be explicitly discussed in the text (the results section would be appropriate). Without such a clear, concise discussion in the results section: a) the rationale behind Figure 2's construction is unclear; and b) the readership is left with outstanding methodological questions. The single sentence provided in the rebuttal letter is sufficient explanation and an important result. 2) Similarly, the authors explain in the rebuttal letter that percentage of CD8+ TILs was estimated as a percentage of total cells. That methodology is definitely acceptable (to me, at least). However, this has not been made explicit in the manuscript text. I suggest modifying Lines 89-90 as follows: "based on the [estimated] percentage of positively stained cells out of total cells." (Adding "estimated" to existing text; brackets indicate my modification/suggestion). 3) Figure 2 still uses TCC (transitional cell carcinoma) in lieu of urothelial carcinoma (UC) which has replaced all other uses of TCC in the text. 4) A minor point: there is inconsistent use of "Figure X" vs "Fig X"; please compare lines 109 and 123 and choose one for consistent style. Experimental design: No concerns. Validity of the findings: No concerns. Additional comments: I believe this paper is deserving of publication and congratulate the authors on their efforts. However, I believe the four really minor corrections in Part 1 (above), particularly the first two points, really are necessary corrections prior to publication and will better enable colleagues' interpretation of the data and results, and the TIL community's ability to replicate the methods in the paper.

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: TUMOR-INFILTRATING CD8+ LYMPHOCYTES PREDICT DIFFERENT CLINICAL OUTCOMES IN ORGAN- AND NON-ORGAN-CONFINED UROTHELIAL CARCINOMA OF THE BLADDER FOLLOWING RADICAL CYSTECTOMY Review round: 2 Reviewer: 2

Basic reporting: No comment. Experimental design: No comment. Validity of the findings: No comment. Additional comments: The authors have adequately addressed the reviewers' comments and have presented an improved version of this 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: A RETROSPECTIVE STUDY: DOES UPPER AIRWAY MORPHOLOGY DIFFER BETWEEN NON-POSITIONAL AND POSITIONAL OBSTRUCTIVE SLEEP APNEA? Review round: 1 Reviewer: 1

Basic reporting: I think the article can be published with corrections. Given the limitations of a retrospective study, the reported data and the conclusions still make sense. The authors used routine polysomnography and CT scanning in the appropriate manner and the data were unsurprising. The fact that NPOSA , compared to POSA, is worse and due to anatomical abnormalities is not at all a surprise. And the reasons appear totally logical in that there was, on the average, a lengthened soft palate and a reduced or narrower glossopharynx. My concern is with the clinical significance with respect to treatment regarding these observations. If one has OSA it requires treatment regardless of whether one has POSA or NPOSA due to the well-known debilitating consequences of untreated OSA. One possible conclusion is that NPOSA would probably almost always require PAP therapy whereas POSA might be more susceptible to a mandibular repositioning device. Some discussion along these lines might be helpful. Wrt data reporting, there is no need for 2 decimal points. For example, patient age should be 30.2 +/- 9.4 years not 39.23 +/- 9.41 years. And the same applies to BMI, AHI, and so on. Experimental design: They authors used routine Procedures to assess OSA and to measure the patients' anatomical characteristics. And the statistical evaluation followed standard and correct procedures. Validity of the findings: The findings are valid while not being very surprising. However, nice to have meaningful statistical data. Additional comments: The comments above should suffice but happy to make further comment if the review is unclear in any way.

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 RETROSPECTIVE STUDY: DOES UPPER AIRWAY MORPHOLOGY DIFFER BETWEEN NON-POSITIONAL AND POSITIONAL OBSTRUCTIVE SLEEP APNEA? Review round: 1 Reviewer: 2

Basic reporting: The background needs to demonstrate the importance of this work. Experimental design: The method of research needs to be detailed. Validity of the findings: no comment Additional comments: This study is to investigate the differences in upper airway morphology between positional (POSA) and non-positional (NPOSA) in 75 patients with obstructive sleep apnea. But please consider the following questions: 1.What is the significance of exploring this difference in the progress and treatment of the patients with OSA? 2.How to judge POSA and NPOSA? You should provide for the method of analysis of these concepts. 3.How to consider the relationship between nocturnal rostral fluid shift and the severity of OSA? How to avoid this kind of confounding factors? 4.Why all of these patients only have OSA? How about MSA or CSA these patients?

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 RETROSPECTIVE STUDY: DOES UPPER AIRWAY MORPHOLOGY DIFFER BETWEEN NON-POSITIONAL AND POSITIONAL OBSTRUCTIVE SLEEP APNEA? Review round: 1 Reviewer: 3

Basic reporting: Jiao and colleagues retrospectively described the differences in upper airway morphology between 30 patients with positional and 45 with non-positional OSA. Various measurements and diameters calculated on CT scan are compared between the 2 groups. The paper is overall well written and shows some significant results, however there are some major and minor revisions to address. In particular, the authors use too many abbreviations, and some of them are not so easily interpreted, which makes the article sometimes difficult to read, since the reader is obliged to check the meaning of the abbreviation quite often. Furthermore, The discussion is poorly structured, I suggest to briefly report the main results and then discuss each of them comparing with prior literature. Major revisions: - Materials and Methods: the authors state that they performed a PSG, but they did not include electroencephalographic data. I suggest to add this among the study limitations. - Results: Mean age of study population was 39 years. Differently from other studies, The study population is very young, can the authors justify and comment on this? - in the Results, line 119, the authors do not write if BMI was different between groups. This data is written in table 1, however, given its importance, I suggest to add it in the text, too. - discussion, lines 154-157: the meaning of this sentence is not clear, I suggest rephrasing. - discussion, Lines 159-166: I suggest to deepen this point. - discussion, line 178-179: 'specifically...35.41mm'. This result is not present in the result section. I suggest to add it to the section and then discuss. - discussion, line 196-199: ' while there... the two groups': I suggest to better explain this point. - discussion: add to the limits of the study the retrospective nature. Experimental design: Major revisions: - CT test is not routinely asked in patients with OSA. The authors should explain why the exam was requested in this group of patients. If it was requested for research purposes, patients should have signed an informed consent. Validity of the findings: no comment Additional comments: Minor revisions: - introduction line 59: few studies on the Chinese population. Line 60: Chinese patients using... - Materials and Methods line 68: treated in the hospital - Materials and Methods line 93: would have had calming - Results line 133: GP was significantly smaller. - conclusions, line 217: were in SPL, and the CD... - figure 1: the position of the head during CT was not in the correct position - figure 1: >25% of all.

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 RETROSPECTIVE STUDY: DOES UPPER AIRWAY MORPHOLOGY DIFFER BETWEEN NON-POSITIONAL AND POSITIONAL OBSTRUCTIVE SLEEP APNEA? Review round: 2 Reviewer: 1

Basic reporting: I acknowledge that the authors have made some corrections and improvements to the original manuscript, however most of the statements that were not clear in the first version are still not clear. The manuscript absolutely needs to be revised by an English translator, in fact, many statements sound incomplete and without the main verb. Minor comment: - Materials and Methods: the authors state that the 105 patients underwent surgery. I suggest to add the reason why they required surgery (OSAS treatment) Experimental design: no further comments Validity of the findings: no further comments 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: MULTIGENE PHYLOGENY OF THE SCYPHOZOAN JELLYFISH FAMILY PELAGIIDAE REVEALS THAT THE COMMON U.S. ATLANTIC SEA NETTLE COMPRISES TWO DISTINCT SPECIES (CHRYSAORA QUINQUECIRRHA AND C. CHESAPEAKEI) Review round: 1 Reviewer: 1

Basic reporting: The text presents an important contribution to the understanding of the diversity of jellyfishes from US east coast, by presenting new and detailed data on a well-known thought to be a single species (until now named Chrysaora quinquecirrha). I am not a native English-speaker, but the manuscript is well written, and the language used is very clear throughout the text. “Introduction” section provides background on the topic and included traditional and updated literature. The structure of the text is in accordance with the standards, but I suggest the authors to move the “Systematics” section before the “Discussion”. Figures and tables are of high quality and essential for helping to clarify the results, unless when noted. Experimental design: The methods were clearly described and are updated with the available literature. Conclusions are well supported by the data presented. Validity of the findings: Although there was an indication in the literature about the occurrence of two varieties of the jellyfish studied, the authors provided robust data to identify and sort them out. The nomenclatural issues were carefully followed and the main result (species name validation) is sustained based on the articles of the Code. Summing up, my evaluation of the manuscript is that it corresponds to an important contribution to Marine Biology in general as it provides advances in the field, especially unravelling the identity of a significant jellyfish species of the Atlantic US coast. Additional comments: In general the authors were cautious in not proposing new names for the different groups found among the genus Chrysaora. I think that additional data must be gathered to perform such big changes in the classification of the family. There are some minor corrections and suggestions performed directly on the PDF file, summarized as follows in order of importance: 1) holotype specimen must be deposited in a well-known collection, as well as other comparison individuals; 2) provide further data on the Senegalese specimens (images as supplementary material would be welcome); 3) correct some references regarding format and some data.

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: MULTIGENE PHYLOGENY OF THE SCYPHOZOAN JELLYFISH FAMILY PELAGIIDAE REVEALS THAT THE COMMON U.S. ATLANTIC SEA NETTLE COMPRISES TWO DISTINCT SPECIES (CHRYSAORA QUINQUECIRRHA AND C. CHESAPEAKEI) Review round: 1 Reviewer: 2

Basic reporting: No Comment Experimental design: No comment Validity of the findings: No Comment Additional comments: PeerJ Multigene phylogeny of the scyphozoan jellyfish family Pelagiidae reveals that the common U.S. Atlantic sea nettle comprises two distinct species (Chrysaora quinquecirrha and C. chesapeakei) (#17919) Brief summary This study seeks to reconstruct the phylogenetic relationships among 4 pelagiid genera and 11 of 13 species of Chrysaora. In addition collections were made along the US Atlantic and Gulf coast of C. quinquicirrha, from which molecular, gross anatomical and cnidomic data were collected. The stated goals of the authors was to clarify taxonomic status among genera and species of this group of nuisance species. The molecular approach used nuclear 28S and mtDNA COI and 16S. Morphological analyses focused on tentacle and lappet number, oral arm length and nematocyst dimensions. Results suggest that C. quinquicirrha is polyphyletic, and comprises two distinct lineages one from the Gulf and the other from the Atlantic. This conclusion is supported by molecular as well as morphological evidence. This paper is certainly interesting scientifically as well as provides an important contribution to applied resource management. The phylogenetic analysis presented is technically sound, and the resulting systematic claims are convincing. Figures are of sufficient quality, including maps, photos and trees. The systematics sections is about 11 pages, raising the editorial question of whether this section is appropriate for the journal PeerJ, or might be included as an appendix, otherwise this could be more appropriate for a taxonomic journal. I have made a number of minor grammatical suggestions and minor corrections of typos. Specific Comments L 122 Please italicize “Sanderia” L153 Use of the small unprimed italicized letter implies that the heme prosthetic group is in a hemochrome linkage, and a lower-case italicized letter, e.g., c', should be used. L184 Italicize “c” L201 and L203 Please write out the NIH DNA database as “GenBank” L219 “jModelTest” L328 I’d suggest rewording “two distinct clades that were highly diverged” “two distinct, highly divergent clades” L332 & 335 and throughout Please be consistent: “Figs” vs. “Figures” L337 Replace “highly-supported” with “well-supported” L344 Replace “observed” with more appropriate term, such as “evaluated” or “examined” L345-6 “were observed for 20 quantitative and qualitative macromorphological characters either taken from Gershwin and Collins (2002) or new to this study (maximum oral arm length)” This sentence mentions 20 traits, and lists a single trait that was unique to this study. It remains unclear to the reader why a single character is listed, this could lead to confusion as to whether this single trait listed is the only trait used that was not one from the Gershwin and Collins 2002 paper, whereas if those traits that fall into both categories are indicated in a table, perhaps simply referring to the table would be more clear at this point. L361 In: “<~1.5%” inclusion of the symbol “~” seems awkward, does removing this symbol drastically alter the meaning? If not I’d suggest deleting it. L374 “all those sequenced from” this is admittedly a bit of a pet peeve of mine, but to state that “individuals” are sequenced is inaccurate, strikes me a slang, since DNA, or gene fragments are sequenced rather than jellyfish, or individuals being sequenced. I’d suggest rewording to something like “jellyfish included in the molecular analysis” or something along these lines that more precisely reflects the author’s intended meaning. L423 Replace “a paraphyletic Chrysaora.” with “paraphyly of Chrysaora.” L434 Should a question mark be added following “limited” L475 “therefore, conclusions based on these sequences should be made with care.” Why? Perhaps consider explaining, for instance “because the geographic provenance of these individuals is unclear”, or what ever the reasoning. L484 The following sentence: “C. quinquecirrha occurred in two well-differentiated” opens with an abbreviation of the genus, whereas starting a sentence with an abbreviation should be avoided. This is an editorial, and grammatical decision. L596 “and” not italicized L604 and 608 Include “(MBA)” after first mention of “Monterey Bay Aquarium”. L634 “(Figure 4 and 6)” please follow journal guidelines and be consistent in the manner in which two figures are referred to, there are instances where the following is used “Figures 4, 6”

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: SEDIMENT MICROBIAL TAXONOMIC AND FUNCTIONAL DIVERSITY IN A NATURAL SALINITY GRADIENT CHALLENGE REMANE’S “SPECIES MINIMUM” CONCEPT Review round: 1 Reviewer: 1

Basic reporting: The authors test the suitability of a macrobenthic ecological observation due to salinity gradients, on sediment microbial community structure and function. The authors provide sufficient background on the original ecological concept, their study system, and their methods. Raw data are available. Data and figure reformatting is suggested. Minor grammar and word changes in the manuscript are recommended. Experimental design: The authors use 16S rRNA gene sequencing of sediments from a fresh-to-marine transect, and typical metrics to analyze the role of habitat and environmental influences on microbial communities. The study seems to build off a previous publication from the same group, that evaluates the influence of salinity on bacterial community structure in lagoons in the same sampling location (http://www.sciencedirect.com.proxy.lib.umich.edu/science/article/pii/S1874778716300058), but the current study incorporates a broader salinity range than previously evaluated. The authors infer functional characteristics of observed bacterial communities, but functions for 38-75% of taxa in each sample cannot be obtained. The authors also tentatively evaluate archaeal community composition, of which this study design is not optimal. Validity of the findings: The authors conclude that Remane’s “species minimum” concept does not translate to sediment microbial community structure and function. Their main supports are 1). A decrease in species richness over the salinity gradient; and 2). Strong correlation between salinity and community composition, and salinity and function. The authors rightly suggest caution when interpreting archaeal relative abundances and functional profiles from bacterial 16S rRNA gene data. The authors use a paragraph in the introduction discussing the unknown impacts of sea level rise and salinity changes on freshwater systems. Their study system could represent a microbial community in transition from fresh to saline conditions, and functional signatures could be evaluated in that context; the authors could address this compelling ecological consequence in their discussion. Additional comments: Please review paragraph breaks for clarity and cohesion. There are examples of unexpected paragraph breaks at lines 114-115, the methods section for “DNA extraction, PCR amplification and 16S rRNA sequencing”, 340-341, and 369-370. Please check grammar and flow. For example, lines 224-225 could be clearer: “The number of sequences assigned to an OTU in each sample represents the relative abundance of the OTU in a sample.” Suggestion for lines 201-203: “The raw sequence reads retrieved from all the sediment samples were quality trimmed using sickle (Joshi & Fass, 2011), to where the average quality score dropped below 20 (-q 20) as well as where read length was below 10bp (-l 10).” Methods: Lines 133-139 detail two different instruments to measure dissolved oxygen, salinity, and temperature. Were these instruments cross-calibrated? Please see comment about Supplementary Table 6. Lines 169-172: This statement implies that the authors used multiple primer sets on different samples, and compared resulting community profiles. However, primers impart community biases, and the use of different primer sets in the same study is puzzling. Please provide background research on or results comparing bias from the selected primer pairs. Lines 224-226: Are your analyses on each station using averaged representations of OTUs across the replicates? If so, do you have tests on the similarity of the replicates? Figure 2: Are there any significance values attributed to the ellipses, or are they just encircling the station points? Figures 3, S2, and S3: Please clarify if each bar represents the relative abundance of phyla in one sample at each station, or if the relative abundance profiles of samples at each station were averaged to get a mean representation of phyla at each station. If they were averaged, see above comment about methods. Graph axes need labels. Figure 4: Vertical lines separating each triplet per KEGG would make interpreting the graph easier. Axes need labels. Figure 6: Would error bars on OTU abundances be visible on this graph, or be obscured by the station points? Supplementary Table 1: Please change “paired-end reads” to “read pairs”. One overlapped read is the result of two paired-end reads. Supplementary Table 6: Chlorophyll a is reported for all stations, however the methods state that fluorescence ~ chlorophyll a was measured at only Arachthos delta and Kalamitsi stations. How was fluorescence or chlorophyll a measured at all the other stations? Also, the chlorophyll a values for those latter locations have a different format than the rest. Discussion: Your study has a clean and simple design, of using the sediment microbial communities and relevant environmental parameters to test Remane’s species minimum in other systems. I think the results will have broader significance if you can relate them to benthic ecological succession due to rising sea level and climate change.

