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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: MECHANISMS OF OAT (AVENA SATIVA L.) ACCLIMATION TO PHOSPHATE DEFICIENCY Review round: 1 Reviewer: 2

Basic reporting: The topic of the article is interesting and it is suitable for publishing in Peer J. The presented studies concern on growth parameters and physiological adaptations which enable four commercial cultivars of oat to grow under Pi deficiency. The authors postulated that extracellular phosphatases play significant role in acclimation of oat plants to low Pi regime. Although the method of acid phosphatases assays is quite old-fashion, the concept and results are valuable and might be good base for application in agriculture. A better understanding of the mechanisms of plants responses to Pi deficiency is essential to develop strategies to increase crop yield. It would be worth to carry out some genetics approaches to elucidate mechanism of oat responses to Pi starvation at molecular level (i.e. identification of AP sequences in oat’s genome, analyses of AP genes expression in different Pi nutrition, Western Blot to determine AP proteins in more quantitative way). The introduction is written well and purpose of the study is clearly presented. Also results were analyzed properly with adequate statistical methods. The discussion was based on updated literature and supports the research hypothesis. Experimental design: No comment Validity of the findings: No comment Additional comments: Minor changes have been suggested: Line 198-200: Please explain why you used different concentrations of inorganic (1 mM KH2PO4) and organic (0,1 mM phytic acid) as a P source? Is 1 mM concentration of Pi enough optimal for crops? Line 425: “…oat cultivars could effectively utilized phytates, organic P forms generally not easily available to other plants”. This sentence should be rewritten. It seems that only oat might use phytates as a P source? Are there any specific phytases from oat that mobilize organic P reserves in soil? A reference is needed. Line 613: Abbrevations: PN – is missing PM – is missing PAM- is missing

You are one of the reviewers, your task is to write a review for the article. You will 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: CAN 9Q34.2 RS633862 POLYMORPHISM PREDICT SURVIVAL IN EPITHELIAL OVARIAN CANCER? Review round: 1 Reviewer: 1

Basic reporting: Overall several parts of the manuscript- especially the introduction and discussion section needs to be rewritten to address several grammatical and punctuation errors. Several aspects needs to be rewritten to address the technical aspects and to ensure the right rationale and message is getting across The results need to be addressed again to ensure the statistics used are well explained and the correlation is well described Experimental design: Line 141- p value – report the exact p value form the gtex Kindly expand on the results obtained from SNPinfo Kindly expand on the overall follow up time line. Detailed clinical and pathological information was retrieved for all the patients however, no information is provided. Including a demographics table including all this information is pertinent For the multivariate analysis- kindly mention in the methods exactly which covariates have been included in the analysis including if continuous or catergorical How was the bioinformatics analysis done to determine that the SNP was a TFBS. Also kindly expand on the actual bioinformatics analysis done vs. information look up using databases such as GTEx and UCSC genome browser. Along the same lines “Genotype-phenotype correlation analysis” is merely looking up the expression on the GTEX database or was any further analysis using the samples in the study done? The gene-expression of the ABO bases on the genotyped should be determined (if samples permit) in the study samples itself to check the correlation with Validity of the findings: Rationale for picking ABO vs others reported in the previous paper by the authors should be expanded upon Conclusion/Discussion needs more clear writing to tease apart the message Lack of greater follow up time was reported as one of the short comings of the paper. However no details on the actual follow up done was provided Some kind of replication is warranted especially for the gene expression reported usingGTEX Further information is needed regarding the SNP in question - rs633862 - minor allele frequency especially in the Han chinese population - rationale for the grouping of the SNP - AA+AG vs GG instead of carriers vs. non-carriers of the SNP Additional comments: The paper needs to address some big questions and several parts need to be readdressed to make it clear and lucid. More elaboration is needed on the SNP itself and the reason for not addressing the gene dosage effect and grouping both carriers and non carriers together. If G allele is being purported as the protective allele then additional analysis with just the GG genotype is needed to support this claim.

You are one of the reviewers, your task is to write a review for the article. You will 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: CAN 9Q34.2 RS633862 POLYMORPHISM PREDICT SURVIVAL IN EPITHELIAL OVARIAN CANCER? Review round: 1 Reviewer: 2

Basic reporting: Literature references, sufficient field background/context provided Data missing for study group as detailed below Experimental design: Study group design- 1. Separating loss of follow-up and death from the disease may better clarify survival analysis 2. Time at enrollment is not stated. Importance arises weather neoadjuvant chemotherapy was introduced to advanced stage disease at time of enrollment. The same pitfall for discussing survival analysis is also true for treatment with Bevacizumab as it is a factor for prolonged survival in a subgroup of ovarian cancer patients. If so, this needs to be discussed as an optional explanation for survival difference. 3. There is a need for sub classifying EOC by histological subtypes as the prognosis is different. HGSOC comprised 71% of EOC in this study. Are the results true for other subtypes of EOC apart from HGSOC? 4. There is missing data regarding how many patients had reached to follow up and was there a difference between the groups in that matter? Validity of the findings: See above Additional comments: Research question well defined, relevant & meaningful dealing with front research in oncology In summary, representing the study group by histologic subtype, number of patients reaching to follow-up, stage of the disease and novel treatments relevant for accepted treatment of ovarian cancer today my explain survival difference ruling out the hypothesis of 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: CAN 9Q34.2 RS633862 POLYMORPHISM PREDICT SURVIVAL IN EPITHELIAL OVARIAN CANCER? Review round: 1 Reviewer: 3

Basic reporting: The report is clear and well written and professional English is used through out . References are up to date and relevant. There is a few typos to be corrected in subediting. Reference 1 Angelov et al. should be 2014 not 1900. Tables and figures are relevant and clear. Experimental design: The research question is well defined, relevant and original. Methods appear to be well decribed. Validity of the findings: The data is well presented and appear statistically sound. Limits of the applicability are acknowledged. Additional comments: Congratulations on an excellent paper. I support the publication 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: CAN 9Q34.2 RS633862 POLYMORPHISM PREDICT SURVIVAL IN EPITHELIAL OVARIAN CANCER? Review round: 2 Reviewer: 1

Basic reporting: Several grammatical and spelling errors need to be rectified in the manuscript. Experimental design: Comments have been addressed Validity of the findings: Comments have been addressed Additional comments: The authors need to address the discrepancy regarding the expression data. In the manuscript text they report that AA genotype has increased mRNA expression whereas the Gtex data (figure 2) states that GG genotype has increased mRNA expression. Also, in the text they report "rs633862 G→A variant may regulate ABO expression" whereas it technically should be A->G since All other comments have been addressed.

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

Title: WATER ABSORPTION THROUGH SALIVARY GLAND TYPE I ACINI IN THE BLACKLEGGED TICK, IXODES SCAPULARIS Review round: 1 Reviewer: 1

Basic reporting: Please see comments to author. Minor adjustments are needed. Experimental design: No Comments Validity of the findings: Please see comments to author. Minor adjustments are needed. Additional comments: Reviewer comments to the submitted manuscript entitled, “Water absorption through salivary gland type I acini 1 in the blacklegged tick, Ixodes scapularis” by D Kim and colleagues. In the present study, the authors perform a number of molecular and physiological analyses to demonstrate the mechanisms of water absorption and rehydration through the tick salivary gland. The authors go on to suggest that type 1 acini are responsible for water balance, which is a nice addition to Y. Park’s previous work focused on the neuroendocrinology of tick salivation. The authors suggest the Na+-K+-ATPase protein may represent a target site for tick control, but this reviewer thinks the potential for exploiting this target as a site of control is limited. This reviewer does not see any glaring errors in the research design or the presentation of the data. The manuscript is well written with very few grammatical errors, repetitions, or incorrectly cited references. The data are clearly presented. There are a few minor revisions that are suggested: 1. Previous work by D Kim and Y Park has shown evidence that the Na+-K+-ATPase pump is involved in ion secretion rates. It is likely that the water secretion is due to the development of an osmotic gradient that is derived from an electrochemical gradient that facilitates the transport of anions. However, the authors show the Na+-K+-ATPase is expressed in the type II and III cells for ion secretion whereas this study suggests the Na+-K+-ATPase is responsible for water balance in type 1 cells. This reviewer suggests discussing the expression patterns of Na+-K+-ATPase and the potential implications for osmotic regulation and maintenance. 2. Line 85: This reviewer suggests including the final concentration of rhodamine in the feeding studies versus the volume and starting concentration. 3. Line 117: was this water labeled with Rho123? 4. Methods: It would be beneficial to include a statistics section that highlights the number of replicates, statistical analyses used, etc 5. The data presented in lines 174-184 assume that oaubain is inhibiting a membrane expressed Na+-K+-ATPase pump. However, this molecule is not completely specific for Na+-K+-ATPase and has been shown to alter calcium cycling, mitochondria functioning, etc. Either of these will most certainly alter the function of the salivary gland acini and should be addressed with additional studies or, at a minimum, a more thorough discussion. 6. Lines 231-240: Historically, RNAi has had difficulties penetrating through the basement membrane of tick salivary glands and has been shown to be marginally effective at reducing mRNA constructs. Therefore, the assumption that the inability to reduce Na+-K+-ATPase mRNA due to a long half-life is a large assumption and should be softened. Also, it would be of great benefit to include a figure showing the RNAi knockdown in the synganglion lading to reduced feeding.

You are one of the reviewers, your task is to write a review for the article. You will 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: WATER ABSORPTION THROUGH SALIVARY GLAND TYPE I ACINI IN THE BLACKLEGGED TICK, IXODES SCAPULARIS Review round: 1 Reviewer: 2

Basic reporting: The manuscript by Kim et al., "Water absorption through salivary gland type I acini in the blacklegged tick, Ixodes scapularis" is well written and addresses an important topic, water balance in ticks. This work builds on these authors previous studies. However there are concerns to this current version. The general concern is that its unclear in this manuscript why this was conducted. Authors should clearly state the objectives of this study, how data in the manuscript accomplished these objectives. What is the implication or contribution of these data to tick physiology? Some specific objectives include 1. Explain the formula that was used to calculate the volume of water that was imbibed by ticks. 2. Clearly state controls 3. Results section lines 195-199 did not make sense, what does natural behaviors in drinking water? Experimental design: Experiments not well described, please explain your controls Validity of the findings: Difficult to determine in absence of well explained controls Additional comments: Explain objectives and how data here met those objectives

You are one of the reviewers, your task is to write a review for the article. You will 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: WATER ABSORPTION THROUGH SALIVARY GLAND TYPE I ACINI IN THE BLACKLEGGED TICK, IXODES SCAPULARIS Review round: 1 Reviewer: 3

Basic reporting: no comment Experimental design: This research is properly designed and described Validity of the findings: Conclusions are well stated otherwise I have no comments Additional comments: An important contribution to the literature on salivary glands of ixodid ticks. The only error I could find is the incorrect authorship for a cited paper which should read as follows: Needham G, Rosell R. Greenwald L. and Coons, LB

You are one of the reviewers, your task is to write a review for the article. You will 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: LAND SNAILS OF LEPTOPOMA PFEIFFER, 1847 IN SABAH, NORTHERN BORNEO (CAENOGASTROPODA: CYCLOPHORIDAE): AN ANALYSIS OF MOLECULAR PHYLOGENY AND GEOGRAPHICAL VARIATIONS IN SHELL FORM Review round: 1 Reviewer: 1

Basic reporting: This preliminary study of Leptopoma from Sabah is interesting and important because: a) because studies on the molecular systematics of Cyclophoroideans and the Familt Cyclophoridae are few and far between and this study is the first of its kind for the genus Leptopoma and b) because it seeks to explore the utility of traditional shell-based taxonomic characters in a quantitative way. Overall it meets all 5 of the basic reporting requirements, but I recommend that the following changes should be made: - The sampling for the molecular component of their study is limited so they need to state in the discussion that this is a preliminary study looking at a small individuals at a small number of sites and adapt their discussion accordingly - e.g. clearly highlight the localities sampled in the phylogeny on the map in Figure 1 so that readers can differentiate those localities from all other sites. - Avoid repetition – don’t repeat what has been said in the introduction in the discussion and conclusions - Standardise and explain/refine terminology and use it in a consistent way. - e.g. Explain what is meant by shell form right at the beginning of the article by stating that it includes all shell morphological characters (shape, colour pattern, ornamentation). Actually, I would avoid using the word ‘form’ in this context because it often implies shape/size or colour pattern. Instead I would use shell morphology here and use form in the context of the 8 colour patterns - you can refer to these as colour forms. In this way you avoid confusion with your other use of shell colour pattern (i.e. one of the 3 characters used to define the 8 colour forms) - e.g. replace the term ‘apertural ring band’ with ‘apertural ring’ or ‘apertural band’ - e.g. Don’t use alternative terms such as characters/traits – stick to one option – seeing as this is a taxonomic paper I would use characters and drop the use of traits. Experimental design: Overall it meets all 5 of the experimental design requirements Obviously a lot of thought has gone into the experimental design and an attempt has been made to use some very recently developed tools/ approaches – that’s very commendable as is the effort that has been made to explore morphological variation in a quantitative way – the authors are really trying to tackle a difficult problem boldly. That said, they shouldn’t overemphasise the broader relevance of their findings, but more on this below. Validity of the findings: While the authors’ data/results do support their conclusions, they shouldn’t overemphasise the generality of their results. There are three issues here: 1) Having only sampled 4 of the c. 100 species in the genus they shouldn’t conclude that their results are generally applicable to the whole genus. Clearly their findings indicate that there may be a wider problem, but this issue has still to be investigated. 2) Nowhere is it made clear what the global ranges of the 4 Leptoma species. Are these species endemic to Sabah? Or are these species more widely distributed? I think it is important to provide a little information on this in the methods or even in the intro. e.g. Species X has a range encompassing the Malay Peninsula, and the islands of Borneo and Sumatra. Provide references as appropriate/if available. If most of what is currently known is largely based on the BORNEENSIS dataset the authors should make this clear. 3) As with 2), it is not clear how representative this study is in terms of the overall species richness of Leptopoma in Sabah. What proportion of Sabah’s Leptoma species are included in this study? In the discussion the authors need to briefly lay out what they would do to improve the design so that they can improve their understanding of the Sabah Leptopoma and broaden the relevance of their findings e.g. improved sampling from across the ranges of the 4 species, improved population sampling. Additional comments: I have made lots of suggestions directly on the pdf of the MS - please have alook at these. Please also re-write the abstract to reflect the changes I have suggested. Make it clear in the abstract that the study is based on a new/recently gnerated datatset, that your study deals with the taxonomy of 3 species in Sabah and that your findings necessarly reflect this - i.e. avoid extrapolating from your study to the whole genus. I look forwad to seeing more from the authors on this very interesting and intriguing system and it is great that they are trying to quantify shell characters even though it is an extremely challenging thing to do.

You are one of the reviewers, your task is to write a review for the article. You will 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: LAND SNAILS OF LEPTOPOMA PFEIFFER, 1847 IN SABAH, NORTHERN BORNEO (CAENOGASTROPODA: CYCLOPHORIDAE): AN ANALYSIS OF MOLECULAR PHYLOGENY AND GEOGRAPHICAL VARIATIONS IN SHELL FORM Review round: 1 Reviewer: 2

Basic reporting: Species delimitation is known to be problematic in the genus Leptopoma, which is traditionally classified based solely on shell morphology. This is an interesting study to investigate the molecular phylogeny of similar-looking species in Sabah, and examines the reliability and suitability of some conchological characters for species delimitation in two polymorphic species that occur sympatrically. The figures and tables are clear, and as a whole, the manuscript is well structured. Nevertheless a slight change in the title is suggested because there is insufficient evidence to suggest that the variations are due to geography. Experimental design: Adequate and methodical. Validity of the findings: The findings are sound. The unreliability of certain characters, notably shell colour and patterns, used in shell morphology based taxonomy are well elucidated by the results. Additional comments: I suggest that the introduction include a little explanation regarding the presence of a “dark ring band” in the aperture of some specimens of both Leptopoma pellucidum and L. sericatum. It is not apparent whether this character is only found in the Kinabatangan population and nowhere else. Is this found only in gerontic shells with a much thickened outer lip? As the authors have mentioned in the discussion, this interesting character has not been noted in other works. Hence some details will be informative. The problem of species delimitation with intermediate shell forms has not been properly treated? Were intermediates sequenced or included in this study? I would be interested to know the status of these intermediates. Most experienced taxonomists are well aware of the limitations of using shell colour and patterns for species identification. For example many fossils and subfossils, especially of extant species, without discernible colour can usually be quite easily identified by the relevant experts. Students however, often use colour and patterns in their initial attempts at classification. Perhaps a sentence could be added in the conclusion to emphasize the reliability of the ‘hard’ characters such as shell surface sculpture, spire profile, and shape, over colour and patterns. Overall, the MS is clear and well written. However the language still requires a bit of attention. This is nevertheless a very minor issue and does not affect the quality of the study. I have made some slight edits on the marked PDF attached.

You are one of the reviewers, your task is to write a review for the article. You will 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: PROTEOMICS RESEARCH AND RELATED FUNCTIONAL CLASSIFICATION OF LIQUID SCLEROTIAL EXUDATES OF SCLEROTINIA GINSENG Review round: 1 Reviewer: 1

Basic reporting: I found that the manuscript had a large number of sentences that need to be rewritten in clear and unambiguous English, and would recommend that the authors get some help in writing the manuscript. Some of the sentences that I found to be unclear can be found on lines 38, 121,123, 150, 174, 190, 276, and 290. I found that the materials and methods section was very difficulty to follow. In multiple instances important information was lacking. For example, it's unclear whether multiple gel slices were used for the proteomic analysis or if the entire protein mixture was included in a single analysis. Items like the composition of Buffer B (line 150) or what's in the solutions 1, 2 and extraction buffer (lines 139 - 145) need to be added. To summarize, the entire materials and methods section needs to be rewritten. The authors did not include all of the references from the text in the References. For example they cite an article from Georgios et al, 2012 (lines 254 and 315) on Neurospora chitinases, and don't include the reference in their references cited. In line 315 the reference is given as evidence that the chitinases are used during sclerotia formation. While I don't doubt that chitinases are expressed during sclerotia formation, the reference is for Neurospora crassa, which does not make sclerotia - the reference doesn't apply to the statement being made and should be deleted from line 315. I would also strongly request that the authors include a figure of the gel electrophoresis. This is a vital piece of data that needs to be included. Another very important element that is lacking is a much more detailed analysis and discussion of how their results for S. ginseng exudates compares to analysis of Liang et al on the proteins in S. sclerotiorum exudates. The authors practically ignore the excellent proteomic analysis for S. sclerotiorum exudates even though they are using the S. sclerotiorum genome as their basis for identifying their proteins. A close examination of the Liang et al paper will be instructive to the authors on how they need to present their findings. My examination of their findings would indicate they haven't found anything new or novel. I also think that the authors need to re-examine their discussion section and the references they are using to make sure they more accurately present the research literature and how the findings add to that literature. For example, the work of Colotelo and it's relationship to their work would be a good topic to discuss. Experimental design: It is difficult to assess the experimental design for the proteomic analysis because the materials and methods section lacks key information about how the experiments were conducted. The basic approach is sound and the type of information they obtained seems to be what one would expect. The research question is well defined but a more rigorous comparison with what is known about exudates in other Sclerotinia species would be helpful. One major flaw in the analysis is that the S. ginseng genome is not available and so the S. sclerotiourm data-base was used. Although I understand that they couldn't use the data-base for the organism being studied, the problems with using another data-base are not discussed or addressed in any way. Validity of the findings: The data does seem to be robust and statistically sound. Additional comments: Although I think a proteomic analysis of Sclerotina exudates is a good research project, I can not recommend that the article be published. I would recommend a total rewriting of the article with the inclusion of key information that is currently lacking (like the gel picture, and a comparison to other exudates). I think the materials and methods section needs to be completely rewritten to include a clearer narrative of how the experiments were performed. The current discussion section is lacking key analyses. As mentioned above, the Liang et al article covers the same type of analysis and would be a good model for the authors to follow.

You are one of the reviewers, your task is to write a review for the article. You will 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: PROTEOMICS RESEARCH AND RELATED FUNCTIONAL CLASSIFICATION OF LIQUID SCLEROTIAL EXUDATES OF SCLEROTINIA GINSENG Review round: 1 Reviewer: 2

Basic reporting: The article is sufficiently concise and well-written, and provides necessary background an experimental details. The raw data has not been shared. At a minimum, the peptide sequences should be made available for the matched peptides. Experimental design: Experimental design is acceptable. Although there are no independent replicates, the objective is merely to identify proteins present, not quantify them. Exudates from >600 plates were collected, so this provides some pseudoreplication that is sufficient for this purpose. Validity of the findings: Findings are valid and sound, except as noted in the General comments for the author (below). Additional comments: If the journal format allows it, the Results & Discussion sections should be combined. In any case, most of the Discussion is not relevant, and does not draw on or reference the Results. For example, lines 300-311 (about the droplet morphology) are in fact new results that were not presented in the results section. Regardless, the information lines 300-311 is not particularly useful or relevant, or well-supported and thus should be removed. Lines 287-293 provide very general information about proteins and could also be deleted. A list of peptide sequence for the identified peptides should be provided, if not the raw spectra.

You are one of the reviewers, your task is to write a review for the article. You will 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: PROTEOMICS RESEARCH AND RELATED FUNCTIONAL CLASSIFICATION OF LIQUID SCLEROTIAL EXUDATES OF SCLEROTINIA GINSENG Review round: 2 Reviewer: 1

Basic reporting: The revised manuscript has been greatly improved. I find the English and the referencing of the research literature to be acceptable. Experimental design: The revised Material and Methods section has been rewritten and is much improved. The only comment I have is that the authors used a Bradford assay to determine the protein concentration in their concentrated exudate sample (line 118), which contained 2% SDS. Generally the Bradford is not used for protein determination with SDS containing samples since SDS affects the assay. However, they might have used the Bradford assay and included SDS in their standard curve. I would simply ask the authors to double check whether they used the Bradford or some other assay for protein determination and if the Bradford assay was done and the standard curve was generated in an SDS solution to state that this was the case. Validity of the findings: The data presented clearly identifies a number of proteins in the S. ginseng exudate. Since a similar study with S. sclerotiorum exudates has been done, I do not see their findings as being especially novel, but their report does add to the information about fungal exudates. Their conclusions are well stated and supported. Additional comments: The manuscript has been substantially improved by the rewriting process.