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: SEDIMENT MICROBIAL TAXONOMIC AND FUNCTIONAL DIVERSITY IN A NATURAL SALINITY GRADIENT CHALLENGE REMANE’S “SPECIES MINIMUM” CONCEPT Review round: 1 Reviewer: 2

Basic reporting: no comment. Experimental design: no comment. Validity of the findings: -Data is robust, but statistical analysis could be improved to specifically test the role of salinity in driving community patterns. -Conclusion are well stated, but not linked to original research question as novel aspects (community variation and functional profiles) were added and not justified at the first place. Additional comments: I praise the authors for their extensive data set and for their thorough statistical analyses. The manuscript is clearly written and there are only minor points to be checked for improved clarity. Yet, some points should be addressed to make the study stronger as follows: 1) The study would gain from being more focused on what the title and abstract described, i.e. the testing of the theory that "taxonomic diversity of microbenthic organisms is lowest within the horohalinicum". The manuscript should have thus consisted of testing local microbial richness (alpha diversity) as a function of the salinity gradient. The authors extended their analysis to community turnover and to the functional relevance of their observed diversity patterns, which in my opinion, analyses that dilute the main message of the study or at least have not been justified with respect to Remane’s theory. I would suggest that the authors either change their title and abstract to be broader in scope, or fit their text content to the testing of Remane’s theory as main objective. 2) Some technical points are not clear: 2a. Line 213: why did the reads have to be mapped back to the OTUs? AT this stage of the procedure, all reads should be clustered into OTUs. Please clarify. 2b. Line 209 the singletons were removed, and few lines later (line 213), they were added back. Is this a normal procedure (please provide references then)? It seems that this procedure may add sequences that will not fit to any OTUs because those reads were not considered in building the OTU matrix at the first place. 3) One main point that the authors touched upon is whether salinity is the main driver of community patterns or not. The current analysis based on correlations does not account for factor confounding and for the amount of variation each factor might explain. To address this point, I suggest that the authors perform a variation partitioning analysis (see vegan package varpart function for instance) in order to quantify the amount of variation in community that is due to salinity uniquely while taking other environmental and spatial factors into consideration. Hence, factor confounding on community variation can be disentangled. The same approach can be applied to changes in richness and would be a strong point to be made in this study. 4) The salinity gradient (e.g. Figure 6) does not include samples at the 5-8 psu that are mentioned in Remane’s concept. This would suggest that the authors should reinforce the idea that the authors should refocus their study on the effect of salinity and biogeography on community variation, instead of addressing Remane’s concept. Minor comments 1) Line 197: The reference Leinonen et al., 2011 could be removed or put after "ENA" on line 196. 2) Figure 2. The legend "nMDS of the microbial OTUs" is not a valid legend, as nMDS applies to a distance matrix. Please rephrase. 3) Lines 325-326: please rephrase as the sentence is intrinsically redundant.

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: SEDIMENT MICROBIAL TAXONOMIC AND FUNCTIONAL DIVERSITY IN A NATURAL SALINITY GRADIENT CHALLENGE REMANE’S “SPECIES MINIMUM” CONCEPT Review round: 1 Reviewer: 3

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: General comments: 1) Lines 41-42: In the Abstract, the authors provide an important statement that “the sediment microbial OTUs in this study area do not follow Remane’s concept which could be attributed to the different life strategies of micro- and macroorganisms”. Although the idea of differences in life strategies between unicellular plankton and large multicellular bottom-dwelling organisms is highly relevant to the results of this study, it is, unfortunately, neither mentioned in the Introduction nor discussed elsewhere in the manuscript. Moreover, this idea was originally published by Telesh et al. (2013) and then referred to in several other articles, e.g. by Herlemann et al. (2016), who wrote: “Telesh et al. (2013) suggested that the life strategies of unicellular planktonic organisms differ substantially from those of large multicellular bottom-dwelling organisms, resulting in deviations from the species-minimum concept, which is supported by the results from our study of bacterioplankton” (Herlemann et al., 2016, page 7). Therefore, I definitely recommend considering this viewpoint in the manuscript and discussing it, providing the appropriate citation(s). Alternatively, this statement should be deleted from the Abstract since it is not a result obtained by the authors of this submission. 2) Lines 101-103: It is very difficult to agree with the authors’ statement that “…confirmation of Remane’s concept for prokaryotes would mean that small-bodied, fast-developing and rapidly evolving microbes respond in a similar way as benthic macroorganisms in a salinity gradient”, mainly for the following reasons. First, this is likely not so because of specific morphological, physiological and ecological characteristics of these groups of organisms. Thus, the differences in their reaction to salinity gradient will always be present due to dissimilarities in size, mode of life, osmoregulation etc. These issues are discussed in many details in Telesh et al. (2011, 2013, 2015 and 2016). Second, even if diversity of sediment prokaryotes happen to follow the Remane’s curve, one should look for more appropriate (maybe even unknown yet) reasons for this pattern. In this respect, a good example was provided in the paper by Schubert et al. (2011, Mar. Pollut. Bull., Vol. 62) where the authors revealed the Remane’s curve-like pattern in the diversity of macrophytes in the salinity gradient in certain cases; however, the reasons for the species minimum of those macroalgae were the lack of suitable hard substrates but not at all the critical salinity (horohalinicum) in those habitats. Therefore, I suggest that the authors provide deeper analysis/explanations of the obtained results and a broader discussion of the possible mechanisms that underpin those findings. 3) Lines 313-321: The result which showed that for certain microbial OTUs a KEGG profile could not be retrieved, thus these OTUs constituted the fraction of unexplained taxonomic units (FTU), and that this fraction was the highest in the lagoon samples (67.51 %) and lowest in the marine samples (38.91 %) is of special interest. I agree with the authors that this issue needs further investigation which might shed more light on the reasons and mechanisms behind the natural prokaryote diversity distribution pattern in the salinity gradient. However, I believe that the existing knowledge on microbial diversity in the other large water bodies with salinity gradient (e.g., the San Francisco Bay, or the Chesapeake Bay and its sub-estuaries) allows for certain generalizations already now. The paper will benefit from the enlarged discussion of these important issues. Specific suggestions: 1) Line 69: peak (not “pick”). 2) Lines 280-281 and elsewhere in the text: I suggest to change “it seems” to “it is likely” or “we believe that” etc. 3) Legend to Figure 4: Please consider salinity gradient while compiling the legend and change the order of symbols in Fig. 4 to follow the gradient: river -> lagoon -> sea.

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: SEDIMENT MICROBIAL TAXONOMIC AND FUNCTIONAL DIVERSITY IN A NATURAL SALINITY GRADIENT CHALLENGE REMANE’S “SPECIES MINIMUM” CONCEPT Review round: 2 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: I accept all authors' responses to my critical comments and suggestions. One minor 'technical' correction should be made: in Line 725 of the MS, the format is corrupted (two references are not separated by the line break).

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: EXTERNAL VALIDATION OF CUT-OFF POINTS FOR FOVEAL THICKNESS TAKING INTO ACCOUNT THE INTRARETINAL FLUID USING OPTICAL COHERENCE TOMOGRAPHY TO DIAGNOSE DIABETIC MACULAR OEDEMA Review round: 1 Reviewer: 1

Basic reporting: . Experimental design: . Validity of the findings: . Additional comments: 1 pdf line 41 “ist (No authors listed, 1985; Kinyoun et al., 1989). Screening for diabetic retinopHY “ This is not a correct refeence. Why not use numbers as is now almost invariable in reference list and put the numbers in the text? The actual rerence ( pdf line261 is missing the year 2: lines38-41 grammar poor The reviewer re-writese: ….is indirect stereoscopic biomicroscopy using a (60)D lens.after mydriasis. ( ETDRS report – 3: line 43 ? Retinography? 4: line 46 “ due to “ implies a causal relationship. Has this really been established? 5: lines 49-50 “in the ophthalmological consultations. “ replace by a full stiop – cut it out 6: Line 54 “. In other words, the influence of intraretinal fluid has not been considered.” This is not logical and also it is not “ in other words”. If there is intraretinal fluid then either the retina is thicker or retinal cells must have vanished. I think this requires much more explanation. Much of what is referred to here is usually thought of as retinal cysts. Thickening without cyst formation would be an important variable 7: Foveal thickness. The foveal is a very small area, and the macula is much bigger. The authors should make a distinction between clinically non significant macular oedema and clinically significant macular oedema.. I think a figure illustrating OCT findings in the various categories they propose would make things clear 8: Line 60- I do not know what is meant here by bootstrapping. The area under the ROC curve is a method of obtaining specificity and sensitivity values It is not a calibration. Its setting a criterion. Is this the word the authors wanted? It permits one to obtain optimal values for dividing categories. These depend upon several factors and one is the proportion of patients who have the condition. Its no use having high specificity if very numerous patients have t be examined- the number of false positives wil be so large that the criterion cannot be used-the service will be overwhelmed. I think up to line 68 should be revised. 9: Line 72 The “who” implies that after “Elche” there should be the word “were” 10: Line 76 an area of 169555 inhabitants” Catchment area perhaps? Wrong power! inhabitants number =x1 while area =x2 11: line 89 But early Alzheimer causes reduction in the nfl layer! The authors should check the liturarture. 12 :lines 91-96 I have already commented that a very similar sentence is poor English. 13: lines 105 107 The authors must understand “ an area of 1000 cetrnal microns “ is not normal scientific use. “valuation” is used incorrectly. 14: line 124 What does accuracy mean here? Given the strange way s quantitative data have been handled, the reviewer suggest that that the authors state the number of false positives and false negatives per 1000 referrals, 15: line 147 The statistical package may have worked but what is written is slightly nonsensical. Putting in decimal points when dealing with individuals is illlogical. See table1 Table 1 caption What does this mean? “n(%), absolute frequency (relative frequency)”; Why not insert words into the table ? The revewer has used have used medical statistics for over 50 years, and have never seen S used as an indicator of standard deviation. If the figures given aree mean values, don’t we want to know the standard error of the mean? This is usually indicated by s.e. or s.e.m. The variables important to diagnosis include age and weight Surely these were recorded? And nephropathy? And crdiac nsfficiency ? Other interesting differences found in our work is country of origin. Do migrant workers form part of the sample? 16: line 169 The reviewer feels that given what the authors describe as intraretinal fluid is what he would call a cyst 17: The reviewer cannot comment on this bootstrap technique . Figs 1-5 are meaningless to him. He would like to see the ROC curves 18:line 181 “However, we must bear in mind that we are validating a diagnostic test through the calculation of sensitivity and specificity, independent parameters of disease prevalence (Lalkhen & McCluskey, 2008). In other words, the use of a sample obtained in specialised care consultations is not a selection bias “ The reviewer does not agree The optimal specificity and sensitivity of a test does depend upon the severity and frequency of a condition in the population which hs been recruited, 19: line 185 “ In addition, our sample was selected completely at random.” How can this assertion be made without providing evidence? Screening of populations is tricky. And the present sample of patients is so different clinically to the one provided by table 1 column 1 that small differences between optimal results is not surprising or even noteworthy. 20: for all the references If DOIs or other unique codings are not given (and they are not) then the year of publication must bre inserted.

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: EXTERNAL VALIDATION OF CUT-OFF POINTS FOR FOVEAL THICKNESS TAKING INTO ACCOUNT THE INTRARETINAL FLUID USING OPTICAL COHERENCE TOMOGRAPHY TO DIAGNOSE DIABETIC MACULAR OEDEMA Review round: 1 Reviewer: 2

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: The manuscript is well written and the scientific english language is adequate. References, figures and structure of the article are correct and meet the objective of the authors and the journal's recommendations. In the discussion section, I recommend to clarify the clinical benefit that can have an ophthalmologist reading this paper and to implement the introduction section analyzing in more detail the purpose of the article. Data and statistical analysis are correct, and the value of the research may have globally a good level of impact.