You are one of the reviewers, your task is to write a review for the article. You will 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: PROTEOMICS RESEARCH AND RELATED FUNCTIONAL CLASSIFICATION OF LIQUID SCLEROTIAL EXUDATES OF SCLEROTINIA GINSENG Review round: 2 Reviewer: 2

Basic reporting: Revised manuscript is now acceptable. Experimental design: Revised manuscript is now acceptable. Validity of the findings: Revised manuscript is now acceptable. 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: RECORD BREAKING ACHIEVEMENTS BY SPIDERS AND THE SCIENTISTS WHO STUDY THEM Review round: 1 Reviewer: 1

Basic reporting: Overall, the paper is clearly innovative and has the potential to provide valuable contribution to the field of arachnology. However I believe that its main focus can become ambiguous, since some of central terminology used can be misinterpreted. Particularly the term "record", which in arachnological research is often linked to geographical occurrences of species. (eg. Estol, N. and RODRIGUES, E.N.L., 2017. A new species and a new RECORD of the spider genus Nesticus (Araneae, Nesticidae) from southern Brazil. Zootaxa, 4231(4), pp.564-566. Malamel, J.J. and Ambalaparambil, S., 2017. First RECORD of Epidius parvati Benjamin, 2000 (Araneae: Thomisidae) from Pathiramanal Island, India. Check List, 13(3), p.2114. DEMİR, H., SEYYAR, O. and NAJİM, S.A., 2017. Thomisus citrinellus Simon 1875 is a new RECORD (Araneae: Thomisidae) for spider fauna of Iraq. ) This does not mean the title should loose it's outreach message, or its focus towards reaching and engaging with a broader audience. In fact the title can be rewritten to reinforce this objective, while making it clear what "record" means in this context (eg. Record breaking achievements by some of the world's smallest champions: A scientific frame work for the first database of World Record Holding Spiders). The title section "a resource for using organismal biology as a hook for science learning" is neither intuitive to understand (e.g. organismal biology), nor a part of the publication analysis. I don't disagree with the statement, and potential areas towards which the publication can be impactful should be mentioned, but since they were not a part of the analysis, it is my personal opinion that they should not be referred in the title. Literature references provide a good background in the context of this research, but were neither exhaustive, not systematic. It is perhaps impossible, or unfeasible to produce on a comprehensive scale, but it would be clearly valuable to produce a systematic one (I mention this approach in more detail at the "validity of the findings" section). Experimental design: The primary research fits well within the area of "Literature Review Articles"and this was how I accessed it's scientific and methodological merit. The work is relevant and meaningful, but I don't believe the research question was well defined, and it doesn't state clearly how it fills and identifies knowledge gaps. I believe this is mostly due to to a lack of a rigorous approach, and a lack of information on how to replicate the study. Although this was not (neither can it realistically be) a comprehensive review of spider record holding achievements published in the literature, it is my opinion that the authors should: a) have built a structured scientific framework for the set of traits that can be measured or analysed in a standard way, and b) attempt to produce a comprehensive or representative systematic bibliographic review. An approach which would not only enhance the manuscript but also allow for it's reproducibility. Therefore, I would strongly encourage the authors to use their "Brief Introduction to Spiders", as base point to define which metrics can be considered for record achievement (including the ones which were considered but no scientific data was found). Generating a framework that methodically revises the current knowledge and pinpoints unstudied fields of research, allowing other arachnologists to built upon it. In practice, I am suggesting revising the proposed "four general categories" in a methodical manner. (E.g. Category II - Morphology Section a - Prossoma Characters 1 - external; Traits: Overall traits (biggest/smallest; proportionally larger/smaller; flattest/tallest carapace, most/least colourful...) Eyes (largest/smallest; proportionally larger/smaller...; for best eyesight see cat.III physiology) Chelicerae/fangs (longest/shortest; proportionally larger/smaller; wider/thinner...; for venom toxicity or wider opening angle see cat.III physiology; for quicker closing time see cat. IV behaviour) Ornaments (hairiest/baldest; most cuticular spines - Aphantauchilus... Characters 2 - internal; Traits: Venom gland (smallest/biggest; proportionally larger/smaller; etc...; for venom toxicity see cat.III physiology) Brain (smallest/biggest; proportionally larger/smaller; ... for best memory see Portia fimbriata cat IV behaviour) Section b - Opistossoma Characters 1 - external; Traits: etc... ) This sort of methodically approach (eg. comprehensively listing all morphological traits that can be feasibly measured) would facilitate the analysis of the database, and the detection of unstudied/unrecorded areas, and when broadly applied to all the categories explored would not only include more physiological traits (eg. longest/largest/smallest male/female reproductive organs), but also: - more environmental features (e.g. the hottest/coldest habitat dweller); - more behavioural traits (eg. the loudest spider, see Allard, 1936, Davis, 1904; Edwards, 1981, Lahee, 1904; Prell, 1916; Rovner, 1980; Uetz& Stratton, 1982); - physiological achievements (eg. longest period under freezing temperatures, longest time under water, see Pétillon et al. 2009) - more behavioural achievements (eg. organic projectiles other than silk, since spiders are able to project venom - Scytodes, or excrete liquid from their anus - Hummidia, to considerable distances); - other traits (eg. best body mimic: of leafs -Arachnura, of toads - Poecilopachys australasia; of ants - Aphantauchilus; most realist body double - Cyclosa sp.; etc...) All of which clearly fit the publication premise but were not assessed, potentially because these traits were not researched methodically, but rather collected out of availability. No such list can ever be truly comprehensive, but I believe this approach, even if it generates multiple unknown record holding positions, would clearly allow for gaps to be made clearer, and incentivise other to either provide information about those gaps, or research those fields. Another way to tackle large data gaps, might also be the analysis of local record holders (eg. the most venomous spider in Europe, or the most venomous in the US). Besides producing a rigorous analysis of traits, I would also suggest the authors to perform a systematic bibliographic review on all, or at least on one of the category keywords (as a sampled approach). Using a reference-based search strategy, which would be both reproducible and insure that all the bibliography currently available (in the selected database) was comprehensively revised. As a guideline I recommend analysing the methods used in: Savilaakso, S., Garcia, C., Garcia-Ulloa, J., Ghazoul, J., Groom, M., Guariguata, M.R., Laumonier, Y., Nasi, R., Petrokofsky, G., Snaddon, J. and Zrust, M., 2014. Systematic review of effects on biodiversity from oil palm production. Environmental Evidence, 3(1), p.4. Reed, J., van Vianen, J., Foli, S., Clendenning, J., Yang, K., MacDonald, M., Petrokofsky, G., Padoch, C. and Sunderland, T., 2017. Trees for life: the ecosystem service contribution of trees to food production and livelihoods in the tropics. Forest Policy and Economics. Validity of the findings: As it stands the results can be considered inconclusive, however the suggestions made above should allow the publication to obtain robust data that provides statistically sound conclusions on which specific areas or research fields have been the most, and the least investigated in arachnological literature. 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: RECORD BREAKING ACHIEVEMENTS BY SPIDERS AND THE SCIENTISTS WHO STUDY THEM Review round: 1 Reviewer: 2

Basic reporting: This paper does not necessarily take a form of hypothesis-test setting. Various interesting records about spider are presented, but these records are not related to each other, and are not well integrated in discussion. Therefore, I feel that this paper was somewhat descriptive and distracting. In review thesis, the authors should find new perspectives from heterogeneous findings and phenomena. Experimental design: No comment. Validity of the findings: This is a very useful review that allows ordinary people to access interesting records of spiders. However, as it is a list of known facts, it seemed that there is lack of heuristic elements. I felt that it is necessary to find synergistic findings obtained by gathering various records of spiders. As the authors say, it would be better to provide examples of creative discussion (L.708-709). Additional comments: This paper is an article that summarizes interesting records of spiders with World Record format, based on academically correct information to remove the misunderstanding concerning spiders. Because it is an unprecedented type of paper which is different from the typical review article, it is very difficult to evaluate, but I felt that it could be useful literature to inform the spider world. On the other hand, from the viewpoint of academic novelty, I felt this paper is somewhat descriptive, and the new perspective obtained by gathering many records is unclear. Also, from the viewpoint of generality, since this paper only focuses on the spider topic, I got a somewhat specific impression. In order to emphasize “spider’s greatness”, I think that it would be better to have a comparison with world records of other taxonomic group such as insects. I think that this paper does not necessarily need to be published in the general science journal, and recommend submission in another journal dealing more specifically with entomological or arachnological issues.

You are one of the reviewers, your task is to write a review for the article. You will 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: RECORD BREAKING ACHIEVEMENTS BY SPIDERS AND THE SCIENTISTS WHO STUDY THEM Review round: 1 Reviewer: 3

Basic reporting: Review of Peer J #18552 This is an interesting paper that brings together information about spider biology and taxonomy with the intend to inspire the public and educators to engage with the natural world. I agree that spiders have a privileged position as most people have some opinion about spiders and often react positively when provided with more information. Overall, I believe this paper will make an interesting contribution and only have few comments to make about its readability. Writing style – overall the paper is very well written and entertaining to read. My comments are: The terminology used throughout the paper is confusing and makes it unclear who the intended readership is. Is it biologists? Is it lay-people? If it is the later, then terms need to be explained. Throughout (e.g. 60, 62) phrases are placed in parentheses, but they do not have to. 75-76: sentence too long and convoluted – please rephrase Overall the introduction is quite lengthy and could be shortened and sharpened. Eg. Is 87-92 necessary? 106: Here we build on records from the scientific… 140: The body of a spider is divided…. 140 and onwards – please refer to Fig 1 146 & 147 & 149: ‘behind’…some of the terminology used in this paper is quite sophisticated, so I do not see why the term ‘posterior’ cannot be used instead of ‘behind’ 152: from the ophistosoma 156: terminology already explained earlier 162: remove ‘so called’ 163: explain terminology earlier 174: why what? 177: use the term ‘collaboratively’ 178-181: repetitive 187-190: repetitive 223: First and last listed spider 238: what do the terms in the parentheses mean? 237-239: sentence complex and convoluted, please rephrase 244-245: please explain these obscure terms (nomen dubia; synonymized) 313: explain GB 340: explain holotype 375: the least number… 376: explain anophthalmic 384: capable of spinning… 386: ‘dimension’ – do you mean ‘diameter’? 394: explain MJ 397: explain GPa 418: why ‘sic’ 419: explain hypogean 426: greatest longevity 482: in what year? 547: what do you mean by conscious? 581: a small number… 620: plant products 688: I found the discussion quite repetitive, with arguments that have already been presented in earlier sections. Rather than repeat this information, maybe the discussion could provide some examples of how educators might utilise this information Experimental design: no applicable Validity of the findings: not applicable Additional comments: The idea of this paper is great, I really like it and it almost works, but for the Discussion which needs some work.

You are one of the reviewers, your task is to write a review for the article. You will 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: RECORD BREAKING ACHIEVEMENTS BY SPIDERS AND THE SCIENTISTS WHO STUDY THEM Review round: 2 Reviewer: 1

Basic reporting: The revision of the Discussion section by the authors seems to solve the problems I was concered about in the previous review. English expression and cited references are also appropriate. Experimental design: no comment Validity of the findings: I think that the purpose and perspective of this paper became clear by the revision of the Discussion section. No problem. Additional comments: In the previous review, I was concerned about whether this MS fits the concept of the "PeerJ", but I changed my mind that there is no problem with this point after reading the author's response. In my country, there were no such literature which summarizes interesting records of spiders, so it seems very useful for widely communicating the attractiveness of spiders to people. Additionally, I think that this review is meaningful in presenting a new framework of the review paper. I judge that this paper is worth publishing.

You are one of the reviewers, your task is to write a review for the article. You will 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: RECORD BREAKING ACHIEVEMENTS BY SPIDERS AND THE SCIENTISTS WHO STUDY THEM Review round: 2 Reviewer: 2

Basic reporting: Review Peerj-18552 I have seen the original version of this manuscript and the authors have addressed my concerns. In particular, the discussion is much improved. I only have picked up a few minor items. Line 61-62: remove – unsubstantiated claim 333: the term ‘orb-web spider’ is more accurate. Spiders don’t weave 361: are fangs different from chelicerae? Please check terminology 402: so there are no species that have lost the venom gland? Can you clarify this point? See line 454 466: there are plenty of original papers that attest to the visual acuity of jumping spiders (e.g. work by Zurek) 499: eggs and sperm Experimental design: no applicable Validity of the findings: not applicable Additional comments: Review Peerj-18552 I have seen the original version of this manuscript and the authors have addressed my concerns. In particular, the discussion is much improved. I only have picked up a few minor items. Line 61-62: remove – unsubstantiated claim 333: the term ‘orb-web spider’ is more accurate. Spiders don’t weave 361: are fangs different from chelicerae? Please check terminology 402: so there are no species that have lost the venom gland? Can you clarify this point? See line 454 466: there are plenty of original papers that attest to the visual acuity of jumping spiders (e.g. work by Zurek) 499: eggs and sperm

You are one of the reviewers, your task is to write a review for the article. You will 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: COMPARATIVE GENOMIC ANALYSIS OF THE PKS GENES IN FIVE SPECIES AND EXPRESSION ANALYSIS IN UPLAND COTTON Review round: 1 Reviewer: 1

Basic reporting: The MS (##18119) reported the PKS genes and its expression profile in upland cotton. The results are overall interesting and could be published. However, the MS is poor prepared. Please see my comments and suggestions below! Overall, I think there are three major problems should be addressed. 1. The PKS genes were found in many plants. why are the PKS genes selected from these species (Populus tremula, Vitis vinifera, Malus domestica, and Arabidopsis thaliana) compared with cotton PKS genes? 2. Most figures are too small to see clearly. 3. There are many mistakes in the MS, including abbreviations, spaces between words, singular and plural, etc. The English language should be improved. I suggest that you have a native English speaker review your manuscript. 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: COMPARATIVE GENOMIC ANALYSIS OF THE PKS GENES IN FIVE SPECIES AND EXPRESSION ANALYSIS IN UPLAND COTTON Review round: 1 Reviewer: 2

Basic reporting: The manuscript needs extensive editing to improve its language and grammar. For example, the first abstract seems to be less well edited and states “Plant type III polyketide synthase can catalyze into a series of secondary metabolites …”. I believe the authors meant to write that PKS can catalyze reactions forming secondary metabolites. Line 25 should read “ … candidate genes involved in brown cotton fiber synthesis …” rather than ““ … candidate genes involving in brown cotton fiber synthesis …”. I think that the column heading in Table S2 should read “No. of exons” rather than “No. of extrons”. There are other sentences throughout the manuscript that need to be edited. The introduction should lead the reader to understand why the problem is important and why certain genes are studied. The article aims to study the plant polyketide synthase genes in upland cotton and compare them to other plant species. This is a valid and appropriate question but the introduction could be improved to state any specific and additional reasons as to why this question is important. For example, is the pigmentation of brown cotton fibers of great commercial importance because it commands a higher price? Is the study of PKS genes in upland cotton also important because PKS genes might be implicated in stress response and tolerance, thus helping to produce sturdier versions of a very widely grown crop? All acronyms should first be introduced with their full names and then used thereafter. For example, CHS is first used in line 41, and the reader is only introduced to the full term in line 60. Also, it is initially unclear why CHS is discussed until about lines 70-80 where it is explained that CHS is an important PKS gene involved in many processes including the regulation of pigmentation. All previous results and data mentioned in the manuscript should refer to the appropriate publications. For example, line 83 mentions that the upland cotton genome has been sequenced and assembled but no citation is given. In addition, appropriate scientific names/accession names of the upland cotton and brown cotton lines. All the software tools mentioned such as DNATOOLS, MEGA, Plantcare etc. should also be accompanied by the citations of any respective publications. For some of the previous results mentioned, it is not clear which publication is the relevant citation. For example, two papers are simultaneously cited in line 197, but it is not clear whether they both mention the increase in chalcone synthase activity or if one of them is about the four PKS genes mentioned next. Some of the figures are too small to be legible. Specifically, Figures 4, 5, and S1 are not legible in the current resolution and size. Line 342 mentions Li T 2012 but this publication is not listed in the references. On line 411, Cao et al., 2016 is cited for the discussion of comparative tandem duplication and fragment duplication evolution rates. Is this the first or most prominent publication finding this result? Figures 5 and 6 represent the experimental data generated in this manuscript, and the raw data for these figures has been provided. Experimental design: I do not have any major objection to the scientific validity of the methodology of the experiments and the bioinformatics analyses carried out by the authors. This review is not supposed to judge the impact or novelty of the results and findings. However, it will be very useful to the reader that the results presented here are discussed to illuminate the broader scientific questions they answer or raise. For example, line 234 makes a broad statement that the different motifs found in the different sub-families may be important for the different functional roles performed by the proteins. This seems like a truism, and I suggest the authors try to discuss whether anything is known about the functional role of the motifs they find in the PKS genes studied. Currently, all that the manuscript mentions is that the motif annotations were taken from SMART and Pfam databases. The division into general and specific motifs is due to their abundance in the genes studied, but Cao YP, 2016. I believe the same paper is elsewhere cited as Cao et al., 2016. Is this the same paper? Why is this relevant to the differentiation between general and specific motifs? The method used to differentiate between tandem and fragment duplication should be clearly mentioned. The authors discuss the relevance of the Phe sites in the PKS genes, and line 258-260 mentions that these sites are related to the decarboxylation reaction of malonyl CoA. Please provide a reference for this statement. Subsequently, it is mentioned that the second Phe is substituted by Tyr in certain genes and Thr, Gly, and Ser are also replaced in some genes. For these and other substitution sites, is it possible to differentiate between highly conserved sites, background drift, and positive selection? Can the authors calculate site-specific substitution rates or use some other appropriate method to comment on this? Validity of the findings: As mentioned earlier, I would strongly suggest the authors to discuss the broader scientific context of the specific and quantitative results presented. For example, what is the importance of studying the chromosomal locations of the PKS genes? Is this used to identify the genes as the result of tandem and fragment duplication? I do not have any major objection to the data quality and the conclusions drawn. Additional comments: Overall, the manuscript uses appropriate experimental and analytical tools for the questions it studies. The study of genes involved in pigmentation in brown cotton fibers and the PKS genes in upland cotton have scientific and commercial importance. The manuscript needs major editing for language and presentation. In addition to correcting grammar, some paragraphs, especially in the introduction should be rearranged for better logical flow. This will help the reader and also allow for a better peer review once any ambiguities and distracting language problems are removed. I have suggested one additional analysis: assigning the site substitutions discussed in the manuscript as conservatory, neutral or positively selected. If the authors feel this should not be done, please mention why it is so.

You are one of the reviewers, your task is to write a review for the article. You will 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: COMPARATIVE GENOMIC ANALYSIS OF THE PKS GENES IN FIVE SPECIES AND EXPRESSION ANALYSIS IN UPLAND COTTON Review round: 1 Reviewer: 3

Basic reporting: The paper has a clear structure, sufficient field background, and professional figures and tables. 1. The English writing needs to improve. For example, line 222-224, "in addition to GhPKS3 lack of motifs6,7 and GhPKS1 lack of motifs6 almost all PKS family members include motifs1,2,3,4,5,6,7 these 7 motifs." in addition to sth -> in spite of sth. line 224-225, two predicates in "this is less Chal_sti_synt_N and Chal_sti_synt_C domain part of the phenomenon is very obvious." line 272-273, no predicate in "In upland cotton, PKS gene distribution in A2_chr6 273 (1), A2_chr8 (2), A2_chr9 (1), At_chr11 (1), Dt_chr8 (1), Dt_chr10 (1), Dt_chr11 (4)." 2. For some unknown reason, in many sentences, two individual words are concatenated without spaces in between, making the sentences difficult to understand. For example, line 37, PKSIand PKSIIonly -> PKS I and PKS II only line 41, for examplechalcone -> for example, chalcone, line 110, domainswas -> domains was line 116, weightof -> weight of line 113, thalianawere -> thaliana were line 139, databaseof -> database of line 187, anddivided -> and divided line 307, geneswere -> genes were line 318, functionand -> function and line 348, onlyexists -> only exists 3. Some abbreviations are not defined. For example, line 47, CHSL is not defined. line 81, Proanthocyanidins should occur before PA. Experimental design: The research fills the knowledge gap of the PKS genes in upland cotton. Some analysis needs to describe in details. 1. For example, it is not clear how did the authors derived the conclusion that no direct homologous genes between the species in line 200-201. Is there any p-value or hypothesis testing that support this conclusion? 2. The authors claimed that "gene duplication is the main driving force of the amplification of PKS III gene family" in line 447-449. However, it is not clear how the conclusion was derived based on the facts that (1) "10 gene replication events were identified in the 52 genes" in line 283, and (2) Ka/Ks<0.4 indicates negative selection in line 292. Is 10/52 significantly high? How is negative selection associated with amplification of PKS gene? Validity of the findings: In this paper, the authors identified 11 PKS genes in the genomes of upland cotton species. The 11 PKS genes in cotton were compared with PKS genes in other plants. The expression of the PKS genes was quantitatively measured at various growth stages and tissues. The data is solid and the analysis is sound. 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: COMPARATIVE GENOMIC ANALYSIS OF THE PKS GENES IN FIVE SPECIES AND EXPRESSION ANALYSIS IN UPLAND COTTON Review round: 2 Reviewer: 1

Basic reporting: The manuscript has been edited and the language has improved greatly. Line 19 reads “(a)ccording to phylogenetic tree, a total of 52 PKS genes …”, which should be “according to the phylogenetic tree, a total of 52 PKS genes …” or “according to the constructed phylogenetic tree, a total of 52 PKS genes …”. The supplementary tables S1-S5 reported in the Excel file do not have column headers. On line 333 it says“(f)our pairs of duplicated genes belonged to tandem duplication and seven pairs of duplicated genes belonged to fragment duplication”. I understand that these genes can be identified from the figure showing the chromosomal locations of the genes, but it would be helpful if a table lists which genes result from tandem duplication and which genes result from fragment duplication. Experimental design: Concerns raised in the review have been addressed. Validity of the findings: Concerns raised in the review have been addressed. 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: EARLY SKIN-TO-SKIN CONTACT BETWEEN HEALTHY LATE PRETERM INFANTS AND THEIR PARENTS: AN OBSERVATIONAL COHORT STUDY Review round: 1 Reviewer: 1

Basic reporting: please see attached report. Experimental design: Please see attached report. Validity of the findings: please see attached report 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: EARLY SKIN-TO-SKIN CONTACT BETWEEN HEALTHY LATE PRETERM INFANTS AND THEIR PARENTS: AN OBSERVATIONAL COHORT STUDY Review round: 1 Reviewer: 2