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: EXTERNAL VALIDATION OF CUT-OFF POINTS FOR FOVEAL THICKNESS TAKING INTO ACCOUNT THE INTRARETINAL FLUID USING OPTICAL COHERENCE TOMOGRAPHY TO DIAGNOSE DIABETIC MACULAR OEDEMA Review round: 1 Reviewer: 3

Basic reporting: Innovative Experimental design: Well conceived Validity of the findings: Good Additional comments: Over all a worthwhile manyscript. One major spelling error in abstract: Optimal Coherence Tomography should read as Optical Coherence Tomography

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: EXTERNAL VALIDATION OF CUT-OFF POINTS FOR FOVEAL THICKNESS TAKING INTO ACCOUNT THE INTRARETINAL FLUID USING OPTICAL COHERENCE TOMOGRAPHY TO DIAGNOSE DIABETIC MACULAR OEDEMA Review round: 1 Reviewer: 4

Basic reporting: no comment Experimental design: no comment Validity of the findings: the sample size was 134 eyes. To estimate whether this sample was sufficient to validate the diagnostic test, the Authors computed the specificity estimation using the following parameters: an accuracy of 6.36% was obtained. However, since the sample is not big, I suggest discussing it as a potential, not strict, limit. I agree with the conclusion that given that the validation was performed in the health area covered by their hospital, the Authors encourage other researchers to perform validation in their own communities and only if similar results are found, these cut-off points for foveal thickness can be used in routine clinical practice Additional comments: This study contains interesting observations

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: EXTERNAL VALIDATION OF CUT-OFF POINTS FOR FOVEAL THICKNESS TAKING INTO ACCOUNT THE INTRARETINAL FLUID USING OPTICAL COHERENCE TOMOGRAPHY TO DIAGNOSE DIABETIC MACULAR OEDEMA Review round: 2 Reviewer: 1

Basic reporting: . Experimental design: . Validity of the findings: . Additional comments: General It seems to this reviewer that what the authors have done is to analyse their OCT results in their diabetic patients, and correlated these with clinical opinion derived from slit-lamp microscopy. From this analysis they have obtained values for OCT -derived measures of maximal retinal thickness which can occur in diabetics who do not have clinically significant diabetic macular oedema. The work seems well done. There are a number of mostly linguistic problems in the writing. There is no way of knowing whether these results are applicable to any other hospital or to other maker’s instruments. The MS refers to previous work (2015) published by the same group which seems to the reviewer very similar to the present MS. What is new is that the 2015 results are reproduced by the data of 2017 In detail Title Would “independent” be better than External? Better word order is “ ….for foveal thickness using optical coherence tomography in the presence or absence of intraretinal fluid in patients with diabetic macular oedema” Line 41 delete “by the ophthalmologist” Line 41 Wrong method of citation Possibly “clinical manual Boston “ This is a matter for house style to decide Line 49 Better word order would be “ Optical coherence tomography (OCT), which has been available for several years, allows us to perform a quantitative and qualitative study of diabetic macular oedema by providing a cross-sectional image of the retina, at high magnification, and automatically measures the thickness of various retinal layers. The OCT also shows the abnrormalities in structures such as the presence of cysts snd the accumulation of fluid “. Line 53 “ two parameters” The reviewer does not understand what is meant. DMO most often begins extrafoveally, and the retina near the macula may contain cysts etc while the fovea is unaffected. Do the authors mean “ macula” where thy write “fovea”? Line 54-57 “trying to determine a cut-off point from which we can say that there is diabetic macular oedema. In other words, the influence of intraretinal fluid in the OCT has not been considered, as when evaluating a diagnostic test it is possible that the presence of intraretinal fluid on OCT does not agree with the Gold standard” The reviewer dos not really understand what is meant. In assessing an individual, OCT is so much more powerful than slit lamp with a70D lens that there is no point raising this. We differentiate between sight-threatening and non-sight threatening macula oedema. Is this what the authors are attempting to discuss? Line 62 The reviewer suggests that an illustration showing retinas with and without intraretinal fluid would make things plain! Line 63-64 The fovea is the region where the OCT shows a pit, because there are only photoreceptors, and a thickness of the retina at this point of 90 microns is reasonable. But what is this 310 micron figure? Does this mean that if the fovea is not involved a thickness of 310 microns in extra-foveal retina indicates clinical significance? ( sight-threatening) Line 66 Why use AUC for the Receiver Operating Charcteristic? Its almost invariably referred to as ROC for obvious reasons Line 66 The sensitivity and specificity of a diagnostic test depends on the ROC but also on the values taken. This is in no sense a “calibration” The cut off points are arbitrary. If one wants to include almost all patients, ( that is, to have a high sensitivity ) then the specificity will decrease. The value finally adopted depends on various factors which include the prevalence of the condition, the rate of progress and the severity and also the resources available to track and treat the patients after the intial examination. Line 67. The reviewer is going to accept the statement that there is only one publication giving statistical data about OCT measurements in patients with DMO , and he agrees that confirmatory data in different centres with different patient populations is required. Thus the results presented below are publishable. But a quick search of PUBMed gives 321 papers with some information about this subject ( even if not formalised ) since 2005! Line 72 “ in our community” It is certain if you have a new instrument-of any sort- it is a good idea to collect your own normal values, even if the instrument has been well tested and comes with normative results. But such data are not necessarily for publication! Line 88 The authors can determine whether their results are in agreement with those of Hernadez-Martinez : to call this ‘externally validating’ is a bit presumptious! What would the authors have done if their analysis did not agree with this previous paper? Line 92 “ wanted to participate” -the reviewer rather worries about this. It is conceivable that this could result which was biased and was no generally applicable. . Line 93 Ot is usual to say “exclusion criteria were : dementia. Recent cataract surgery…..”, This is a linguistic point- the conditions cannot be criteria ( in English) Line 100 dilated pupil indirect ophthalmoscopy would read better Line 103 These examinations were carried out by expert… Line 104 Does this mean that the same doctors were used as in the previous cited publications?If so this whole project is scarcely independent! This is important! Line 123 Was renal function measured? Statistical methods as described seem standard, but they cannot produce what is referred to ss a calibration ( line 142) Line 158 This data has been given previously see line 124 There are other such repetitions which shouldbe cleaned up. Line 193 If the patient population equipment and operators were exactly the same as those in the 215 paper, then there would be no point in doing the work at all because it would not establish any generality: all that could be said would be that for these limited conditions th test was reliable. Line 198 Earlier it was stated that randomness was not achieved. Line 218 “externally validated” see reviewers previous comments Line 223 This suggests to the reviewer that this MS deals with patients passing through the same hospital, seen by the same doctors, and examined with the same equipment, as in the 2015 project: and despite there being minor differences between this patient group and the one in the 2015 project, the results are very similar. Bujt what information in this paper would be of immediate use to a hospital in, for example, Venezuela? Figures The reviwer suggests an additional figure with OCT images It would be nice to see an ROC graph. Are any of the other figures really required?

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 COMPLETE CHLOROPLAST GENOME SEQUENCE OF STRAWBERRY (FRAGARIA  × ANANASSA DUCH.) AND COMPARISON WITH RELATED SPECIES OF ROSACEAE Review round: 1 Reviewer: 1

Basic reporting: Sufficient field background information was provided. In general, writing was clear. Experimental design: This work will provide valuable information through the complete sequence of the chloroplast of the cultivated octoploid. This will allow to improve chloroplast genetic engineering and to discard this genome in whole genome sequencing done on nuclear DNA or RNA. The research question was clear, relevant and performed accordingly. The applied methods were well described and detailed. Validity of the findings: Data was well analyzed, statistically sound. Conclusion were well stated, and linked to original research question. Additional comments: The cultivated strawberry (Fragaria x ananassa Duch.) is one of the youngest domesticated plants, developed from chance hybridization between two western hemisphere octoploid species (F. chiloensis and F. virginiana), which becomes one of an important fruit crop in the world to date. However, little is known about the chloroplast genome of the species that gave rise to this important fruit crop. In the manuscript, the authors present the complete sequence of the chloroplast genome of the cultivated strawberry. For generating the complete genome, they performed a combination of de novo assembly and reference-guided mapping of contigs. The complete genome sequence provides a new reference for the evolution and genetic diversity of plants. Then, they compared the chloroplast genome structure and organisation of F. × ananassa cv. Benihoppe with ones of other Rosaceae species. Finally, they performed a cpDNA markers and sequence polymorphisms analysis using data from ten species belonging to Fragaria genus. This work will provide valuable information through the complete sequence of the chloroplast of the cultivated octoploid. This will allow to improve chloroplast genetic engineering and to discard this genome in whole genome sequencing done on nuclear DNA or RNA. The research question was clear, relevant and performed accordingly. The applied methods were well described and detailed. The article is in the scope of the journal Peer J. Minor version is required to considered the following comments. Compared with the nuclear genome, the chloroplast genome is small, and the rate of nucleotide substitutions is so low that the chloroplast genome is considered to be an ideal system for studies on phylogeny and population genetics (Wei et al., 2005). I do not think low rate of substitution is good for population study, definitely is good for phylogenetic analysis. There are 130 genes in the chloroplast genome of F. × ananassa cv. Benihoppe, 112 of which are unique. What do you mean by the “unique”? Six fast-evolving DNA regions were individually and combined to construct phylogeny of ten Fragaria species. More discussion on the phylogenetic analysis with previous ones is suggested to be expended. Please explore the basis for disagreement between some of their own findings and a highly relevant prior study involving Benihoppe.

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 COMPLETE CHLOROPLAST GENOME SEQUENCE OF STRAWBERRY (FRAGARIA  × ANANASSA DUCH.) AND COMPARISON WITH RELATED SPECIES OF ROSACEAE Review round: 1 Reviewer: 2

Basic reporting: An additional chloroplast assembly exists that is not mentioned which provides results of a partial 130 kb chloroplast assembly of F. vesca as submitted GenBank GU363535.1 and described in Davis TM, Shields ME, Reinhard AE, Reavey PA, Lin J, Zhang H, Mahoney LL, Bassil NV, Martin R. 2010. Chloroplast DNA inheritance, ancestry, and sequencing in Fragaria. Acta Horticulturae 859: 221-228. Line 13 Fragaria chloroplast exhibits low variation on what basis? What is the evidence? Or is this based on work of Wei and al 2005 for chloroplast genomes in general or based on Zhang and al 2017 for Rosasceae? The statement is not clear to this reviewer. Line 24 What did the comparative analysis consist of briefly? Line 29. How were F. chiloensis and F.virginiana compared to cv Benihoppe and which F. chiloensis and F. virginiana? For ease, the PI# should be used to indicate each of the accessions used in the study for Fragaria and for all of the Rosaceae species. Introduction Line 57 Hirtula rose as distinct from rose? Line 58 Assembly of five Fragaria species (Shulaev et al., 2011; Hirakawa et al., 2014). There should be more such as Tennessen et al including octoploid and F. vesca ssp bracteata publications. Line 60 Assembly of nuclear genomes are still a work in progress and not finished. Maybe state that drafts assemblies have been released? Line 78-79 Provide references to support that Fragaria has limited variation in chloroplast sequences. Line 86 cp genomes of F. vesca by TMD. The following reference also provides results of a partial 130 kb chloroplast assembly of F. vesca and reference to a GenBank submission. Davis TM, Shields ME, Reinhard AE, Reavey PA, Lin J, Zhang H, Mahoney LL, Bassil NV, Martin R. 2010. Chloroplast DNA inheritance, ancestry, and sequencing in Fragaria. Acta Horticulturae 859: 221-228. Line 98 Transgenic Benihoppe nuclear genome released versus reported? Line 239 - 244. Sampling of 8x and 2x species is too limited to make broad statements on the species, statement and comparisons should be made to those specific accessions that were compared. Line 334 comparison to how many F. virginiana and F. chiloensis and which ones? Experimental design: Inclusion of additional accessions of especially F. virginiana and of F. chiloensis to represent the diversity of the species would permit the kind of conclusions drawn in the study. As only one cultivar was sequenced, the ancestry of the cultivar might be useful as insight into the possible similarity to cultivars in use other countries. Validity of the findings: Any plant accessions used in the study should have Plant Introduction numbers and if Plant Introduction numbers do not already exist, the authors should submit them to a germplasm repository and obtain plant introduction numbers. There are subspecies of both F. virginiana and F. chiloensis and variation within the subspecies, so the specifics of what material was used is critical. There is also variation in F. vesca and F. iinumae and so conclusions should be limited to the comparison made with the specific accessions used in this study and not be drawn to encompass the entire species. Additional comments: If the above comments are addressed, I recommend the article for 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: MOUSE SPERMATOZOA WITH HIGHER FERTILIZATION RATES HAVE THINNER NUCLEI Review round: 1 Reviewer: 1

Basic reporting: The article is clear, comprehensible written. Despite the literature was well referenced and relevant, I consider that the author should refer to the 2010 version of The World Health Organization, and not to the one that was published in 1999. Experimental design: no comment Validity of the findings: no comment Additional comments: The present study is an interesting work applying geometric morphometric to analyze sperm head contours within a population and between mouse strains and/or fertilization stages. The authors identified morphological indicators of highly fertile spermatozoa, and developed a pipeline that might also be applicable to human normal sperm. However, there are some aspects that the authors should take into consideration. - The authors said that their method would greatly facilitate and enhance current reproductive technologies in an objective, high-throughput manner. It is not clear how this method can be used in the clinic laboratory to select a better sperm to perform ICSI. Authors should explain with more details this aspect. - The percentage of sperm hyperactivation correlates with high fertilization rates and is different depending on the fertilization stage (cauda sperm vs PVS sperm). It is possible that the difference in fertilization rates between the 2 strains is due to differences in the percentage of hyperactivation. The author should also perform analyses between motility patterns and aspect ratio in BDF1 (in different fertilization stages) and B6N sperm populations. - In Figure 4, there are only 3 animals analyzed, I recommend at least 4 to increase the number of cells per groups. - It would be interesting if the authors perform what they proposed in the discussion: to test dithiothreitol on caput/cauda sperm and analyze head minor axis and aspect ratio of sperm. These results will explain the changes in these parameters during sperm maturation. - Supplemental Figures: there is not any Figure legend and they are necessary to understand those figures. - Supplemental Figure 2: It is not clear if they stimulated the acrosome reaction such as using progesterone or ZP or if the authors evaluated spontaneous acrosome reaction. It is possible that acrosome reaction by zona pellucida change the sperm aspect ratio. - Line 97, authors wrote Fig.S4C, and there is not such panel in Fig.S4. - Line 165, the authors should define also PC4, PC5.

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: MOUSE SPERMATOZOA WITH HIGHER FERTILIZATION RATES HAVE THINNER NUCLEI Review round: 1 Reviewer: 2

Basic reporting: This manuscript's premise is that the fertilizing potential of spermatozoa is correlated with their overall morphology. The authors focus in subtle differences between sperm, not in dramatic differences in morphology. The manuscript is well written and the assumptions made to compare fertilizing potential are sound. From the biology point of view, the results are interested and have the potential to be relevant in clinical practice. However, this reviewer's expertise in cell imaging is not sufficient to give an opinion on the imaging strategies as well as on the mathematics used to produce the data. I hope other reviewers of this manuscript are experts in cell imaging. Experimental design: As said, assumptions made to choose sperm for analysis are sound. However, the conclusions heavily rely on the validity of the cell imaging methodologies used. Another reviewer should comment on this. Validity of the findings: Nothing to add Additional comments: Nothing to add

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: MOUSE SPERMATOZOA WITH HIGHER FERTILIZATION RATES HAVE THINNER NUCLEI Review round: 1 Reviewer: 3

Basic reporting: The authors presented a clear introduction with citations relevant to the topic and a clear research question. 1) In the line 83 of the introduction, the authors state that the sperm head contours of spermatozoa at different “fertilization stages” was analyzed. This is confusing to the reader, because the authors are using a broader definition of fertilization than the classical (the ability of a spermatozoa to penetrate and fuse with an egg (Yanagimachi, 1994)). In their definition, they are including sperm maturation in the epididymis and sperm capacitation in the female tract as part of fertilization (Okabe, 2013) instead of as required steps to acquire fertilizing-capacity. Therefore, it would be useful if the authors clarify this point by adding a sentence in which this broader definition of fertilization is stated. Experimental design: The experimental design is not completely clear. 2) About the in vitro fertilization experiments: a) Line 105: The authors state that the eggs were placed in TYH medium drops. Were the cumuls cells previously removed? If not, this line should state that “Cumulus-enclosed oocytes were placed in 100 µl drops of TYH medium”. b) For this reviewer is not clear why the authors chose 8 hours of co-insemination between spermatozoa and oocytes before collecting sperm in the perivitelline space. It has been shown that after half an hour fertilization sperm has already fused with the oocyte (Tateno et al, 2013). In addition, Storey et al (1984) reported that mouse capacitated spermatozoa penetrated the zona of cumulus intact eggs in less than two hours after insemination. Therefore, after 8 hours of fertilization it seems difficult to find sperm in the perivitelline space unless fertilization and the cortical granule enzymatic reaction was inactivated to avoid polyspermy blockage and allow more spermatozoa to penetrate the zona. Is this the case? Please clarify this point. As a minor comment, in the line 116: the world “eliminated” should be replaced by “removed”. 3) As this work is based in imaging analysis, it is important in the section of imaging and analysis to specify the camera and the settings that were used for taking the images. The specifications of the objective lens used are also important (for example, numerical aperture). 4) Minor comment. In the section of statistics, please state if when applying parametric tests, the assumptions of normality and homogeneity of variances were evaluated, and if any transformation of data was required. Validity of the findings: The data is robust, statistically sound and controlled. Some minor changes would improve the quality of the work. 5) Figure 4 a. The axis Fertilization stage is misleading because the graph is including different stages of maturation in the epididymis together with spermatozoa recovered from the female reproductive tract. 6) As a minor comment, in line 218 would be useful to disclose the fertilization rates of BDF1 and B6N cauda epididymis-isolated spermatozoa obtained in your laboratory. Additional comments: The paper entitled “Mouse spermatozoa with higher fertilization rates have thinner nuclei” proposes a new way of morphometric and statistical analysis of mouse sperm heads as fertility indicator. The work presented combines imaging analysis with a multivariate statistics method that is intended to develop a new way of classifying sperm and determine fertility potential. The data presented is good however the results section flow was not the best. The text guides the reader to start from supplementary figure 2 , then jumps to Figure 1 A, jumps again to supplementary figure 3, before going back to Figure1B and then continues with all the supplemental information before describing figure 2. I suggest to re-evaluate which information to include in the main figures and which one in the supplementary materials, in order to make the reading and understanding easier for the readers. Finally, it is important to remark that the manuscript shows that the Principal Components Analysis was useful to identify the main variables that represent the morphological variability of the sperm head nucleus: width, and the hook shape of the tip.