Basic reporting: pass. see comments below Experimental design: pass see below Validity of the findings: pass see below Additional comments: Thank you for allowing me to review this very well written manuscript on a population that is often ignored but have significant morbidity. Some minor editing is needed. I have questions and concerns related to the retrospective nature of data collection during the first 24 hours. Additional information is needed regarding the retrospective data – how was it obtained, from where and by whom. It appears that you are comparing two different data sets (first 24 hours – retrospectively collected) and (2nd 24 hours prospectively collected by a parent diary). Please clarify and discuss what the problem with this may be (needs to be in the limitation section) and also included in the discussion section. The discussion also needs to include possible reasons why BW only a predictor for SKS care in mothers and fathers during the 2nd day as well as why primiparity may affect the amount of SSC the father participates in. Please include a limitation section which should included lack of data concerning other comorbidities that may affect lactation (ie pregnancy induced hypertension, diabetes etc) Clarify why you consider the sample size to be satisfactory (line 297). Was a power analysis performed? Line 28: I suggest using a different word than advice (perhaps recommend?) Line 74: add the word “during” after respectively. Line 79: I suggest using a more traditional description of the study design Line 82: it would be more appropriate to report the number of LPI infants born at this hospital Line 84: Define assistant nurse and midwifery care Line 88: who offered the individualized support? Line 91: please clarify whether placing infants STS immediately after birth was part of the research protocol or a standard of care. How did you verify this occurred? What if there were no father or partner? Was data collected regarding how soon after delivery SSC began? Line 103: I would change twins for multiples to include triplets ect Line 128-130: this is confusing and needs clarification Information regarding consent and approval by the ethical review board needs to be presented earlier in the methodology Line 151: define threatening fetal asphyxia Line 178 to 180 is confusing and needs to be rewritten for clarity Line 244: a definition of SUPC is needed Line 277: “facilitated” should be substituted for another word

You are one of the reviewers, your task is to write a review for the article. You will 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: EARLY SKIN-TO-SKIN CONTACT BETWEEN HEALTHY LATE PRETERM INFANTS AND THEIR PARENTS: AN OBSERVATIONAL COHORT STUDY Review round: 2 Reviewer: 1

Basic reporting: Thank you for the opportunity to review the manuscript entitled: Early skin-to-skin contact between healthy late preterm infants and their parents: An observational cohort study. I commend the authors for the improved clarity of the manuscript presented. Abstract Background; the term breastfeeding you could quantify this for the reader as you report exclusive breastfeeding in the conclusion. I perhaps would add : “its associations with breastfeeding (exclusive and any) …. Results section Abstract; I suggest that the authors: use confidence intervals (CI) and not p values in the abstract. If using p values report with up to 2 significant digits and a maximum of 3 ( p=0.12, p=0.001) see: Lydersen, S. (2014). Statistical review: frequently given comments. Annals of the rheumatic diseases.  Abbreviate the term standard deviation (SD). Results section Abstract: it is quite difficult to make sense of so I would suggest that it is rewritten perhaps something like this; Sixty-four mothers spent a mean 14.7 hours, 5.6, standard deviation (SD), during the first 24 hours after birth. While Fathers (n=64) experienced a mean of 9.2 hours 3.3 (SD) …. Conclusion: line 53. Replace the word ‘it’ with SSC Perhaps leave the reader with an important message about SSC and the timing of it and the importance of fathers and mothers both participating rather than restating the key results. What should be done next? Main text Line 211- 214 – reference needed for the statistical methods used line 215- please state if you adjusted for confounding factors/covariants and if so which ones and why? Results Tables: table 1 is not clear. I suggest you combine table 1 (demographics) with 2 and 3 respectively. This will make it easier for the reader. P values - report with up to 2 significant digits and a maximum of 3 (p=0.12, p=0.001) Day Variable n (%) Mean (SD) Unadjusted 95% CI Pvalue Full model 95% CI pvalue Trimmed 95% CI pvalue Conclusions A take home message is needed rather than just repeating the key results What are the implications to practice / policy Experimental design: no comment Validity of the findings: Please review the conclusion; a take home statement is needed what is the significance of the results -clinically and to policy? 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: EARLY SKIN-TO-SKIN CONTACT BETWEEN HEALTHY LATE PRETERM INFANTS AND THEIR PARENTS: AN OBSERVATIONAL COHORT STUDY Review round: 2 Reviewer: 2

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: Thank you for allowing me to review the revised version of this manuscript. The vast majority of the reviewer’s comments were incorporated into the manuscript. There continues to be minor editorial changes needed. Other comments include the following; Line 159-162 belongs in the introduction and not the methods section Substitute the word children for infants. The definitions of the demographic characteristics were defined previously in the methodology section and are redundant in the data analysis section. Line 249: fetal asphyxia during delivery should be changed to fetal asphyxia. I am assuming that cardiotocography is fetal heart rate monitoring and should be stated as such. I also assume fetal PH is umbilical pH and should be changed to make the terms more easily understood. Although use of a breast pump, cup, nipple shield were collected, they are not in the results section. Thus should be removed from the methods section. No need to repeat content found in the table in the text

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

Title: EARLY SKIN-TO-SKIN CONTACT BETWEEN HEALTHY LATE PRETERM INFANTS AND THEIR PARENTS: AN OBSERVATIONAL COHORT STUDY Review round: 3 Reviewer: 1

Basic reporting: generally clear however could be more succinct throughout. See comments in the document provided Needs to be edited carefully and consistent use of terms and abbreviations is needed. Experimental design: see comments in table provided Validity of the findings: low impact Additional comments: See table provided for specific 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: EARLY SKIN-TO-SKIN CONTACT BETWEEN HEALTHY LATE PRETERM INFANTS AND THEIR PARENTS: AN OBSERVATIONAL COHORT STUDY Review round: 3 Reviewer: 2

Basic reporting: No comment Experimental design: no comment Validity of the findings: no comment - these have been addressed in the limitation section Additional comments: Thank you for allowing me to again review this manuscript on a very important topic regarding the late preterm infant who as we know is a high risk of morbidity. Although there are still minor editorial errors that need correcting the authors and adequately and appropriately responded to the reviewers comments and concerns. I just had a few comments/questions 1. Line 144: was phototherapy an exclusion criteria (would be rare but if the infant had a hemolytic process?) 2. Line 239: “minus” is confusing 3. Line 370: ethnicity is also important to rate of breastfeeding

You are one of the reviewers, your task is to write a review for the article. You will 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: ESTIMATING THE POTENTIAL BIODIVERSITY IMPACT OF REDEVELOPING SMALL URBAN SPACES: THE NATURAL HISTORY MUSEUM’S GROUNDS Review round: 1 Reviewer: 1

Basic reporting: Clear, unambiguous, professional English language used throughout. This is an exemplary example of a clearly written, structured and produced piece of work. There are very few typographical errors, errors of omission or of expression/fact. I have annotated comments onto the PDF (attached to this review). Intro & background to show context. Literature well referenced & relevant. The introduction has a decent review of the literature but there are a couple of points that require some thought. Firstly, I am not persuaded that by the ‘neglected status’ of urban ecology (p.1, line 44ff). There has been a great deal of recent activity in this field. Perhaps rephrase to say just that. I think the lengthy diversion into the history of the grounds redesign at the NHM has too much detail; it might better sit in the methods of supplementary materials. It needs slimming down to about a third picking up just the salient points. The key point of the paper is that it introduces a means of assessing the impact of site-based developments, which is an area where little robust work exists (aside of classic impact assessments). This is the section that requires emphasising. Structure conforms to PeerJ standard, discipline norm, or improved for clarity. This is fine. Figures are relevant, high quality, well labelled & described. The Figures are nicely produced but would benefit from some modification: Fig 1: rotate by 90 degrees Fig. 2: needs scale and north arrow. Not all data sources are listed (in comparison to Github sources). Fig. 3: Maybe use a 2nd Y-axis to improve clarity and remove the grey text as you describe what it means in the figure caption. Fig. 5: A bit small to read easily on the screen - increase figure and font size. It is clear on retina-type displays but not everyone will have access to one of those. Raw data supplied (See PeerJ policy). Raw data, R code and latex all available on Github @: https://github.com/helenphillips/GroundsRenovation. The code indicates possible sources of problems but looks fine. I have not tried to emulate the analyses. Experimental design: The design is clearly outlined but there are a few areas where the paper would benefit from more methodological detail and support/justification (all minor stuff): 1. unpack what is meant by the dose-response modelling framework - p.4, line 138 2. I understand why going for a Species|Area-type model is sensible given what you are trying to achieve but lots of variables will impact species richness/density (e.g habitat structure, site context ie. situated in areas with lots of similar habitat or isolate, disturbance histories, time and the like) You allude to this at the end of the introduction but I think you need a develop this a little more and revisit to more directly as a critique in the discussion. I was a little puzzled by line 157 where you mention time limitations; what were they? I cannot see that in the discussion either. 3. Data collation: You need to document what data was extracted using ImageJ. 4. GLMM is a sensible approach but it's a small dataset for inference outside the realms of the study. Some justification of that is needed. 5. Your assumptions are sound given the data but why did you carry out sensitivity analysis on only one of the three? 6. Why were both the density and richness models simplified by removing the additive effects? Elsewhere you mention data paucity as a reason why interactions were not undertaken. Is that the reason? Validity of the findings: The authors have used an appropriate and robust approach to the analysis and they are careful not overextend the reach of their findings. I like the central idea of this paper as it allows an evaluation of outcomes of site developments thus chimes well with UK planning policy which is essentially site-based at the point of decision. The analytical model / approach I believe to be robust enough to be used elsewhere but it may need nuancing for each specific location. Some guidance on what form this might take in the discussion would be helpful. I think also that a fuller review of the issues surrounded a focus on area is needed as a critique in the discussion. 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: ESTIMATING THE POTENTIAL BIODIVERSITY IMPACT OF REDEVELOPING SMALL URBAN SPACES: THE NATURAL HISTORY MUSEUM’S GROUNDS Review round: 1 Reviewer: 2

Basic reporting: 57-109: This is far too much detail for a study of this type, strongly recommend cutting this down significantly. You are trying to argue that your study has value beyond being a case study, this much detail on the history and specific challenges of your site does not help make this argument. Experimental design: Line 190: I think this is a central limiting factor of your research. You used data from urban areas “when possible”, but we cannot tell when this was, and a huge wealth of literature tells us that relationships between biodiversity and habitat in urban areas are altogether different than in natural landscapes. This point needs to be made, and strongly. This is a major assumption that casts some doubt on your findings. Line 227: When scaling in this matter, you completely ignore edge effects, unless I am missing some part of your methods. Given how small your habitat patches are, it seems these effects may be extremely important. And generally, since you are using values from the literature that are collected across much, much wider scales, I don’t think the relationships are likely to scale down to the level of the patches you’re working on. Validity of the findings: The findings are valid, I think, but the inference space is extraordinarily narrow. You have applied a simulation modelling exercise to a small urban patch using non-urban data, and you haven’t verified it with any field sampling whatsoever. Is this a promising approach for other systems? It might be, though I have some grave concerns about edge effects and scaling as I describe previously. But to test that you’d need to validate it with field data, which this study fails to do. So what we have in the end is a simulation study, one that tries to use possibly unrelated data to predict biodiversity outcomes for land management on an extremely small scale, and finds those changes to be minimal for total species richness. It’s intuitively obvious that by adding new habitats, and high quality habitats, we would expect these values to go up. Of course, even species richness isn’t usually what we’re interested in, compared to things like diversity of rare species. The method has promise, but I’d like to see it explored over a much wider area, and with some field data to back it up. At any rate, I think you need to discuss these limitations more fully in the text—you are way out on a speculative limb, and I think the text needs to work harder to acknowledge that. Additional comments: Thank you for the opportunity to review this manuscript. It is very well written, and in general I applaud studies that investigate the relative value of small patches of urban habitat, which are often much more valuable than people realize. Technically the paper is sound, nothing was done that is misleading or that reaches erroneous conclusions. So for the purposes of this journal I suspect it is publishable. Unfortunately, I found the potential benefits of the paper for management or conservation lacking. This is a fairly simplistic modelling exercise, applied only to a single case site, that makes a variety of unrealistic assumptions to try to make a relatively simple final observation about how wildlife species might use habitat. I appreciate what the authors are trying to do, but I think the study needs to do at least one or two of the following to be of much use to anyone: 1. Apply their method to more sites 2. Validate their predictions with field data 3. Create a more complex model that incorporates things like edge effects, connectivity, and the like. I do realize that potential impact is not a determining factor for publication in this journal, so I bring this up largely out of completeness, and in the hopes that the authors would like their work to have a wider impact. I hope this review is useful to 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: MOLECULAR PHYLOGENY AND EVOLUTIONARY HISTORY OF MORICANDIA DC (BRASSICACEAE) Review round: 1 Reviewer: 1

Basic reporting: This manuscript explores the phylogenetic relationships of Moricandia species (seven spp.) Sampling seems to be adequate with several specimens per species and nuclear ITS and plastid trnT-F and ndhF sequences. Author also tested the position of the closely related Rytidocarpus moricandioides. Overall, analyses are adequate, and results obtained were interesting, revealing that M. foleyi is not included in Moricandia. Moricandia s.s. (without M.foleyi) is composed by two geographically structured clades, the African and the Iberian clades. Additionally, Iberian specimens of R. moricandioides are included in the Iberian clade, while African specimens (Rytidocarpus s.s) were placed sister to Moricandia. Result from divergence time analyses related divergence of Moricandia with the geologic history of the region; however I am not an expert on the geological evolution of this area and cannot give a robust evaluation of these findings. I think that this manuscript is adequate for its publication in PeerJ after applying minor changes (see comments for the author) Experimental design: no comment Validity of the findings: no comment Additional comments: This manuscript explores the phylogenetic relationships of Moricandia species (seven spp.) Sampling seems to be adequate with several specimens per species and nuclear ITS and plastid trnT-F and ndhF sequences. Author also tested the position of the closely related Rytidocarpus moricandioides. Overall, analyses are adequate, and results obtained were interesting, revealing that M. foleyi is not included in Moricandia. Moricandia s.s. (without M.foleyi) is composed by two geographically structured clades, the African and the Iberian clades. Additionally, Iberian specimens of R. moricandioides are included in the Iberian clade, while African specimens (Rytidocarpus s.s) were placed sister to Moricandia. Result from divergence time analyses related divergence of Moricandia with the geologic history of the region; however I am not an expert on the geological evolution of this area and cannot give a robust evaluation of these findings. I think that this manuscript is adequate for its publication in PeerJ after applying minor changes (see comments for the author) some suggestions/ corrections: Maybe it will be better if results from individual analyses of ITS and cpDNA are showed before the combined analyses Authors could indicate on Fig. 4 (the chronogram) the node used for the calibration based on the geological information (uniform 5.39-5.33 mya) I am not an expert in the tribe Brassiceae, but the authors transferred M. foleyi to the genus Eruca because this species resulted sister of Eruca. However phylogenetic analyses included only four Eruca species, Brassica rapa, and Raphanus sativus. Is this sampling adecuate? Perhaps, in analyses using a broader sampling of Brassiceae M. foleyi could be recovered related to other genus of the tribe. It would be interesting to analyze this dataset using species tree inference under the multispecies coalescent model Line 182: GTR+G instead GMT+G

You are one of the reviewers, your task is to write a review for the article. You will 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: MOLECULAR PHYLOGENY AND EVOLUTIONARY HISTORY OF MORICANDIA DC (BRASSICACEAE) Review round: 1 Reviewer: 2

Basic reporting: Francisco Perfectti et al. reported a research to reconstruct the phylogenetic relationships of Moricandia species using the traditionally marker genes of ITS and trnT-trnF. I think this paper is ok and I only have several following concerns. 1. To better present the results, the genus name and clades name of 'African clade' and 'Iberian calde' should be labeled on the Figure 1. 2. I can not understand 'The subspecies M. moricandioides pseudofoetida appeared inside the branch of M. moricandioides but well separated from the other M. moricandioides subspecies' on page 13 line 240-241. Actually, two M. moricandioides pseudofoetida species are sister group of other three M. moricandioides subspecies. 3. on page 13 line 245, the author should specify the exact samples name of the R. moricandioides. 4. The Fig. 4 should be Fig. 3 on page 14 line 271. Experimental design: 5. Normally, the support value of Bayesian tree pretend to be higher than ML tree. The author should not only label the support value of Bayesian tree, but also label the support value of ML tree on Figure 1 by only presenting the topology obtained by the Bayesian method. 6. The authors only listed the Bayesian trees of separated results on Figure 2. How about the ML tree of separated cpDNA and ITS? Validity of the findings: 7. The authors claimed that two Rytidocarpus moricandioides subspecies are located into the clade of Moricandia and is indeed a new Moricandia species. But how about the statistical possibilities of other topologies? I suggested the authors to do the AU-test of other potential topologists of Moricandia species using the same dataset as in Figure 1. Unless the result can reject the possibility that the Rytidocarpus moricandioides g01-13 and g01-14 are sister groups of all other species in the pink filled region, I can trust that these two species are actually new Moricandia species. Additional comments: no 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: MOLECULAR PHYLOGENY AND EVOLUTIONARY HISTORY OF MORICANDIA DC (BRASSICACEAE) Review round: 1 Reviewer: 3

Basic reporting: The paper provides some updates on the evolution and systematics of the genus Moricandia. Results and conclusions look reliable and the paper is in focus of the journal. On the other hand, the quality of the text is poor and needs substantial English edition (eg. p 6 line 98. Please replace "their" by "its"). I strongly suggest that you have a native English speaking colleague review your manuscript. Additionally some technical terms including bp (base pair) used differently as "nt" or autor use the terms its1, its2 (page 8 line 140) instad of ITS1, ITS2 (these are commen usage of these regions and also the name of the relevant primers). Materials used in this study look proper for such kind of phylogenetic studies as well as literature references. Experimental design: Design of study looks good and proper for filling the gaps regarding phylogeny and systematics of the Moricandia with the exception one analyses. For details, please see comment in "Validity of findings " section. Regarding model selection, i have never heard the GTM model that autors used in Bayesian analyses. Please check it. Validity of the findings: Data looks robust but i do not understand why Ancestral Area Recounstruction analyses did not include this comprehensive study. Without these analyses all discussion about origin of the Moricandia species will be speculative. 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: RESPIRATORY SYNCYTIAL VIRUS GENOTYPES NA1, ON1, AND BA9 ARE PREVALENT IN THAILAND, 2012–2015 Review round: 1 Reviewer: 1

Basic reporting: This manuscript provides additional information on RSV genotypes circulation in Southeast Asia Region. As the RSV molecular epidemiology data from developing countries especially from long term studies are still lacking, this manuscript contributes on the data needed to determine the global RSV circulation and transmission. . There are some drawbacks of this manuscript: 1. There are several grammatical errors and some sentences need to be reframing. The flow of idea of the sentences within the paragraphs and between the paragraphs are unclear. There are some repetitions of vocabulary used in the sentences. 2. The structure of the manuscript should be revised based on the PeerJ standards. 2.1 The abstract should be revised as it did not follow the PeerJ standard. 2.2 There is no conclusion paragraph. As the PeerJ standard, the author should add the conclusion section. 3. The title of this manuscript is too general. The authors may change it to the more specific title which related to the contents of the article. 4. Figures and tables: Figure 1 needs a revision (left vertical axis ?) Raw data is not provided. I am unable to verify the RSV sequences obtained from this study from NCBI website as they mentioned that these sequences have not yet been released. Experimental design: 1. The topic of this manuscript falls within the scope of the journal 2. Research questions should be more well-explained, as it seems inconsistent from the abstract, introduction and the discussion. 3. Methodology: 3.1. The author should clarify the methodology regarding the samples as in methods section (line 82) the samples were from a retrospective study while in the discussion (line 1993) the author mentioned about multi-year surveillance study. 3.2. The author should add the screening diagnostic methods used for the samples as it is mentioned in the abstract but it is not well-explained in the methods. 3.3. Detailed methodology on DNA sequencing should be provided. Validity of the findings: 1. The authors do not provide the demography data of the samples. 2. The discussion on the first and second results should include the comparison with the results from the previous RSV studies in Thailand, Southeast Asia countries and other countries in tropical region. 3. The authors should discuss the results of amino acid substitutions (line 161-181) as the discussion is limited to N-linked glycosylation analysis. 4. The authors should provide more discussions on the third and fourth results. In the discussion (line 239-258), it is not clear whether the authors discuss their results or the results from the references. 5. As previously mentioned (point B.2.2), the authors should provide a comprehensive conclusion section. Additional comments: The authors should improve the manuscripts (language, grammar, and the clarity of the presentation) before publication. Furthermore, the authors should provide more detailed discussions on the results presented in the article. Overall, the authors should consider the manuscript to be reviewed and edited by a native English speaker to improve the clarity.

You are one of the reviewers, your task is to write a review for the article. You will 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: RESPIRATORY SYNCYTIAL VIRUS GENOTYPES NA1, ON1, AND BA9 ARE PREVALENT IN THAILAND, 2012–2015 Review round: 1 Reviewer: 2

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: RSV infects lower respiratory track of, especially, children worldwide. In the current study, Thongpan et al. used molecular techniques to describe epidemiological features of RSV Thai strains, detected in several thousands samples, collected over the past couples of years (2013-5). The study is well described, and provides updated data on RSV infection in Thailand. I have some comments, below: Line 29-30: It will be helpful to give a brief description of 'RSV-A' and 'RSV-B' in the abstract, for readers who are not familiar with these terms. Line 33: Perhaps the results reported here could describe only in the context of Thailand, not Southeastern Asia. Line 53: Should it be 13 RSV-A genotypes (not 11)? Line 65: Any info to describe how the particular strain of virus did spread around the world? Line 83-91: Were the 3,306 specimens derived from 3,306 subjects/patients? If not, from how many subjects/patients? How many samples were from subjects with flu-like illness and probable RSV infection? How many samples were from King Chulalongkorn Memorial Hospital and from Chum Phae Hospital? Such info should be mentioned in the manuscript. Line 108: There were 278 positive samples. I wonder whether a total of 212 strains (118 RSV-A and 94 RSV-B) were identified from all these positive samples, or there were just some positive samples that failed to identify the strains? Line 112: What are the genotypes of the RSV reference strains B1 and A2? Line 171 and 176: duplication of which gene or region? Line 211: It will be more interesting to see if there is any info about the percentages of co-infection by both RSV subgroups. Figure 1: Is it possible to adjust the scale of the Y-axis to 0 - 100%, to clearer describe % positive samples, in relation to the number of samples, at any time points? Figure 2 and 3: Can the Figures 2 and 3 be combined into one tree, to see overall phylogenetic relationships of all RSV Thai strains? In the legends, adding some info regarding about number of samples, where they were derived, how many samples in each year, and indicators of which are outgroup/reference strains, would be helpful to readers. Figure 4 and 5: the symbols '.' and '~' and any others, marked in the figure, should be given a short description in the legend. Can these two figures be combined into one sequence alignment of both RSV subgroups?