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: MOUSE SPERMATOZOA WITH HIGHER FERTILIZATION RATES HAVE THINNER NUCLEI Review round: 2 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: The authors took care to answer to my questions and make this revised version of the manuscript easier to follow. I apologize for not finding the supplementary figure legends in the first round of revision.

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: MOUSE SPERMATOZOA WITH HIGHER FERTILIZATION RATES HAVE THINNER NUCLEI Review round: 2 Reviewer: 2

Basic reporting: The authors have addressed all comments from reviewers. Experimental design: No Comment. Validity of the findings: No comment. Additional comments: The authors have addressed all 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: MOUSE SPERMATOZOA WITH HIGHER FERTILIZATION RATES HAVE THINNER NUCLEI Review round: 2 Reviewer: 3

Basic reporting: The authors have carefully addressed the issues that were pointed out by this reviewer. The revised version of the manuscript is improved and no further corrections are needed. Experimental design: No further comments. Validity of the findings: No further comments Additional comments: No further 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: CELLOBIOHYDROLASE B OF ASPERGILLUS NIGER OVER-EXPRESSED IN PICHIA PASTORIS STIMULATES HYDROLYSIS OF OIL PALM EMPTY FRUIT BUNCHES Review round: 1 Reviewer: 1

Basic reporting: The manuscript was well and clearly written (with very little grammatical errors), and was backed up by relevant literature references Experimental design: Experimental design was suitable and robust for the research objectives spelt out. Validity of the findings: Results and findings obtained were substantiated by raw data, and conclusions drawn were in accordance with the objectives outlined. However, results on biochemical characterization of the purified CbhB should also be highlighted in the conclusion as an answer to one of the objectives set out (line 115). Additional comments: 1. The English language can be improved, especially for lines 127 (growth of the yeast were performed), line 246 (incongruent sentence), line 273, 279, 296, 300, line 336- mentioned, not mention, line 353- Insensitivity of CbhB rather than CbhB insensitivity, line 370, line 379, line 382, line 384, 2. Please include a close bracket after mL-1, in line 144 3. line190: sugar produced, not product 4. Line 230- state the equipment used 5. Line 346- use of "of course" not quite appropriate 6. Line 353- was statistical test carried out? Cu and Ba seemed to affect at 10mM concentrations, as seen by the reduction of relative activity to less than 80%. 7. line 358- Gurgu and Barby, not Gurgu et al. 8. line 367- rephrase. The loadings were optimised, not the hydrolysis. 9. line 386- was, not is. Also line 391, 396 10. line 390. Use primary reference 11. line 416. please add a qualifier 12. References: please spell out all the authors's names to replace the et al. (lines 441, 466, 475, 481, 524 13. line 521: italicise genus and species names 14. Table 1, line 2- specific activity, not spesific 15. Table 2: table should be self-explanatory. Please write the names of the substrates in full in the notes below the table 16. Figure 4, Lane 2. IMAC- purified CbhB deglycosylated using PNGaseF, rather than deglycosylated IMAC- purified CbhB using PNGaseF 17. Figure 8. Range of Cellic CTec2 in the title was 1.5-30%, but in the actual figure was 5.50 -25%

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: CELLOBIOHYDROLASE B OF ASPERGILLUS NIGER OVER-EXPRESSED IN PICHIA PASTORIS STIMULATES HYDROLYSIS OF OIL PALM EMPTY FRUIT BUNCHES Review round: 1 Reviewer: 2

Basic reporting: None Experimental design: The procedures were efficient to produce and purify an Aspergilllus niger CBH in P. pastoris. Produced enzyme was properly evaluated and used as an additional enzyme during hydrolysis of a model substrate hydrolysis (pretreated oil palm empty bunch). Hydrolysis intervals in this substrate were shorter than usual (5h), but results were adequate for evaluating the recombinant enzyme mixed with commercial CellicCtec. Experimental design was adequate Validity of the findings: Experimental data are well interpreted and discussed Additional comments: The work is a general contribution to the area related to enzymes used in biomass conversion. Production and purification of a recombinant CBH was developed. Over glycosylation of the recombinant enzyme could be one of the causes of non-action on complex and insoluble substrates such as crystalline cellulose. In general, the results are sound and merit 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: CELLOBIOHYDROLASE B OF ASPERGILLUS NIGER OVER-EXPRESSED IN PICHIA PASTORIS STIMULATES HYDROLYSIS OF OIL PALM EMPTY FRUIT BUNCHES Review round: 1 Reviewer: 3

Basic reporting: The manuscript by Woon et al., describes the heterologous expression and characterization of a CBH from A.niger, as well as its ability to increase the saccharification yields of oil palm empty fruit bunches when added to a commercial preparation. The manuscript is well-written and literature references and scientific background are provided. Experimental design: This study is within the Aims and Scope of the journal. In general, the experiments are described with sufficient detail, were performed correctly and the conclusions follow the results. Some parts of the experiments and results needs more clarifications, such as the cloning of the cbh2 gene (PCR amplification, primers and polymerase used) and the experimental design by response surface methodology (enzyme supplementation, choice of upper and lower limits). Validity of the findings: In general, conclusions are well stated and supported. There are some parts that need clarification, such as the low recovery yields of CBH2 and the imprortance of ATR-FTIR data on evaluation of CellicCtec2 supplementation with CBH2 and the changes on substrate morphology. Additional comments: - Lines 62-63: Keywords need to be more specific and representative of this work, for example bioethanol and lignin are not within the scope of this study. - Line 81: “Aspergillus niger is a filamentous fungus well-known for its ability to produce copious amount of lignocellulose degrading enzymes”: this paper by Zoglowek et al., 2015 mainly describes how A.niger has been used as a host for heterologous production of CBHs and other cellulases. - Line 89: T.reesei has low extracellular β-gucosidase activity, but this fungus expresses many intracellular and cell-bound CBHs. - Lines 90-92: Supplementation is also used because different types of biomass have different properties, therefore they require a set of enzymes that has to be tailor-made. For example, endoglucanases are needed for amorphous substrates, whereas CBH supplementation is preferred for substrates with high crystallinity. - Lines 93-95: cellulases consortium has been reported much earlier than 2016, so another reference is suggested. The same for line 103, for CBHs acting on reducing and non-reducing cellulose chain end. - Line 106-107: Apart from glycosylation, there are other post-translational modifications that affect the enzyme performance and activity, ex. N-terminal pyroglutamate fo. rmation that is commonly observed in crystal structures of Cel7A enzymes and is related to enzyme stability, is not possible to occur in the Pichia pastoris expression system. This makes the recombinant enzymes less active and less stable than their native counterparts (Biotechnol. Bioeng. 2014;111: 842–847). - Line 130-131: A full description of the cloning procedure is missing, i.e. information about the gene (exons, introns), the primers that were used and the PCR conditions, as well as the polymerase used. This part is also missing from the Results and Discussion Section (Line 262-263), where cbh gene should be analyzed for glycosylation sites, signal peptide and homology with characterized CBHs and an accession number for Genbank should be given. There is some information on Lines 295-305, but this part should be re-written again carefully. Line 302, “molecular mass of CbhB to ~ 70 to 100 kDa”: it is not clear what authors mean. - Line 170-172: After the digestion with PNGaseF, how was the extent of N-glycosylation estimated? Did authors perform an SDS to compare the molecular weight of the enzyme before and after the deglycosylation? If so, this should be mentioned here. - Lines 197-198: Did authors checked the stability/autohydrolysis of MUF at high temperatures, such as 70-80οC? Did they run control reactions without the addition of enzyme? - Line 206: What was the CBH concentration that was used for calculating the kinetic parameters? How was the end product inhibition estimated? - Line 217: Authors should choose another title, more specific than “complementary effects”. Same for Line 366. - Line 217-220: When a monoenzyme (for example a CBH) is added to a commercial mixture, then there are two types: one is when the enzyme loading of the commercial preparation remains the same and CBH is added, so the final enzyme loading increases (this is called addition/ supplementation), and the other one is when one part of the commercial preparation is replaced by the CBH and the final enzyme loading is constant (this is called replacement/complementation). It should be mentioned which type is being used here. 0 - Line 226: Did authors check the composition of the material (glucose, xylose, lignin) before and after the pretreatment? - Line 233-235: Apart from %TRS rate, the %hydrolysis/saccharification rate could be also estimated by taking into account the glucose and/or xylose content of OPEFB according to the analysis of solid fiber fraction from the following equation (WIREs Energy Environ. 2013; 2, 633-654): % hydrolysis rate=[TRS(mg/mL)/DM(mg/mL)*glucose content*1.111] *100 Did authors check glucose or xylose yields after enzymatic reactions? - Line 245-247: How did the authors chose the limits of the enzyme and Cellic®CTec2 relative abundances? In general, it should be avoided not only working within a wide domain, as this may impact the reliability of predictions, but also limiting in a narrow domain, since extrapolation outside the borders is impossible. The lower and upper limits of each component should be chosen by combining data from the literature with rational consideration. - Line 284-285 and Table 2 data: it is obvious that the enzyme is quite unstable and this can be attributed to many reasons, including the absence of post-translational modifications, such as the pyroglutamate formation ect. CBH is degraded and gradually loses its His-tag and this is the reason why it is not possible to be purified by IMAC and the purification fold decreases. Authors should give an explanation. Did they perform an SDS during the cultivation in order to check whether the molecular weight of the protein remains stable? What is there on the lowest part of the SDS-PAGE shown at Figure 2 (it is cut and not shown)? - In order to check whether the addition of CBH reduced the substrate crystallinity or not, study of the X-ray diffraction spectra could give more accurate results than ATR-FTIR. - According to gene/protein nomenclature, genes are denoted by three italicized lower case letters, while Protein names are the same as the gene names, but the protein names are not italicized, and the first letter is upper-case (see "Guidelines for Formatting Gene and Protein Names", http://www.biosciencewriters.com/Guidelines-for-Formatting-Gene-and-Protein-Names.aspx), so it should be cbhB gene and CBHB protein. - The authors should also check carefully for references' format, as well as typing/editing and punctuation errors in the text and in the figure captions. All organism names should be written 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: CELLOBIOHYDROLASE B OF ASPERGILLUS NIGER OVER-EXPRESSED IN PICHIA PASTORIS STIMULATES HYDROLYSIS OF OIL PALM EMPTY FRUIT BUNCHES Review round: 2 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comments Additional comments: The manuscript was greatly imporoved after revision and now is accepted for publication in PeerJ.

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: NESTS OF RED WOOD ANTS (FORMICA RUFA-GROUP) ARE POSITIVELY ASSOCIATED WITH TECTONIC FAULTS: A DOUBLE-BLIND TEST Review round: 1 Reviewer: 1

Basic reporting: The authors have conducted a double-blind test to test the hypothesis that red wood ants occur in proximity to active tectonic faults. Former publications of some of the co-authors have shown this already. I have to admit – I was not aware that this hypothesis or the published results were criticized by the myrmecologist community. In this case I welcome this study with its rigorous experimental approach if it helps to corroborate former results in an unbiased way. Experimental design: A potential weakness of the study is the polydomous colony structure of one of the ant species studied here (see comment 1). I have two points that I think the authors should clarify in this otherwise well-performed and well-presented study. 1. The authors state that the RWA nests were spatially clustered. Especially for F. polyctena this is no surprise as this ant is known to be highly polydomous (and polygynous) (e.g. in Seifert 2007 or the edited book on RWA ecology and conservation by Stockan and Robinson). Thus, the clustering of nests can to my mind be considered a pseudoreplication as nests are (at least in part) not independent of each other. What are the results for F. rufa alone in comparison to F. polyctena? Here this problem should not be as pronounced. Are the results comparable? Could you potentially do the analysis with the grid cells themselves (RWA present or absent)? 2. One point that is to my mind not made clear enough in the methods section is how random points in the same region were generated for comparison. Please add one or two sentences. Validity of the findings: no comments 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: NESTS OF RED WOOD ANTS (FORMICA RUFA-GROUP) ARE POSITIVELY ASSOCIATED WITH TECTONIC FAULTS: A DOUBLE-BLIND TEST Review round: 1 Reviewer: 2

Basic reporting: no comment Experimental design: Line 20-22: How conducted study in this manuscript does help us to apply (design) double-blind ecological studies in “distribution” and “abundance” studies. As far as I understand the presented study is not related to any of these groups in their classical definition, at least. An example in the discussion/conclusion can help to understand the point authors try to address. Validity of the findings: Line 94-100: How do authors deal with spatial autocorrelation in this analyze. Line 125-149: I miss any discussion/conclusion on why ant nests are significantly closer to faults? Additional comments: The manuscript has overall been written well. It seems authors have not been able to decide if the main topic is testing double-blind approach in the Ecology or an ecological experiment on relationship of ant nests and tectonic faults. Authors can the re-organize the structure in a new way that readers can follow the paper better.