You are one of the reviewers, your task is to write a review for the article. You will 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: IMMUNOFLUORESCENCE CHARACTERIZATION OF SPINAL CORD DORSAL HORN MICROGLIA AND ASTROCYTES IN HORSES Review round: 1 Reviewer: 1

Basic reporting: This is a reasonably clearly written manuscript.Overall the English is quite good, and only occasional sentences are unclear (as pointed out below). References are appropriate and reasonably up-to-date, and the manuscript overall comply with guidelines regarding structure etc. Experimental design: In this manuscript, the authors (Meneses et al) describe their studies of distribution, cell sizes and other characteristics of spinal cord dorsal horn microglial cells and astrocytes in horses. The studies are based on material from (only) 5 supposedly healthy, adult horses. However, the selection criteria for the horses are a bit vague and, while that may be due to the “opportunistic” sampling at an abattoir, it is still a concern. Information should, as a minimum, be provided about the estimated age of the horses, their sex and breed. The description of the morphometric assessments (lines 216-226) is somewhat vague and could be greatly aided by some illustrations of the approach (this could be provided in the supplementary file). Moreover, as detailed below, the illustrations of some of the features described cannot be appreciated in the figures provided, notably Figure 2. Validity of the findings: The authors have a good point about the way the literature regarding shape of microglial cells, as a reflection of activation state, is largely based on dogma originating in some of the early description of these cells, and that the amoeboid shape tends to be the “resting state” in many species rather than the active or reactive state, as originally described. This is certainly the case for species as disparate as mouse, rabbit, ruminants and horses (e.g., J Virol 82: 755; Microb Pathog 103:71). In contrast, the statement that GFAP is not present throughout the astrocyte cytoplasm (line 310-11) is not correct for all species either. It is actually present in such species as mouse and rabbit. What can be concluded from all this is that generalizations cannot be made regarding either of these cell types and that for each species under study appropriate controls must always be applied. The authors discuss at length the size differences of astroglia between horses and humans (lines 317-25), but do not take into account regions/locations (e.g., the Oberheim paper cited describes neocortical astrocytes). Moreover, differences could be influenced by fixation method used (type of fixative, time of fixation etc) as well as other preparatory methods that could influence tissue shrinkage. This aspect should be commented on. Additional comments: The manuscript could be further improved by addressing the following points: • It is misleading to call the approach “immunohistochemistry”, since a chromogen was not used for visualization of the binding of antibodies. Rather fluorochromes were used and the read-out was immunofluorescence, whether by conventional fluorescent microscopy or confocal. This should be corrected throughout the manuscript, starting with the Abstract (line 31). Furthermore, sections are not “stained” (e.g., line 206), but immune-labeled. Please correct this throughout the manuscript. • Line 160: what do the authors mean by “mounted in xylene embedded slides”? Please clarify. • Lines 176 & 178: it is very confusing when the authors say that the primary antibodies were “conjugated” with an [fluorocrome] goat anti-rabbit or donkey anti-goat secondary antibody. The use of the word ‘conjugate’ is misleading in this context, as the term is normally used to describe the joining of two or more chemical compounds, notably the conjugation of an enzyme or a fluorochrome to an antibody. Thus, the secondary antibodies are conjugated with fluorochromes, and the secondary antibodies are used to visualize the binding of the primary antibodies by binding the former to the latter. This should be corrected in the text. • Figure 2: some of the panels are mis-labeled, i.e., there is no panel D, causing some confusion when reading the results section. In general panels E, F and G (which should be D, E, and F) and in particular panel G (i.e., F) are difficult to interpret. It is not possible to appreciate “a tight connection between end-point processes and capillary walls” (line 268) in the figures provided. • Lines 41: correct to “amoeboid or spherical” • Line 58: correct to “found in rodents, with …….” • Line 108: correct to ‘major’. • Line 114-115: what do the author mean when saying “that highly coupled these cells”? • Line 122: correct to “activation at the dorsal horn level” • Line 128: correct to “under either normal conditions or during painful….” • Line 130: correct author name to “Bielefeldt-Ohmann” as in the reference list. • Line 134: replace “disposition” with ‘distribution’. • Line 141: delete “has”. • Line 281: delete the second “in”. • Line 284: replace “stain” with ‘label’ • Line 294: correct to “resting and active states” • Line 297: replace “o” with ‘and’. • Line 308: correct “immunostain” to “immunolabel” END

You are one of the reviewers, your task is to write a review for the article. You will 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: IMMUNOFLUORESCENCE CHARACTERIZATION OF SPINAL CORD DORSAL HORN MICROGLIA AND ASTROCYTES IN HORSES Review round: 1 Reviewer: 2

Basic reporting: No comment. Experimental design: No comment. Validity of the findings: No comment. Additional comments: The authors described the morphological studies of microglia and astrocytes in horse spinal dorsal horn with immunohistochemistry in this manuscript titled 'Immunohistochemical characterization of spinal dorsal horn microglia and astrocytes in horses'. Since the whole study was based on Iba1 and GFAP antibodies, therefore I would recommend authors to use the best well-known antibodies for Iba1 (WAKO) and GFAP (DAKO) instead of the antibodies from Santa Cruz. And the staining for glia cells in the manuscript was not good. As we know that the fixation could also affect the glia cell staining, where Iba1 and GFAP antibodies show perfect staining with mild fixed tissues. And the authors used around 10% PFA for immersion fixation tor two days, which would definitely reduce the staining quality (usually 4% PFA for 2 hrs of immersion fixation).

You are one of the reviewers, your task is to write a review for the article. You will 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: IMMUNOFLUORESCENCE CHARACTERIZATION OF SPINAL CORD DORSAL HORN MICROGLIA AND ASTROCYTES IN HORSES Review round: 1 Reviewer: 3

Basic reporting: English is fine and there is adequate background literature presented to justify studying the topic, but that material leads the reader to think a somewhat different study will be done. A bit more background is needed for the specific work that was actually done and a bit more justification is needed for what actually was done. The general structure of the article is fine. There is no concrete hypothesis stated. See General Comments section below for suggested improvements. Experimental design: The research appears to be original within the Aims and Scope of the journal. However, the research question needs to be more well defined as does the research gap being filled. Portions of the Methods need to be described in more sufficient detail for replication. See General Comments section below for suggested improvements. Validity of the findings: Clearer description of some of the Methods would make the data more robust and validity easier to evaluate. The conclusions could be more tightly linked to the major issue presented, which is the substrates of pathological pain states. See General Comments section below for suggested improvements. Additional comments: INTRODUCTION A significant issue with the Introduction is that it leads the reader to believe that the authors will be doing a study that examines the interaction of microglia and astrocytes underlying pain syndromes when in reality they are simply looking at the normal morphology of microglia and astrocytes in horse. In essence, the last paragraph could serve as the principal portion of the Introduction if the authors just talked a bit about the glial markers in that particular paragraph, talked a bit more about the existing work in the horse, and provided a more detailed explanation of how this normal data will be useful in future pain studies. Some more specific comments regarding the Introduction: In line 68, the concept of the term “static” is a bit vague. For the sentences that begin on lines 73 and 74, in-text examples would be useful (aside from simple referencing). Perhaps “differential functionality” might be better at the beginning of line 77. Line 78: maybe “duration” instead of “time”. Line 79: “Glial-derived” might be better. In the sentence beginning on line 83, it’s not clear whether the authors are suggesting pain syndromes cause the microglial response or whether the response underlies the syndrome. So maybe the beginning should read “Painful syndromes involve the triggering of”. Between this sentence and the following one (beginning on line 86), there is unnecessary repetition regarding Iba-1. Line 89: This sentence needs some clarification. Line 100: what do the authors mean by housekeeping function? This sentence seems to have some circular reasoning (e.g. resting astrocytes express basal levels of GFAP, but how do you know they’re basal levels unless you first know they’re in the resting state? By GFAP levels? See what I mean?). Lines 101/102: Please clarify “external stimulation”. Line 103: What do the authors mean by “pre-synaptic space”? Lines 102-106: The sequence of interaction between neuron and astrocyte could be clarified here. Line 106: Perhaps change “at the spinal level” to “at the spinal cord”. Line 108: “major” instead of “mayor”. Line 112: Might add “with GFAP antibody” after “astrocyte”. Line 120-122: This sentence needs to be clarified. Lines 125-126: This sentence sounds like the goal of this study, given the preceding Introduction material. MATERIALS AND METHODS The description of the sectioning and staining are fairly straightforward. However, the description of the image collection and analysis is much less clear/detailed and can use significant improvement, particularly how the samples from a given horse were assigned to different staining and analysis procedures, and the description of the various morphological measures. More specific comments follow: For animal selection, indicators of pain, which would be important in such a study, were of the most general nature. Why were either cervical or lumbar samples used from a given horse and not both? There are no references for the tissue fixation and staining. How did the authors derive those procedures? How were the sections parsed for the different staining procedures? The logic for the double antibody staining should be explained more clearly. Lines 154/155: Transverse sections? 70%, 80% 90%, etc. or 70%, 75%, 80%, 85%, etc.? Line 159: Do the authors really mean sagittal? If so, why? Line 160: Model of Jung microtome? What is meant by xylene embedded slides? Line 162: Please see query for lines 154/155. Line 163: What type of microwave and what were the settings? Lines 164/165: Was there some kind of serum used along with the TCT buffer for blocking? Line 176 and 178: Do authors mean “incubated” instead of “conjugated”. Line 179: Was there any wash after the secondary antibody incubation before DAPI staining? Line 180: What form of DAPI was used? Aren’t there a few different types? Line 188: Regarding “initially processed as previously described”, up to what point? Line 189: It’s not clear what “incubated separately” means regarding the primary antibody double-staining. Line 203: Should probably add, “and then counterstained with DAPI”. Line 210: Might add “was performed” after “laser scanning”. Line 212: Are the 15 sections all the horses combined, or 15 sections from each horse? Line 213: It’s not clear what the authors mean by “the entire spinal cord”. Line 214: Please clarify what is meant by “completely integrated dorsal horns”. Lines 216-226: The description of the morphological parameters that were measured should be explained more clearly and with more detail. For example, how was shape determined? How was the decision made to classify a cell as spherical in form or hyper-ramified? Was the length of all processes of a cell measured? Did diameter of hyper-ramified cells include the processes? A figure would be helpful showing the various morphometric measures that are described. When measuring process length, was bending of the process taken into account at all? Line 224: Should the word “cells” be “processes”? Line 227-234: Please elaborate on what is meant by “distribution” in line 227. How was the dorsal horn and its laminae identified in these fluorescence sections? What was the criterion for an immunopositive cell at 10X magnification? Was it any stained piece that was on the perimeter of the defined square? How thick was the perimeter line? There is no mention of background staining in the omission controls. How was background staining dealt with? RESULTS It is difficult to assess the validity of the data without clarifying some of the issues raised above for the Materials and Methods, particularly with respect to how tissue slices were assigned to the various staining procedures for each animal. A table summarizing the results would be useful. Is there a way to know whether the microglia are all amoeboid/spherical, or whether processes just can’t be detected by the staining method? When talking about differences between any measures (e.g. between numbers of cells in dorsal horn regions), the authors should note they are simply numerical differences as opposed to statistically significant differences, since no statistical comparisons were made. Is there a reason why the microglial/astrocyte branch contacts weren’t quantified? Perhaps they differ between dorsal horn regions. How were primary and secondary branches defined? Many of the descriptions are quite qualitative and it’s hard to evaluate how representative they really are. Lines 252-254: The authors should clarify what they mean when they say, “GFAP was mainly found in astrocytic primary processes”. Compared to what? Small cell bodies relative to what? How were “long branches” defined when the authors say “extended 2-4 long branches”? Lines 254-257: So primary astrocyte processes then give off 2-4 long branches (why weren’t descriptive statistics calculated here?), which each give off 2 small thin branches? Correct? How were the blood vessels and neurons that the authors mention identified? Lines 258-259: How did double fluorescence make it better for defining astrocyte cell bodies and extensions of processes? Lines 259-261: The authors should explain why it was necessary to use diameter measurements for cell body size in astrocytes but area for cell body size in microglia. How was “main process” defined? Lines 261-263: The authors should clarify why they needed to see the whole cell size for the astrocyte density measurement, and how the Cx-43 staining help with that. DISCUSSION The Discussion makes some interesting points regarding differences with other species and with humans. However, the authors do not really explain how these particular findings are relevant to deciphering the substrates of pathological pain states. Also, no limitations of the study are discussed and there is no mention of effects of background staining. Lines 286-287: Microglial cells were also found in the other dorsal horn regions examined. They were just numerically less dense. Lines 288-289: Maybe no processes were observed using Iba-1 because of staining quality. Lines 299-302: The data you talk about in this paragraph actually do seem to support a difference in microglial morphology between healthy and (encephalitic) diseased horses. Why did the authors not discuss the findings on dorsal horn laminar distribution of microglia? Lines 317-325: The horse, rat human comparisons are interesting, but what does it mean for deciphering mechanisms of pathological pain states? Same question for the laminar distribution? Line 326: This statement should be toned down given that no comparative statistics were performed. Lines 330-333: It is hard to see what the authors describe here in Figure 2F, and the statement about “stretch connection” is not clear. Lines 333-336: The authors need to elaborate a bit here in order to clarify the statement. CONCLUSIONS Lines 344-348: Perhaps the authors might say that exploring other species might reveal glial organization that is closer to human than is rodent, and might represent better models for investigating glial substrates of pathological pain. Lines 353-355: So are you saying you feel horse is a better model for human pathological pain states than rodent?