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: PULMONARY TRANSCRIPTOMIC RESPONSES INDICATE A DUAL ROLE OF INFLAMMATION IN PNEUMONIA DEVELOPMENT AND VIRAL CLEARANCE DURING 2009 PANDEMIC INFLUENZA INFECTION Review round: 1 Reviewer: 1

Basic reporting: This is a descriptive study examining the transcriptome changes that occur during a mild H1N1 2009 influenza infection in mice. The manuscript is coherently written and laid out in a logical and cogent manner. However there are several weaknesses to the manuscript that reduce enthusiasm for acceptance. 1) Although the authors claim no research has been done to examine the non-fatal 2009 flu model, this is untrue. There have been multiple studies performed on H1N1 2009 (Itoh et al Nature 460, 1021-1025, Rowe et al 2010 Virology. Jun 5; 401(2): 257–265 ). to name a few. These should be referenced and more emphasis should be placed on what the purpose of this study is and how it differs from the other studies (CA2009 vs Cat2009 etc). 2) There needs to be more thorough editing performed on the paper, for instance: line 51- "this severe" should be changed to "these severe" line 56 "severe patients infected" should read "severely infected patients" line 64- "previously put in relationship" would read better as "previously correlated with" Experimental design: The experimental procedures seem well defined and the authors demonstrate a previously undescribed mouse influenza model. The numbers of mice seem appropriate and the statistical methods are fine. There is a little confusion in the materials and methods section that need to be clarified as well as some figures that need improvement. 1) Figure 2 demonstrates severe pathology at day 1 and 5. It is unclear what the differences are between day 1 and 5 though. This figure would greatly benefit from histopathological scoring. It is stated in the results section, but as the figure stands, it is showing very severe pathology at day 1 and not explaining that this was only from 2 of the mice. 2) it is confusing why they state "data not shown" on line 102 yet, they seem to have shown the data. 3) line 128 "the to" should be changed. line 135: the authors say "as described elsewhere" that is fine, but give a citation showing where. 4) Line 176: Change "significantly higher body weight loss" to significantly greater weight loss" 5) line 182: "one day after mice infection" remove the word mice 6) line 183: Please use scientific notation for the standard deviation. same for line 184. 7) Line 225: change "induced also" to "also induced". Validity of the findings: 1) Since the 2009 H1N1 outbreak there has been extensive studies into this viral strain and the effects in multiple models (in vitro, mouse, ferret, pig) as well as in patients themselves. The discussion would greatly benefit from a better explanation of how this study ties in to the already abundant data that is out there. 2) The authors use a human isolate and it is unclear whether it has been passaged in mice already. The discussion would benefit with a comparison of well characterized fatal (PR8 or multiple mouse passaged 2009) infections. 3) It is unclear why the authors focused solely on day 5 when they have data from 3 different time points. The manuscript would benefit in a comparison of the changes from day 1, to day 5 to day 10. 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: PULMONARY TRANSCRIPTOMIC RESPONSES INDICATE A DUAL ROLE OF INFLAMMATION IN PNEUMONIA DEVELOPMENT AND VIRAL CLEARANCE DURING 2009 PANDEMIC INFLUENZA INFECTION Review round: 1 Reviewer: 2

Basic reporting: This article is reported in clear and unambiguous manner. There are some grammatical errors and I hope I have highlighted them all in my review. There is an exhaustive list of references and an acknowledgement of an article (Pommerenke et al., 2012), which is quite similar in some aspects. The cited literature provides a good background and context to the study. Figures and tables are provided in the main text and as supplementary data. There are clear and well structured. In the legend of Figure 1, statistics are presented in part B, but there is no asterisk on the viral load graph (B). I would recommend that statistics be presented out with the A and B description. In figure 2 would be it possible to use arrows to indicate the infiltrate in the viral infected lungs Figure 3 is a clear representation of differences in GEP, it would be beneficial to relate this figure to a table of genes that are a different within the figure legend. In this way it would be easier for reader to follow the process. Does Table 1 represent only genes that are unregulated? could examples of down regulated genes also be included? Experimental design: This study represents primary research within the aims and scope of the journal. The research question is clear, but I would record highlighting how it advances knowledge in this area and expand on lines 239 and 240 to explain what this study offers. It is good to see microarray data accompanied by histological data. Methods and ethics are clearly described. Validity of the findings: Data is robust and in line with previous literature. Conclusion is well stated and linked to the research question. Additional comments: This study is clear, easy to read and a good example of the importance of microarray data to illustrate a global view of immunological responses in a specific tissue. I would recommend clarifying some of the figure legends to ensure that figures are fully explained in regards to the complexity of microarray data. For example in figure 3, I understand that the GEP profile is changing, but my immediate question is, what is changing? Can the figure 3 legend be connected to table 1 or supplementary table? Can table 1 be coloured to represent how it refers to figure 3 data.

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: PULMONARY TRANSCRIPTOMIC RESPONSES INDICATE A DUAL ROLE OF INFLAMMATION IN PNEUMONIA DEVELOPMENT AND VIRAL CLEARANCE DURING 2009 PANDEMIC INFLUENZA INFECTION Review round: 2 Reviewer: 1

Basic reporting: The manuscript is vastly improved from the last version and the authors were very responsive to the critiques. The discussion would benefit from a more thorough editing for proper english. Experimental design: the experimental design is well defined and meaningful. Validity of the findings: the data is robust and 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: THE EFFECTS OF FAMILIARITY ON ESCAPE RESPONSES IN THE TRINIDADIAN GUPPY (POECILIA RETICULATA) Review round: 1 Reviewer: 1

Basic reporting: All good Experimental design: All good, but see suggestions in general comments Validity of the findings: All good, but see suggestions in general comments Additional comments: Overall this is a good study. My only issue is that the expectations are a little naive. There is every reason to suspect that the magnitude of response is largely due to inherent properties of the fish (size, size of the mauthner neuron etc) thus I fail to see any reason why the authors would expect to see familiarity effects on these traits. It makes sense for other parts of the avoidance strategy (eg time to first detection, reaction distance etc) as these are modified by social interactions. With this in mind I would alter the introduction a little. Secondly it is hardly surprising that size swamps all other aspects of the escape mechanics. I would use some kind of residual to remove size effects completely. It’s unlikely you’ll find anything, but worth a try. Be sure to show us that familiar and unfamiliar groups do not differ in size. *Note I have marked the pdf for editorial suggestions Specific comments: L63: familiarity has other benefits as well, eg enhanced social learning (swaney et al 2001). L108: I can understand the reasoning behind the idea that familiar groups might respond in greater synchrony, perhaps even respond more quickly at longer distance, but I cant imagine how it would effect things like the magnitude of the response. This is largely innate and controlled by the mauthner neuron. You might even argue that familiar fish feel safer in their shoals and therefore respond later. Hard to say. L115: You need to tell us how long ago. Anti-predator behaviour declines rapidly in captivity. L143: what did you do with the fish afterwards? Did you put them back in the home tanks? Reuse them? Was each fish only tested once? We need these details here. L216: when it comes down to it the Ns are pretty low L231: where length is a covariate it will be important to show that familiar and unfamiliar groups did not differ in length. Note you really need to remove length. Perhaps use residuals. L247: If you ask me this is not unexpected at all. See reasons stated above. L273: The authors might like to read Brown 2002 (Journal of Ethology 20, 89-94)

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 EFFECTS OF FAMILIARITY ON ESCAPE RESPONSES IN THE TRINIDADIAN GUPPY (POECILIA RETICULATA) Review round: 1 Reviewer: 2

Basic reporting: In investigating the role of familiarity in influencing group and individual level anti-predator responses the authors seek to understand what details of responses may be influenced by group composition. Overall the manuscript is clearly written and their justification for their study is well supported. However there are two areas in particular where rewording could help better reflect the study they conducted and results they found. 1) In the abstract the authors state that “Using the Trinidadian guppy we examine the effect of different early social conditions in the three main components of predator evasion”, implying that they manipulate early life experience. Instead the paper manipulates recent social experience (i.e. the two weeks prior to the experiments; lines 136-137), which is a different line of study. I would therefore suggest the authors revisit all mention of “early social experience”. 2) In the discussion, the authors state quite strongly that they “unambiguously demonstrated that familiarity plays a significant role in shaping how groups of fish respond to a stimulus”. Since familiarity only predicted the number of individuals that responded, but did not predict several of the other variables they analyzed, this tone appears unwarranted. The statement they include later on in the conclusion, namely that “In combination, our study suggests that, while familiarity affects how groups respond to a visual stimulus, it plays a less meaningful role in determining the quality of the escape response” is a much more measured and appropriate summation of the study. I would suggest that they revisit the tone in the discussion, especially since PeerJ does not require results to be “flashy”, and thus there is no reason for overstating their case. Finally, and on a more minor note, the (Strodl & Schausberger 2012) paper that they cite does not investigate responses to predators, and I think it may have been cited in error (e.g. line 284, 293). The authors of the paper in question wrote other papers that year which the authors of this ms may have instead intended to cite. I would suggest they double check all references they list. Experimental design: While most of the experimental design was easy to understand (Figure 1 helped especially), there are a few minor areas where more detail would be helpful. Line 128-129: Without knowing the origin of the stock in the different tanks, it is hard to assess whether the individuals were related. A little more detail here, such as how long the stock had been separated and from where it was originally sourced would be useful. Additionally, it would be nice to know if the source and experimental tanks were featureless or did individuals have access to cover? (which could influence anti-predator behavior) Finally, were the size differences between individuals (one big, one medium, one small) also maintained in the unfamiliar trials? Validity of the findings: Since the authors only looked at the details of the anti-predator behavior in the first responding fish of any group (as stated lines 189-190), they were potentially sampling a non-random individual from the group. Given that the size of the individual, as opposed to the familiarity of the group, appeared to predict much of their response behavior, it would be very informative to know whether the size of the first responding individual was similar across the familiar and unfamiliar groups. I would suggest that the authors consider this addition to their analysis because it could mask the effects of familiarity on anti-predator behavior. If there are differences between the groups in the size of the first responding individual, then the authors need to consider analyzing the behavior of other group members. Additional comments: (see comments in previous three sections)

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 EFFECTS OF FAMILIARITY ON ESCAPE RESPONSES IN THE TRINIDADIAN GUPPY (POECILIA RETICULATA) Review round: 1 Reviewer: 3

Basic reporting: The manuscript is clearly written, but the overall presentation can be improved in quite a few places, either by rewording or shortening of the prose. Below, I've made specific suggestions to the author. The background and context of the study, as laid out in the introduction, are strong and well-supported by the literature cited. Structure is fine and overall the results speak to the hypothesis (predictions). I believe the authors have generally met the basic reporting criteria. Experimental design: The design is good, but there are analysis issues that need to be addressed. Specifics outlined in comments to author Validity of the findings: no comment Additional comments: Analysis. I would strongly suggest choosing to use either a strict hypothesis-testing approach or a modeling approach, but not both. My inclination would be the former, because you have set your study up as a test of a hypothesis (predictions). Also, you have relatively few parameters and a relatively small sample size (adequate, but small for this kind of analysis). If you choose the AIC approach, I would recommend calculating a AICc for small sample size. I would also provide the variance inflation factor, c, calculated as X2/df, based on the full model, not the reduced models. Check Burnham and Anderson. Make clear on line 201 that you are using a generalized linear model framework.. Yours is a fixed effects model, so although obvious, spell it out for the reader. Also, need to include the link function used, which I assume was a normal log link, but again, need to make these particulars clear. I believe you may have a mistake in calculating K. If your analysis (generalized linear models) used the log likelihood method of model selection, which I’m guessing it did, then K = no of explanatory variables, + intercept + variance. Finally, with the AIC approach you would not be reporting p-values, except possibly for the Wilcoxon test (line 198). Table 1. treatment + length should be “treatment x length” since it is an interaction. The order of model effects (factors) column seems unconventional to me. I would check this against other published tables. Possibly start with the full model that contains all of the factors, including the interaction, then all remaining combinations thereof, putting them in increasing order of the ΔAIC. You could choose to report just those with an AIC < 4.0. That would be one way to do it. Results/Discussion. You might consider the effect of group size in your discussion. It would have been useful, in hind sight, to test for this, because group size could interact with familiarity. Contrary to your predictions, groups composed of familiar individuals were less responsive than groups of unfamiliar individuals, suggesting that groups size may be an important factor. Group size could combine with safety in numbers, so two curves of response plotted against group size may start the same (n = 1), separate around 3 or 4 ( your result) but then converge at higher groups, like around 8 with very low responses. Do you think this is possible? If so, perhaps work it into the abstract in place of 31-32? Additional comments Abstract. 19 please! driver = cause 19-20: Suggestion: Traits that contribute to the avoidance/amelioration of potential threats should be under strong selection during these stages, along with the process of familiarization. 22: “Less, however, is known” suggest “Yet, gaps in our knowledge remain”. 27: suggest: …we compared the number of individuals in each test group that responded to an artificial stimulus, their reactive distance and magnitude of their response (acceleration and distance) in groups composed of…” 30-32. Consider revising, perhaps bringing in the idea of group size, assuming you think it could be playing a role here. 34. suggest: Our experimental approach revealed specific aspects of the escape response that are more likely to be influenced by variable(?) early social conditions. 53 for = to 57 between individuals within a group 64 Ward and Hart (2003) found…. 66 Groups composed of…individuals have been found to be more… 73 “will have an idea” = “may remember” 80 take out “fairly” 81 furthermore = for example 91 “…responses. We used a tropical fish, the Trinidad guppy ( ), because its behavioral ecology and predatory regimes are well known (Magurran 2005).” (This is a suggestion, and you might be able to write it better, but I would try to come up with an explanation along these lines) 105 hypothesis = prediction 127 size? 134 I would take out these sorts of personal observations unless you are prepared to provide some data. A critical reader might be skeptical that really observed this. 139-143 It took me a while to figure out that each test group has 3 fish. You should state here in the methods, clearly, so that the reader has it in mind going forward. 154-155. Again, I don’t think you need to say this. You already have a very artificial set up and the reader knows and appreciates that you are not necessarily trying to exactly duplicate nature. That’s not your point. 162. Take out “gently”. You didn’t do it “roughly”, right? 163. Take out “so no …required” 164 standard 166 escape 166 and = or 167 insert “were” 256 “, escaping may not always be the best strategy” As written, this doesn’t make sense to me if the alternative is not to escape and be eaten. How about writing “responses may be highly variable”? 283-284. I suggest shortening the Discussion, and statements like this could easily come out. 296-307: Good. Keep this paragraph! Overall, Yours is a nice contribution that will be of interest to the many researchers who are studying predator avoidance behavior.. I believe that if you go with the hypothesis testing approach, deleting the AIC material, you will have a successful paper. I would also recommend shortening the Discussion, and possibly even the Introduction. It could be tighten a bit too.

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 EFFECTS OF FAMILIARITY ON ESCAPE RESPONSES IN THE TRINIDADIAN GUPPY (POECILIA RETICULATA) Review round: 2 Reviewer: 1

Basic reporting: The authors have satisfactorily addressed all of the issues that I raised in my previous review. They agreed with my comments in almost all cases with the exceptions being only suggestions, and in those cases I understand their reasoning. Their combination of the hypothesis-testing with information theoretic approaches was a mistake in my view and so I was very relieved to see that they had agreed and omitted the latter. I read the other reviews, which also raised important questions, and again, it appears to me that the authors have addressed them, although it is possible that the reviewers may not agree. Experimental design: I believe that the article meets the PeerJ standards with respect to experimental design. Validity of the findings: no additional comments 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 EFFECTS OF FAMILIARITY ON ESCAPE RESPONSES IN THE TRINIDADIAN GUPPY (POECILIA RETICULATA) Review round: 2 Reviewer: 2

Basic reporting: No further comment Experimental design: I have no specific comments about the experimental design, which was generally ok. Validity of the findings: I have a number of important concerns about the validity of the findings and their interpretation. First of all, it is unclear what specifically the authors analysed to compare the responsiveness between familiar and unfamiliar groups and there was no way I was able to replicate their result with the provided data and only non-significant effects were acquired. Secondly, for the other four analyses the authors used a GLM approach and therefore forgot to account for non-independence between the groups and should therefore have used a mixed modelling approach (GLMM). This is likely to considerably change results. Thirdly, the authors refer to significant effects of familiarity while there are none, to multiple significant effects of length where there is only one, and make wrong interpretations on finding a significant effect. From the data and results quite an extent of the discussion seems not appropriate as the effects described seem to be different than interpreted. Additional comments: Besides my main concerns about the analyses and their interpretation and the other points below, a more important general point is that the study is still lacking is a good explanation of the mechanistic expectations and interpretation of why familiarity would matter in fast startle responses, especially in the introduction. This point should be well thought about and integrated and, together with clarifications to the reasoning for doing this specific study, besides because it has not been done yet, the manuscript will be improved. Line 24: This sentence remains ambiguous and best to remove “during early life stages “ or make clear also the experiments were conducted in the same life stage. Lines 30-31: See points below, I do not think the result holds and that therefore there are no significant effects of familiarity on responsiveness in the specific situation tested. Line 32-35: Not “more” as familiarity had no effect and furthermore size only had an effect on reactive distance! Lines 60-62: Familiarity itself does not have anything to do with individuals, therefore be careful with defining it here. Line 64: Please rephrase this sentence. Repeated exposure to a stimulus can lead to familiarisation, in a social context that may thus be conspecifics with whom an individual interacts, such as during foraging. Line 64-67: This can give greater fitness benefits/this has been shown for a couple species but be careful with making such a general statement as it may be species or context dependent. Line 70-71: Again, don’t generalise too easily. Line 73-75: Same. Line 77-83: This depends strongly on the species and the type of social system, for example much more likely in birds with strong social structures and many repeated interactions than in fish that live in very large fission-fusion populations. Line 86-87: This sentence seems to show the opposite of what is stated in the foregoing sentence. Line 104-107: It remains unclear why exactly familiarity would need to be investigated in the context of escape response and thus why the study was conducted. Stating that “how familiarty shapes predator evasion … remains unexplored” (Lines 85-86) and “Dominici calls for… all espects on an escape response” (lines 103-104) do not give clear reasoning why this would be needed. Line 120-122: But why? What mechanisms are fundamental to your prediction? Line 163-169: So was there was a difference in the way fish were placed in the observation chamber for testing as fish from familiar groups would be placed there together and fish from unfamiliar groups one at a time? This could have a number of effects and should be clarified. Lines 163-169: Thus each group received one test trial? This needs to be clarified. Lines 198-214: These two paragraphs need to be integrated into one, which can be half the length. Lines 227-238: The authors need to make clear if they run analyses at the individual or group level. If at the individual level they cannot do a GLM as they have to account for non-independence within groups by running a mixed model and group id as a random factor. Furthermore, the way how responsiveness is analysed is not correct (see below). Also, if the analyses were run at the group level it needs to be clarified how body length was calculated per group. Lines 244-246: Statistically testing this, such as with a Chi-squared test, shows there is no significant difference between the groups in any fish responding or not. This should be integrated here. Lines 246-248: I don’t understand what the authors analysed here and I cannot replicate their result. It seems the authors want to compare the average nr of responsive individuals per group between the treatment. But running this actual analysis with a Wilcoxson rank sum test yield a non-signifant result. Running this with all groups with zero responders excluded still results in a non-significant result so it is mysterious what the authors actually did. Also, here it can be clarified that for double the number of groups in the unfamiliar groups no individual responded compared to the familiar groups, although that effect was als non-significant. Line 253-258: This section is confusing and seems to make false interpretations. From Table 1 it is clear that neither treatment or length had a significant effect on the reactive distance nor was there an interaction between them. This needs to be stated first of all. Secondly, that the intercept was significant simply means that it for all individuals on average it was significantly different from zero, which is not that informative for your questions. Thirdly, as you don’t find an interaction to be significant there is no difference in slopes, which is now argued on line 255. Line 263-265: Length was only significant for maximum speed, thus again, this statement is false. Line 266-271: The table clearly shows there was no significant interaction between familiarity and body length thus does not support this statement and interpretation! Results: From the model output in Table 1 the (lack of significant) results are clear to me: familiarity and body length did not interact and did not have an effect on the reactive distance, maximum speed, maximum acceleration or total distance. The only significant effect observed was unsurprisingly that larger fish reached a higher maximum speed. Line 278-279: The authors actually do not show this as their only significant result for familiarity cannot be replicated. Furthermore, as the previous reviewers also already highlighted the authors should not make too strong statements and try to oversell their results. Line 280-281: This is purely hypothetical and has no place in the first paragraph of the discussion. Line 283-284: Again the authors make a statement about an apparent significant result that is not signifant (at least to table 1)! Line 285-286: Even if this effect would be significant it does not really say anything about how groups respond but how individuals in such a group respond. Line 287: See other points, the result on which this statement is based needs to be clarified/rectified and may considerably change the discussion. Line 287-316: A likely alternative explanation is that fish in familiar groups are more at ease as they are with individuals they have seen before. Line 342-343: Some more sentences about the relevance of studying such a fast and innate response as studied here would be helpful as it may explain the lack of significant results. Line 354-355: I wouldn’t call one significant effect far greater Line 361: The test used was not novel altought the sentence currently seems to suggest that. Line 363-366: Please see other comments about the underlying analysis and be careful about statements about behaviour being ‘adaptive’ as finding significant relationships between behaviours does not require any adativeness. Comment 7 Reviewer 2: This reviewer makes a fair point that I don’t think the authors address satisfactory. The authors should at least make the suggestion by the reviewer clearer in their discussion. With the current analyses the authors did (i.e. not controlling for group) it is hard to know for certain if it was indeed not always individuals of a certain size responding quicker.

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 EFFECTS OF FAMILIARITY ON ESCAPE RESPONSES IN THE TRINIDADIAN GUPPY (POECILIA RETICULATA) Review round: 3 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: Comments on Wolcott, Barbosa and Ojanguren 9-11-17 I have studied the responses to Reviewer 1 & 2, and generally I’m satisfied that you have adequately addressed their concerns, as well as my own (#3). I believe that statistical analysis is much improved over the original, and I agree with the decision not to use an analysis of residuals. I think you have gotten an interesting result in the finding that familiarity does not affect all components of the escape response. The findings relative to size effects are also noteworthy. I think these will be useful to those working in this field. One of my thoughts on the results is that individuals in groups of familiar individuals ‘feel safer’ and hence react less to stimuli than those among unfamiliar individuals. I might avoid saying that they feel more at ease, which, unlike ‘feel safer’, is not standard in the behavioral literature, as far as know. Below. I have made more suggestions. Most are of an editorial nature. I think the abstract was generally overlooked in the original reviews, because it seems to need a bit more work. 24 I would take out “antipredator”. All you need for clarity is “escape responses” 24 Suggest “the guppy, Poecilia reticulata”. Drop “Trinidadian” 30 ‘surprisingly’ = ‘contrary to the prediction’ 30 ‘by’ instead of ‘of’ ? 31-32 Take out the plausible sentence, as this opinion is better left for the discussion 33 insert ‘rather’ after ‘size’ 35-36. I think you should revise this. Rather than advocating for your ‘approach’, which is not the subject of this study, try a sentence that summarizes or gives your most salient conclusion. How about the last sentence of the Discussion? Something that grabs the reader’s attention. 64 insert ‘and’ after the comma 65 ‘thus’ take out 109-112 Somehow, I think this sentence is out of place. It doesn’t logically lead to 112, as yours is not an integrated approach, right? 113 ‘shaping’ = ‘affecting’ 115-116 Here’s a suggested rewording. Guppies shoal . . . . birth. During this early developmental period, guppies acquire group familiarity and its associated antipredator benefits ( ). Does such familiarity then lead to greater group cohesion, e.g. as in avoid joining a different group? However, the whole paragraph seems not to hang together very well. The points are not flowing logically to me. Suggest revisiting it. I think you need to better set the stage for you important last paragraph, the statement of purpose. 120 doesn’t flow logically from 119. 170 ‘by’ instead of ‘of’ 172-175. Here’s another suggestion for wording. ‘Familiar groups consisted of individuals that were together for two weeks. Unfamiliar groups were comprised of individuals taken singly from different holding tanks.’ You can omit the rest, i.e. …not seen each other before and put them together… 232 typo, check ‘…both response variables, 231-234 The ANOVA model assumes normality and homogeneity of variances of residuals, and also independence of residuals when you analyze responses on the dependent variable. I think you might simply say that normality and homogeneity assumptions about the distributions of residual values on the dependent variable were improved by log-transforming the response variables. The analysis is generally robust to small deviations, and it is usually pretty hard to absolutely meet all the assumptions. I think your results are clear enough. That’s my opinion. 239 Try ‘Individual S.L. did not differ… 240 (SD) 278 ‘quality’. This word seems a little vague to me. Not sure exactly what it means. You might say ‘… some aspects of the escape response’ or something to that effect. 298. Why not ‘feel safer’ rather than ‘at ease’? 367 Again, maybe ‘quality’ should be ‘specific components of’ I trust that you will find these comments useful overall. Doug Fraser

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: EVIDENCE FOR CONTINUAL HYBRIDIZATION RATHER THAN HYBRID SPECIATION BETWEEN LIGULARIA DUCIFORMIS AND L. PARADOXA (ASTERACEAE) Review round: 1 Reviewer: 1

Basic reporting: The grammar needs attention (e.g., the first sentence is a sentence fragment, and the word “still” makes the title of the paper confusing—it suggests that something about the nature of Ligularia x maon… has changed). In some places the grammatical problems make this manuscript difficult to follow, and thus difficult to review. Some of the ideas present may benefit from clarification/substantiation, but that’s hard to assess currently. Considering hybridization “one of the drivers of speciation” is a little bit strong for the first sentence of the paper—that’s a somewhat controversial/complex claim. Ln78 – provide citation for these species belonging to this section. And introduce the family affinities of this genus at some point? The introduction might be more effective if it were more tightly organized/clarified. E.g., a section on the evolutionary significance of hybridization, followed by a section on Ligularia, followed by a section on the specific study system, and its relation to the hybridization question. Ln94 – it’s unclear at this point what the distinction is between “natural hybrid species” and “hybrid offspring.” The voucher information in table S1 is incomplete. Each studied population should be supported by at least one voucher, deposited in an official herbarium (collector number and herbarium of deposition need to both be indicated here). The beginning of the Discussion belongs in the Introduction. Fig. 1 – it would be more informative to present a most-parsimonious tree (rather than a consensus tree) and to then label the branches of that tree with the bootstrap support values. Strict consensus trees don’t have very much information in them. Fig. 2 – Interesting (odd?) that relatively few of the hybrid sequences are the same as the most-common haplotype from either parent. Instead, many of the hybrid sequences are unique. Is this an artifact of the fact that the sequences from the hybrids were cloned (and thus have more PCR errors, etc., in them), and the parental accessions were direct-sequenced? Fig. 3 – Something is wrong with this tree. Is it also a consensus tree? Instead, a single most-parsimonious tree should be presented, with meaningful branchlengths, and bootstrap support values added on. Fig. 5 – This figure would be clearer if each of the parental species got the same color in each of the panels. A figure showing some plants of each taxon would be a welcome addition to the paper. Experimental design: It’s not clear (at least not in the beginning) how the cloned data were used. How many colonies were sequenced? For individuals with two “alleles”, how were they analyzed? ln141 – 1000 replicates for the best tree search? (Rather than for bootstrapping). Validity of the findings: 224 – “almost all individuals” of the hybrid shared haplotypes with one of the putative parents. But not all? What does that imply? 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: EVIDENCE FOR CONTINUAL HYBRIDIZATION RATHER THAN HYBRID SPECIATION BETWEEN LIGULARIA DUCIFORMIS AND L. PARADOXA (ASTERACEAE) Review round: 1 Reviewer: 2

Basic reporting: The English language should be improved to ensure that your international audience can clearly understand your text. I suggest that you have a native English speaking colleague review your manuscript. Some examples where the language could be improved include lines 62-65, 75-75 among others – the current phrasing makes comprehension difficult. Line 103 needs more precision in concepts. “Classification” is part of a different taxonomic task. It should say: “identified” or “determined”. Some grammar errors like spaces, periods, etc. are marked in the text. The rest of the information about literature, figures, data, etc. have no problem. Experimental design: Minor comments and suggestions: The plant materials acquisition must be more explicit. (lines 98-102, and 179). Say how did the authors got the F1, and F2 materials. Do specimens on F1 and F2, and from backrosses presented morphological variation? I suggest to include the percentage of the species individuals, instead of the word "most" (line 124). Validity of the findings: The authors are not correctly interpreting the phylograms. The explanation or interpretation in lines 228-230 of the Fig. 1 are not correct. What the authors consider as "cluster A", it is an unresolved clade formed by three different groups, and a polytomy with 22 unresolved individuals. The nrITS outcome and the provided interpretations based on the tree are erroneous. On the other side, the description of Fig. 3 in lines 253-257 is also incorrect. The tree does not show independent clusters for L. spicata (outgroup), L. lamarum, and L. paradoxa x maoniushanensis. Even more, what they consider a clade for L. lamarum (H3 and H6) it is part of the unresolved area of the tree. You cannot consider that H3 and H6 show a closer relationship than the rest of individuals sampled. The evidence and discussion for nrITS and cpDNA evidence for the Ligularia species hybridization and introgression patterns and processes must be reinterpreted (Some information about this can be found easyli in http://evolution.berkeley.edu/evolibrary/article/phylogenetics_03, in http://tolweb.org/tree/home.pages/treeinterpret.html, or in Lanford, M., et al. 2014. Bioinformatics 30 (17): i519-i526. DOI: https://doi.org/10.1093/bioinformatics/btu463 The rest of the evidence seems to support their concluisons. Additional comments: Tha manuscript present a very interesting study to understand plant hybridization with molecular markers and analysis in an evolutionary context. However part of the phylogenetic interpretations are not correct. If they are willing to reinterpret the nrITS and cpDNA phylograms, the results, discussion and conclusions will be substantially 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: EVIDENCE FOR CONTINUAL HYBRIDIZATION RATHER THAN HYBRID SPECIATION BETWEEN LIGULARIA DUCIFORMIS AND L. PARADOXA (ASTERACEAE) Review round: 2 Reviewer: 1

Basic reporting: Grammar and English usage need further attention. References, figures, data sharing, results/hypotheses -- all seem fine (I suggest removing at least one figure -- see more detailed comments below). Experimental design: yes Validity of the findings: yes Additional comments: This paper is greatly improved over the earlier version, but the grammar still needs considerable attention (at least in my opinion – ultimately this would be up to the journal). For example, in the abstract, it should be “is a hybrid” instead of “is hybrids” in the results section, and it’s unclear what “sufficient for hybrid generation determination” means. In some areas of the paper the language issues are sufficient to make it very difficult to understand what is being said. ln69 I still feel this section is too strong, even with the stated caveat that “not all hybrids form new species.” At the least, I’d suggest replacing “they will” with “they may” on this line 88 What is “family affinity”? 89 “affinities of many species at some point” is exceedingly vague 98 I don’t understand: “An interesting phenomenon in both populations is that some individuals are different from other, and they are typical individuals of the species.” 113 State this last point more clearly; something like: “Is L. xmao a cohesive hybrid species, reproductively isolated from its progenitors, or do the L. xmao individuals instead constitute a recurrently-formed hybrid swarm” 152 The number of clones sequenced for each accession should be included in a table if they haven’t been already. 159 You mean that a consensus sequence was used for closely-related sequences from a single individual? What cut-off was used to decide if sequences were closely enough related to merge? 163 All ITS cloned sequences were used? But then it says later that two sequences were selected. That selection process sounds suspicious – if the authors are deliberately selecting sequences that are close to the parental sequences and throwing out the rest, aren’t they biases the analyses towards the desired result? 265 Inclusion of identical sequences should not reduce resolution or support. 353 I’m not familiar with the introgression program but very small p-values typically indicate highly significant results, which I would have thought in this case would mean strong indication of introgression. That’s not the case? 383 The beginning of the discussion has already been covered in the introduction. The entire first section (“Conditions of…”) could be omitted. 469 Was the germination trail (1000 seeds) part of this study, or the earlier cited one? 475 etc But this mechanism – that largely infertile F1s are formed at high enough rates that a lucky few produce enough seed to allow hybrids to perpetuate – is inconsistent with your data, which shows almost no F1s but a lot of F2s. 508 This section on Pinus etc belongs in the introduction rather than in the discussion Fig. 1 Nice! Fig. 2 This figure is still strange. Why are all the terminal branches exactly the same length? Contrary to the caption, I very much doubt that this is a most-parsimonious tree. (At least, not a phylogram, with the associated scale bar [10 changes]). A single most-parsimonious tree should be presented, as a phylogram. For individuals that had multiple sequence types present, some method should be used to show which sequences come from which accession (e.g., a series of lines connecting sequences from a given individual). Fig. 3 I don’t think this figure adds anything to what is already shown in Fig. 2. It is not a depiction of “reticulate history” unless I misunderstand. Instead it is a haplotype network, where reticulations indicate other possible mutation paths. Fig. 6 Strange and interesting that there’s very little evidence of F1 plants in this sample (whereas F2s seem much more common). I don’t remember that message being made very clearly in the manuscript, but I might have missed it. Fig. 7 I don’t know enough about this approach to be able to follow this figure. What are fAmAm and fAmAd? Table 3 I think this table could safely be omitted.

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: EVIDENCE FOR CONTINUAL HYBRIDIZATION RATHER THAN HYBRID SPECIATION BETWEEN LIGULARIA DUCIFORMIS AND L. PARADOXA (ASTERACEAE) Review round: 2 Reviewer: 2

Basic reporting: Just typographic errors: Line 43: say ecidence; must say evidence Line 235: say maoniushan- ensis; must say maoniushanensis Experimental design: no comments Validity of the findings: Lines 227-230, and 235-141: The tree presented in the Fig 1 is not resolved. When a phylogeny has no resolution, you cannot describe group relationships. In this case, it is not true that the outcome resolved two phylogenetic sections (A and B, even if they resulted in the network at Fig 2, which is not a phylogeny). Hence, what the authors call section B, has the same probability to be phylogenetically related with accession H2, or with the H5-H7 branch or with any other. The order of the branches resulting from the analysis does not gives hierarchical groupings or relationship evidence. If the clade branch is not resolved at the base, you cannot say that you have a monophyletic group. There are not sections to be separated, you can say that most of the times, some samples resulted together forming branches (indicate bootstrap numbers or parsimony branches support), but that you do not know the relationships among them. I cannot agree with the tree description, interpretation, and conclusions presented. Additional comments: Most of the evidence presented in the paper support the proposal of hybridization and introgression within Ligularia duciformis and L. paradoxa. Then I suggest to leave the phylogenetic analysis out of the paper since is not a good evidence to support the hypothesis presented in this study.

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: EVIDENCE FOR CONTINUAL HYBRIDIZATION RATHER THAN HYBRID SPECIATION BETWEEN LIGULARIA DUCIFORMIS AND L. PARADOXA (ASTERACEAE) Review round: 3 Reviewer: 1

Basic reporting: I realize that this isn't particularly helpful, but the English grammar, etc., is still not at a state that I would be comfortable with, were I a journal editor. Perhaps peerJ can do some copy editing? 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: EVIDENCE FOR CONTINUAL HYBRIDIZATION RATHER THAN HYBRID SPECIATION BETWEEN LIGULARIA DUCIFORMIS AND L. PARADOXA (ASTERACEAE) Review round: 3 Reviewer: 2

Basic reporting: no comment Experimental design: no comment Validity of the findings: The problems I found are solved. I think that the paper migth be published as the last version is. 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: ILLEGAL USE OF NATURAL RESOURCES IN FEDERAL PROTECTED AREAS OF THE BRAZILIAN AMAZON Review round: 1 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: I find the overall findings valid, however I believe it would be interesting to add in the analysis the temporal trend of the illegal use of natural resources and if this changes over time i.e. if there is an increase or decrease. Moreover, I wonder if the authors have looked at the correlation between the illegal use of natural resources and law enforcement activity (number of guards, monthly patrol etc) . Beside the PA protection category, does the presence of law enforcement activity have an impact on the number of illegal activities over time? PA protection category is not always correlated with law enforcement effectiveness. These additional analyses would help to understand why there so many illegal activities and if law enforcement and/or the park management is efficient and effective. The take-home message would be beneficial for both managers and decision-makers. Moreover, I have an analytical concern about including both terrestrial and coastal/marine reserves – I expect that fishing activity is more present in coastal/marine areas and hunting activity more present in terrestrial areas. It would be good if this could be explored and included in the analysis. There are many repetitions in the discussion section about the description of the results (the result section already includes it). This needs to be better structured. Moreover, I would suggest to add more literature about protection measures against illegal use of natural resources. Additional comments: This manuscript was nicely written and interesting to biodiversity conservation and protected areas management. I have highlighted a few areas that could benefit from some clarification or rewording, which are listed below: L123. IBGE, could you give the whole word Instituto Brasiliero de Geografia e Estatistica L131. Established is repeated twice L140-141. Can the authors add the total area (in square km) of the PAs studies overall the ones that are managed by the federal government? L142. Can the authors add somewhere the percentage of grassland and savanna per park? Would be good a table with the PAs and the % of habitat cover types L145 Brazilian System of Conservation Units L146-148. Information not necessary L181. 1 Km2 instead of from 1km by 1 km L181. 200 m2 instead of up to 200m by 200m L207. Please add table with results from correlations L215. Would be nice have subtitles like in the methodology section L271. Threatens L294. Reference missing L314…in order to achieve just an effective conservation, it is necessary to involve local… Table S2. Please add a clear description of each category of illegal activity

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: ILLEGAL USE OF NATURAL RESOURCES IN FEDERAL PROTECTED AREAS OF THE BRAZILIAN AMAZON Review round: 1 Reviewer: 2

Basic reporting: The manuscript’s content is relevant given the lack of studies that contemplate the illegal use of natural resources in the PAs. This kind of research is important to understand the PA's needs and to enable some strategic actions. The MS was very well written, and brought a lot of good and recent references. Experimental design: Some of the reviewer suggestions to improve the MS are bellow : Line 43: Change "significant reduction in deforestation in the region" to "significant deforestation reduction in the region" Line 117: Put the Datum used to compose the maps. Line 126: Change "of the territory of the country" to "of the country's territory" Line 131: Take away one the words "established" To all figures: Put the Datum and Projection in the maps, with this anyone that reads your maps will know this information. Figure 1: In the legend: put the units of the “Illegal activities”, like: “Numbers of illegal activities” Divide the legend in two columns, the map’s layout will be improved. Suggestion to the legend order an items: First column: Numbers of Illegal activities 1-23 24-55 56-97 98-199 200-316 Second column: Brazilian Amazon Rivers Protected Areas Sustainable use Strictly protected To all tables:Change “Brazilian reais” to “Brazilian real” Validity of the findings: The results show the importance of your work, however i missed some explanation in the conclusions, that how the government authorities would use this data to improve the threats to the biodiversity in the PAs. 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 MITOCHONDRIAL GENOME SEQUENCES OF THE NORTHERN SPOTTED OWL (STRIX OCCIDENTALIS CAURINA) AND THE BARRED OWL (STRIX VARIA; AVES: STRIGIFORMES: STRIGIDAE) CONFIRM THE PRESENCE OF A DUPLICATED CONTROL REGION Review round: 1 Reviewer: 1

Basic reporting: gel picture really needed? Experimental design: some details could be in an appendix Validity of the findings: no comment Additional comments: when repeatly mentioning a software product or mito-genome, no need to cite again numbers in tables should be right-justified line 101: able to explore

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 MITOCHONDRIAL GENOME SEQUENCES OF THE NORTHERN SPOTTED OWL (STRIX OCCIDENTALIS CAURINA) AND THE BARRED OWL (STRIX VARIA; AVES: STRIGIFORMES: STRIGIDAE) CONFIRM THE PRESENCE OF A DUPLICATED CONTROL REGION Review round: 1 Reviewer: 2

Basic reporting: In general, I found the manuscript to be well written; even some of the denser parts of the Methods section were clearly worded and easy to follow. The figures are of high quality, but some could be cut (or combined, or moved to Supplementary Materials) without significantly impacting the manuscript; see detailed comments below. Raw sequences have been deposited in NCBI’s Sequence Read Archive, but I was unable to find Genbank accession numbers in the text for the two new sequences produced for Strix o. caurina and Strix varia; these should be provided. Experimental design: The research described in this manuscript is original and adds to the growing body of work on avian control region evolution. Extensive detail is provided in the main text on the methods used, with further details given in the Supplementary Materials. If anything, the Methods text could be trimmed, moving more of the nitty-gritty to Supplementary Materials. Validity of the findings: Overall, the authors make good use of their data to address questions outlined in the Introduction. Employment of both next-gen and Sanger sequencing techniques was critical to obtaining accurate mt genome sequences for these owls. In fact, the authors could further emphasize the fact that their study represents a cautionary case in which a next-gen-based assembly resulted in an incorrect mt genome sequence. Given the prevalence of next-gen sequencing, combined with the fact that control region duplications have been found in a growing number of disparate avian taxa, it is important to consider the possibility of control region duplications whenever sequencing avian mt genomes. Additional comments: I would suggest re-thinking the sub-headings used within the Methods section. Since you have separate descriptions regarding the sequencing/assembly of the two species’ genomes, it would be nice if each description were labeled as such, and the two descriptions were structured (divided) into consistent sub-sections. My detailed comments are listed below by line number. l.36 At the end of the abstract, you might want to add a sentence regarding the shortcomings of short-read sequencing/assembly methods in cases like yours. l.42 “which led to” l.43 “of the chicken is representative” l.100 Technically, you can’t cite (Hanna et al. 2017) in this way, since it is not published. More correct: (Hanna et al. unpublished) or (Hanna et al. in prep.) l.160 “amplify a slightly longer” l.184 Did the length of the longer PCR product jive with the (incorrect) Illumina-based assembly? l.209-214 The purpose of this comparison was not clear to me. l.227-256 This section could be moved, so that it is presented after the description of the S.varia assembly. l.227-228 The alligator sequence comparison isn’t mentioned/discussed later on in the text, so it’s not clear why it’s included here. The alligator sequence is included in Figure 6, but as noted in the figure comment below, it could be cut without detracting from the conclusions. l.238-240 See previous comment. l.247-248 See above. l.256 “Nuclear pseudogenation” might be a better term here, since you are referring to pseudogenes that appear in the nuclear genome. There are also pseudogenes of mt genes that appear within rearranged mt genomes (e.g., see Eberhard & Wright 2016). l.258 See previous comment. Related comment: Is there any evidence of mitochondrial pseudogenes, such as those found in some parrots (see Eberhard & Wright 2016)? There seems to be a short stretch of sequence on the 5’ side of CR1, before the hairpin sequence; any chance that it’s similar to tRNA-Glu or ND6? l.287-292 It might be better to present the primary sequence source first, and then describe the secondary one. l.299 It would be helpful to briefly explain that the two contigs are. l.421 Is it possible to discount the hypothesis that the long fragment was a numt? (or, to really play devil’s advocate, is it possible to determine that one or both of the fragments are indeed mitochondrial?) l.446-447 This is interesting (though I don’t have any idea why this might be)! l.453 As someone who works with other (non-owl) birds, I found these divergences to be quite high for taxa that can hybridize. Are these levels of divergence typical for owls? l.461-462 Identical or nearly identical CRs (e.g., as observed in snakes, e.g., Kumazawa et al. 1996, MBE 13:1242-1254) could also cause assembly problems. l.466 “complete and accurate assembly” l.469 “inferred the presence” l.471 “but rather deduced that a duplication was present from the appearance” l.475 “The mitochondrial genomes…” l.502 “as found in some other avian taxa” l.508 Not just Amazona parrots; also found in several other parrot taxa with independently evolved CR duplications (see Eberhard & Wright 2016) l.517 “a proper phylogenetic test of this hypothesis, with increased taxon sampling, is warranted” Table 1 At least one of the primers listed in the table was designed by other researchers (e.g., N1, Barrowclough et al. 1999), and this should be noted. It would be good to add a column for references, or add footnotes, to cite sources for primer sequences, and also note which were designed by the authors. Table 2 Some of the information presented in the table (e.g., Alignment score, Entropy) are not discussed in the text, and don’t seem crucial to the results/discussion. Table 3 Per earlier comment (l.256), “Nuclear pseudogenes (Numts)…” Two of the columns, E-value and Bit score, provide information that is never discussed in the text, so could be eliminated. If they are not cut, then Bit score needs to be defined in the caption. Figures 1&2 Both of these are beautifully drawn, but are largely redundant; could be replaced with a single figure that shows gene order surrounding the CRs. Figures 3&4 The figure caption should also state that the locations of the goose hairpin and primer binding sites are shown. To streamline the figure a bit, the yellow boxes labeled “control region 1” and “control region 2” could be cut, since the regions are already labeled on the left. Figure 5 The description given in the text is probably enough, so it’s not necessary to include this figure. If desired, it could be moved to Supplementary Information. Figure 6 The caption refers to panels B & C, but no such labels appear in the figure. Although the information presented in the figure is not incorrect, it illustrates only a subset of the gene orders that have been thus far documented for birds; the purpose of illustrating the owl gene order within an incomplete context is not clear. I would recommend a figure that illustrates the gene order around the owl CRs (as suggested in lieu of Figs. 1&2), and then cite (and discuss, if you wish) other papers that have provided more complete discussion of mt gene order rearrangements in birds (e.g., Mindell et al. 1998, and more recently, Gibb et al. 2007 and Eberhard & Wright 2016).

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: INFECTION OF ARMY ANT PUPAE BY TWO NEW PARASITOID MITES (MESOSTIGMATA: UROPODINA) Review round: 1 Reviewer: 1

Basic reporting: The writing was clear and easy to understand. The introduction provided adequate background information. There are some minor grammatical errors here and there that should be tended to. Experimental design: • Line 98: If the pupae were collected from Aug 2008 to April 2011, then that spans far more than just 10 months. If the samples were collected from 10 specific months, please specific which months. • Line 130: “Global parasitism rate” is too broad. I’d suggest the phrase “overall parasitism rate among Macrodinychus spp.” • Line 132-133: It’s not clear why results on number of workers are assessed. There is not reporting on this data in the results section in terms of prevalence of infection. • Given that the pupae were collected over a long span of time, perhaps one should look for potential seasonal or temporal patterns in infection rate. • Line 441: Were the differences in prevalence of infection between colonies statistically different from one another? • The behavior “antennated” is very similar to “ignored” except perhaps for the duration of antennation. Is this minor difference biologically relevant? • Some of the methods require minor clarification. For instance, the experimental could have recorded the duration of the ant-mite interactions. Would this additional data not have been useful? Validity of the findings: • I cannot comment specifically on the species descriptions, as this is not my area of expertise. I can comment only on the behavioral assays. • Line 438-441: It’s not clear what value these set of numbers provide. The infection rate of 1.69% is already reported in line 434. What is the relevance of reporting number of workers? • Lines 454-459: The reported values for AI are presumably in percentages, in which case the values seem exceedingly low to be biologically relevant. Can you please clarify? • Line 452-453: Given that there are overall differences between the different myrmecophile species, it would be useful to present a post-hoc analyses to determine which groups are different from one another (as suggested in the methods). • Line 474 and 488: Is there direct evidence that the mite is feeding on the hosts? The presence of a cavity does not necessarily indicate feeding. Even if the mites left behind a scar, this may be a response to the chelicerae pinching the host tissue, and not necessarily feeding by the mite. Still, please specify in the results section if scars were indeed observed in your study. • Lines 483-484: This study does not actually provide experimental evidence that M. hilpertae and M. derbyensis feed on host hemolymph. This assumption is based on circumstantial evidence based on the presence of a cavity and scar, which could be a melanization response to the pinching/tearing of the chelicerae. • Lines 490-509: The authors suggest that the overall low infection rate may be due to that fact that the association is native. But they failed to considered a number of other alternative explanations, e.g. seasonality, spatial (nest) heterogeneity, etc. The low infection rates may also be an artifact of sampling bias. Given that pupae were collected during colony migration, and that migration may serve as a form of counter-defense, then the pupae being transported may be biased towards uninfected pupae (assuming ants can detect the difference and leave behind the infected pupae). • Figure 5A: The behavior “attack” is not included, is this an oversight? • Figure 5B: Please indicate the units for the dependent variable. Additional comments: • Abstract: The statement that “Our results support the hypothesis that the primary habitat of all Macrodinychus mites is the ant nest” is a somewhat of an overstatement and not really testable since one cannot conclude with full certainty that “all” Macrodinychus spp. inhabit nests. This statement is also inconsistent with lines 76-77, which state that only 1/3 of Macrodinychus species are associated with ants. • Lines: 511-512: This statement seems out of place as there is not further reference to the down-regulation of parasitism rates and it’s relevance to the results. • Lines 524-527: The rationale behind this statement is not clear. Please expand. • Discussion: It would be useful if the authors could discuss the potential ecological and perhaps biocontrol ramifications of this very interesting form of symbiosis. • Overall, the discovery and morphological description for the two new mite species is valuable. However, the results on rates of infection need to be more detailed and interpretation of the behavioral assays needs to be treated with more rigour; for instance, the discussion does not address the different behavioral responses of the ants. For instance, the discussion does not provide much context for the results reported in Figures 5A and 5B.

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: INFECTION OF ARMY ANT PUPAE BY TWO NEW PARASITOID MITES (MESOSTIGMATA: UROPODINA) Review round: 1 Reviewer: 2

Basic reporting: AS a reviewer I am making no claim for expertise in mite systematics, but enjoy a solid description of natural history and a fascination with the communities in less known places, and that is what the present manuscript contributes along with a description of two new mite species. The dense populations of eusocial insects provide unusual opportunities for parasites and certainly the conditions where previously undescribed forms may be found. Thus, I believe this paper has clear value and perhaps a dual audience of the few mite specialists out there combined with the more abundant students of ants. I also found the paper generally well written, especially the introduction and methods (for which I have basically nothing to say). they are were very clear to someone tangential to the topic, but I think some organizational shifts in the results and discussion will make the content more accessible for those more interested in the parasitoid system than the necessarily very complex and detailed description of mite anatomy associated with species identification. Overall, I think the changes I suggest should be straightforward. The main point is to be consistent in the order of presentation across sections. You open with life history and focus the discussion on life history (actually you forget to discuss the new species aspects there, largely because I think you used a results and discussion approach for describing M. hilpertae and M. derbyensis, but bury the behavior in the results below the species description. Easily fixed, I think. 1) Start the results with the Life History and nest observations (442-467) and follow them with your description of the two species, and end results with those descriptions 2) The genus description is not your result nor really is a key. Why not move this to the discussion as a section on Generic variation and keys to the species (my one area where I think rewriting beyond moving text is required) as you preface your key with the material at 187-217 prior to presenting 397-431. 3) Still keep 469-527 as the start of discussion, and the Conclusion stays at the end, below the key. Smaller points: in the systematic note (194-198), you need to translate the German group terms first and then simply indicate for precision the term used by Hirchmann, or omit that presentation if this “Gangsystematik” simply hasn’t influenced the modern systematics of the group. At 210, to what the “Diagnosis” applies is needed. I assume Macrodinychus, but in contrast to what other genera? 249-251: adjective first…. Female or male operculum, not operculum female. 270-281, you claim you make an estimate, but report a very precise number, and similarly at 452-467, make sure significant digits are relevant to the means reported, for example 23.60 +/- 10.46 really is just 24 +/- 10, but check throughout your paper for both issues that reoccur. 360: avoid relative terms, “Legs relatively short”, in a species description lacks information without a clear contrast. 376 where you say “2, rather than 3”; to what does the 3 refer? Experimental design: The design came out of a chance discovery, and that's ok. Validity of the findings: solid. Aren't new species by definition "novel." Additional comments: Figures: Hey, just nice photos. When I read two mite species, one thinks really… you can be sure when your behavioral work often lumped the two together, and yet your photos show clearly two different animals, and they fall out separately in the key completing that story. Perhaps most of my comments are all up in the basic reporting section, but that is the most relevant. Shouldn't take more than an afternoons work to make improvements to readability. The paper tells a good story, adds alpha taxonomy on an understudied group, and links the species to a well known host 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: INFECTION OF ARMY ANT PUPAE BY TWO NEW PARASITOID MITES (MESOSTIGMATA: UROPODINA) Review round: 1 Reviewer: 3

Basic reporting: This is a nice and good written manuscript with some deficiency. Experimental design: Acceptable. Validity of the findings: The manuscript contains original and new spientific results. Additional comments: This is a well-written and clear manuscript, with nice pictures and some nice drawings. The found species seems to be new, but the illustrations are not enough for a taxonomic paper. The digital pictures are nice, but not show exactly the small details of the investigated specimens. Need to add more drawings to the manuscript, the most important part of the description of the new species is the exact and clear illustration which can help the identification and recognisation of the species. Kontschan in press paper is already published.

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: CLINICAL OUTCOMES OF RESIDUAL OR RECURRENT NASOPHARYNGEAL CARCINOMA TREATED WITH ENDOSCOPIC NASOPHARYNGECTOMY PLUS CHEMORADIOTHERAPY OR WITH CHEMORADIOTHERAPY ALONE: A RETROSPECTIVE STUDY Review round: 1 Reviewer: 1

Basic reporting: 1) The article should be reviewed by a native English speaker – there are significant grammatical errors. Experimental design: 1) Their inclusion criteria did not require a biopsy confirmation that the residual/recurrent mass was actually tumor. This seems a major limitation of the analysis. They also do not describe how many patients in the surgical group had false positive resections. 2) In the results section it is unclear whether the stage described refers to the original stage (as per standard practice) or the stage at recurrence. Based on Figure 2, it seems they are referring to recurrent Tstage. As a practice, these are referred to with Arabic numerals (1,2,3,4) , not roman numbers (I,II, III,IV). Validity of the findings: 3) The subgroup analysis involved very few patients – I am not sure how much we can rely on the validity of the data at that level. A comment should be made to indicate these findings should be viewed as exploratory. For instance, looking at the OS and DFS curves in Figure 5, the only group that reaches significance is surgery vs CRT for advanced disease. 4) The inclusion of EBV-DNA is fascinating and a clear strength of this paper. Given that the bloodtests were drawn serially, however, efforts should be made to clarify at what time point we are calling someone “EBV-positive”. In addition, more space should be used to emphasize the most impressive finding on these survival curves – the absolutely shockingly poor outcomes among patients with EBV + disease posttreatment. Again, though, does the EV+ in Figure 5 reflect EBV+ before or after treatment? 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: CLINICAL OUTCOMES OF RESIDUAL OR RECURRENT NASOPHARYNGEAL CARCINOMA TREATED WITH ENDOSCOPIC NASOPHARYNGECTOMY PLUS CHEMORADIOTHERAPY OR WITH CHEMORADIOTHERAPY ALONE: A RETROSPECTIVE STUDY Review round: 1 Reviewer: 2

Basic reporting: The paper is well written and the conclusions reasonable. Experimental design: The study design is effective and it is able to investigate the impact of EBV-DNA in patients' prognosis according to a subgroups stratification analyzing the stage of disease and the treatment strategy adopted. Validity of the findings: Data is robust and statistically sound. Additional comments: The paper can be improved by addressing these issues: 1) Please, specify in the material and methods section the histological subtype of nasopharyngeal cancer enrolled in this study. Only non-keratinizing carcinoma (WHO type III) have been included? I strongly suggest you to specify this information. 2) The results herein reported suggest that surgery was more effective than CRT at facilitating the reduction of EBV-DNA viral load and therefore surgical treatment has better therapeutic efficacy than CRT alone. (Discussion, lines 254-256). However, surgery is not feasible in every case of nasopharyngeal cancer recurrence (e.g. internal carotid artery encasement, massive intracranial intradural involvement, orbital content invasion). I would like to suggest the Authors to improve the Discussion section by including the list of contraindications for endoscopic nasopharyngectomy currently accepted. In this regard, I suggest you to refer the study by Castelnuovo et al. where actual contraindications for salvage surgery in recurrent nasopharyngeal cancers have been clearly elucidated (Castelnuovo et al. Endoscopic endonasal nasopharyngectomy in selected cancers. Otolaryngol Head Neck Surg. 2013 Sep;149(3):424-30. doi:10.1177/0194599813493073) 3) A limit of this study is that patients have been allocated between the surgical and non-surgical group arbitrarily, without any randomization. The Authors should discuss deeply this and possibly propose future studies where a randomization can be introduced.

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: CLINICAL OUTCOMES OF RESIDUAL OR RECURRENT NASOPHARYNGEAL CARCINOMA TREATED WITH ENDOSCOPIC NASOPHARYNGECTOMY PLUS CHEMORADIOTHERAPY OR WITH CHEMORADIOTHERAPY ALONE: A RETROSPECTIVE STUDY Review round: 1 Reviewer: 3

Basic reporting: The article contains several English irregularities, especially with the use of adverbs ( see examples in the revised PDF). It would benefit from a revision by a native English speaker. Overall, it is well organized and beautifully illustrated Experimental design: The main flaw in this section is the lack of clarity about how the treatments were chosen. It lacks a description of criteria used in each group. There are other important data points missing (see revised PDF) Include reason for death (local, loco regional, distant mets, side effects of treatment. Validity of the findings: The analysis seems sound; however, the conclusions are flawed in view of the lax methodology, as described above. They will have to be revised. In addition, theaters fail to address complications from each approach of treatment. This can also affect 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: COMPARISON OF HUMAN PAPILLOMAVIRUS (HPV) DETECTION IN URINE AND CERVICAL SWAB SAMPLES USING THE HPV GENOARRAY DIAGNOSTIC ASSAY Review round: 1 Reviewer: 1

Basic reporting: Data regarding the worldwide and local epidemiology is missing. Experimental design: In the statistical section there is no reference to sample size and power calculation Validity of the findings: I suggest avoiding using phrases such as may be useful and could be accurate when concluding. Additional comments: I enjoyed reading the manuscript. It is well written and is quite clear. "Urine samples processed with the HPV GenoArray assay may be useful for the clinical management of HPV infection and the technique could be an accurate, noninvasive method for monitoring HPV infections in women." This sentence should be rephrased. The test does not change management. Something like - Urine sample with the GenoArray is an alternative for women declining to undergo PAP smear however now days and using current technology it is not the first line screening option.

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 OF HUMAN PAPILLOMAVIRUS (HPV) DETECTION IN URINE AND CERVICAL SWAB SAMPLES USING THE HPV GENOARRAY DIAGNOSTIC ASSAY Review round: 1 Reviewer: 2

Basic reporting: The manuscript is written clear english throughout following professional article structure. Experimental design: Research question is clear, well defined, relevant & meaningful. However, as many controls are missing, the results do not fully support authors conclusion. Validity of the findings: There is a minimal impact and novelty in this manuscript. Data are not robust and credible. Additional comments: Developing a noninvasive HPV detection method may be useful and significant, particularly for people in developing countries. However, this manuscript by Nilyanimit et al. provide limited value. The results of HPV prevalence in this study are not consistent with other previous studies. For example, the percentage of HPV16 positive samples is unusually low among the samples showing that less than 20% (12 out of 65) of HPV-positive samples have HPV16 DNA. As the authors did not use any positive and negative controls, it is concerned if the procedures and methods used here were appropriate. Additionally, the overall concordance percentage (65.2%) between the cervical swab and urine samples is not impressive and thus, it is not sure the urine test that the authors suggest is useful at all. It is impossible to understand what the authors exactly did in this study, because many important descriptions about experimental procedures and data are missing. The authors should explain in detail how they extracted DNA, normalized the samples, validated the assays and specimens, etc.

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 OF HUMAN PAPILLOMAVIRUS (HPV) DETECTION IN URINE AND CERVICAL SWAB SAMPLES USING THE HPV GENOARRAY DIAGNOSTIC ASSAY Review round: 1 Reviewer: 3

Basic reporting: No comments Experimental design: 1. It would make the paper stronger if the authors could show an alternate method for HPV detection other than the two they compare, which would show if the the HPV Genoarray is the most superior non-invasive method for detection. 2. It would also be interesting if the authors could discuss the rationale for choosing the current GenoArray method compared to other existing methods. Validity of the findings: 1. It might be better if the authors can provide a clear gap in knowledge and state the necessity and importance of such a study. 2. The authors should also consider discussing the differences they observe with sensitivity to the other existing methods, since these other methods are currently used as well. The authors have discussed the difference with the other GenoArray based study but not with studies involving other methods Additional comments: The manuscript "Comparison of human papillomavirus (HPV) detection in urine and cervical swab samples using the HPV GenoArray Diagnostic assay" discusses the use of non- invasive methods for HPV detection, which is crucial given the rising incidence of HPV. The manuscript is well written and discusses the results and aspects of the assay in required detail. It might make the manuscript stronger to discuss the gap in knowledge with current HPV genearray studies and also discuss the differences seen compared to the other available methods as this might further highlight the importance of this 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: COMPARISON OF HUMAN PAPILLOMAVIRUS (HPV) DETECTION IN URINE AND CERVICAL SWAB SAMPLES USING THE HPV GENOARRAY DIAGNOSTIC ASSAY Review round: 2 Reviewer: 1

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