You are one of the reviewers, your task is to write a review for the article. You will 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: IMMUNOFLUORESCENCE CHARACTERIZATION OF SPINAL CORD DORSAL HORN MICROGLIA AND ASTROCYTES IN HORSES Review round: 2 Reviewer: 1

Basic reporting: Only minor spelling and syntax issues still need to be corrected. Otherwise much imporved. Experimental design: No further comments Validity of the findings: No further comments Additional comments: The Authors should be commended for their attention to the reviewers' comments and criticisms.The manuscript is now notably improved, and only minor issues remain to be dealt with: line 53: correct to 'immuno-label' line 202: correct to 'three' line 216: suggest modify the sentence to the following "GFAP primary antibody binding was visualized by incubation with Alexa Fluor 488 conjugated donkey anti-goat secondary antibody (1:500, Invitrogen, Camarillo, CA, USA)...." line 339: delete "to obtain" line 394: replace "o" with "and" line 397: change "evidencing" to "suggesting" line 406: correct to "used for study of the morphology......" line 507: correct to "focus on exploring...." line 530: change to "immunofluorescence analysis". END

You are one of the reviewers, your task is to write a review for the article. You will 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: IMMUNOFLUORESCENCE CHARACTERIZATION OF SPINAL CORD DORSAL HORN MICROGLIA AND ASTROCYTES IN HORSES Review round: 2 Reviewer: 2

Basic reporting: Please note that this is a re-review of the original submission. The basic reporting is now satisfactory and no longer misleads the reader to think that a different type of study will be done. Experimental design: The descriptive nature of the study has now been made clearer and many of the methodological details lacking in the original have been added or clarified. However, there are still a few details that could use clarification that are noted below. Validity of the findings: As noted, several methodological details have been clarified, improving the perceived validity of the findings. The authors have added a few theoretical connections between their findings and pathological pain states, but the discussion of limitations of the study is still minimal. Additional comments: The authors have addressed the vast majority of the comments made by this reviewer on the original submission. INTRODUCTION: The Introduction now seems adequate. MATERIALS AND METHODS: The reference that the authors cite in their responses, for the Bouin’s fixation procedure, should be added near the end of paragraph 1 of the Methods. The author’s response describing the 3 adjacent sections for the 3 antibodies and the total of 15 sections per horse should be added to the Methods text. In the Methods section it still needs to be made clearer why there seems to be a set of single labeled tissue produced, and then another set of double labelled tissue produced, despite the fact that the authors feel it’s clear. Line 167: The authors should further clarify what “separately” means (is it really sequentially, with first primary antibody on Day 1 and second primary antibody on Day 2?). Line 198: How was a hyper-ramified cell defined? Line 207: When the authors say GFAP and Cx-43, do they mean double labelled, while the Iba-1 is single labelled tissue? Irrespective, this should be clarified in text. Lines 212-215: The authors say in their response letter that dorsal horn laminae were not identified, so how did they make the laminar distinctions mentioned in these lines and how were they able to visualize dorsal horn regions? It’s still not clear how the authors can have confidence in their numbers without a background estimate/correction. For Supplemental File A: the numbers in Part A should be explained. Line 12 should read “using 30 square perimeters”. RESULTS For the qualitative descriptions in lines 225-227, and 234-235, some idea of how representative this is should be presented. Otherwise, it’s hard to interpret the validity of the observations. Line 232: Why is the number of primary processes presented as a range and not a mean? Line 242: Cx-43 alone? The HE staining comment in the author’s response regarding tissue integrity and blood vessels should be added to the Methods. DISCUSSION The author’s response regarding lines 299-302 of the original submission should be placed in the Discussion around line 275. Lines 278-279 need to be toned down because the very limited data available in horse, although inconclusive due to small numbers, seem to indicate there might be a relation between disease state and cell form. Line 306: Should say, “…the highest number of astrocytes were counted in laminae I, II and III…” Line 313: Should say, “…we showed some evidence that astrocytes…”

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

Title: GENOME-WIDE IDENTIFICATION AND EXPRESSION PROFILE ANALYSIS OF CCH GENE FAMILY IN POPULUS Review round: 1 Reviewer: 1

Basic reporting: Figure 6 (RT-qPCR results) should include the raw data or bar plots and the statistical significance of the difference should be discusses. It is not easy to identify the level of difference from heatmaps. In addition, because untreated and treated samples are in separate plots, they cannot be directly compared due to separate normalization. Experimental design: The article will fall within the scope of PeerJ. The analyses are routinely used and are well done. There are some issues however. See specific comments related to experimental design are below. 1. The definition or identity of CCH gene family is not clear and objective. Is the MXCXXC motif and the secondary structure pattern not present in any other gene family? Is there a minimum sequence identity that authors looked for. A single criteria of BLASTp with an E-value seems arbitrary. It should be clearly discussed what makes a CCH family unique, and use that criteria. 2. Is the induction of expression level differences upon Cu stress statistically significant? It is not clear from the heatmaps. If it is not significant, it should be clearly stated and discussed. 3. It is not clear how the A. thaliana, and O. sativa CCH genes were identified. Will a similar sequence search conducted for CCH in these genomes. The criteria should ideally be same for all genomes. 4. It is known that a single query may not be enough to cover all the genes in a gene family and the choice of query also affects the family coverage (see e.g. Kaushik et al. PLOS ONE 2013). It can be tested by a sequence search using all A. thaliana CCH if any new CCH proteins are identified in any of the 3 organisms. 5. Are the functions of the predicted motifs known? And based on the motifs, can a new functions of CCH in Populus be identified? Domain combination analysis can also be used to identify novel functions of CCH if any. 6. Has the lineage leading to the evolution of Populus experienced multiple Whole Genome Duplications? How does genomic distribution of CCH relate to other WGD events in the past? 7. Is there any specific property in ohnolog PtCCH and the ones that are retained from SSDs? It will be interesting to discuss. 8. Has the expression pattern diverged between the leaves in Pt and PnCCH? What could be the reasons and implications for such a divergence. Validity of the findings: The finding would appeal to limited audience interested in CCH family in plants. Most of the article is very descriptive. If the results are put in a broader context, it will make the manuscript more appealing to broad audience. 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: GENOME-WIDE IDENTIFICATION AND EXPRESSION PROFILE ANALYSIS OF CCH GENE FAMILY IN POPULUS Review round: 1 Reviewer: 2

Basic reporting: The manuscript by Xu et al. describes the identification of genes encoding copper chaperone (CCH) family proteins in Populus. Based on the known P. trichocarpa genomic sequence, they perform a bioinformatic analysis and identify 21 proteins with sequence and structural similarity to Arabidopsis CCH, analyze the chromosomal location of the corresponding genes, and perform a phylogenetic analysis including proteins from rice and Arabidopsis. They also analyze the expression of several of the Populus genes in different organs under different conditions (control, copper deficiency, copper excess). Their main conclusion is that the CCH family is expanded in Populus and they suggest that this may be related to the ability to grow on contaminated areas and accumulate metals. The manuscript is correctly written, with references to the relevant literature. The bioinfomatic and experimental analysis was properly performed, although some corrections and clarifications are required, as described below. Figures and figure legends: -Fig. 2 legend: Please, explain the meaning of the different boxes connecting the chromosomes. -Fig. 3C: References to motifs are too small and difficult to read. Please, refer in the legend that the sequences of the motifs are listed in Table S4. Also, are these motifs similar to any known motif, thus giving clues to their possible functions, or are they just conserved sequences between CCH proteins? -Fig. 3C legend: explain the meaning of S1 to S3. -Please, add bootstrap values to the trees, or an indication of how significant the different branches are. Related to this, do the trees shown in Figs. 3 and 4 have the same structure? If not, how do the authors explain this? How do clades S1 to S3 in Fig. 3 relate to clades SI-SIII observed in Fig. 4? Ideally, the labeling of clades should be the same (S1-S3 or SI-SIII). -Fig. 4: I guess there is a mistake in the labeling of some groups (blue and green in the upper left part should be SIII instead of SII). -Fig. 5: The labels in the bar graphs are difficult to see (too small). The legend should be more descriptive, so that the reader can understand the figure without referring to the text. This applies to all figures. Please revise this. -Fig. 6: Relative expression is shown. Relative to what? Untreated controls? Please, clarify this. Also, give a hint of the statistical significance of the results (for example, include an asterisk in those rectangles where changes in expression are statistically significant). Discussion: Lines 425-434, explaining primer design and why the expression of some genes was not measured, should be removed from here and included under Results. Conclusions: The Conclusions are not properly written. English is poor and there is repetition of concepts already mentioned in other sections. Re-write or, preferably, remove this section, since it does not add much to the paper. Experimental design: I find that a basic question that is not correctly addressed in the manuscript is that of the function of members of the CCH family. The authors refer ambiguously to the CCH gene from Arabidopsis, which has been functionally characterized, and then identify 13 CCH genes in this species. Are there any studies on these other CCH genes? Which one of these 13 is the “real” CCH? (I guess it is AtCCH6). These matters should be discussed in the manuscript. Also, I suggest that the authors perform complementation studies in yeast to analyze if representative Populus CCH proteins from different clades are functionally equivalent to Arabidopsis CCH. This would add a functional significance to the expansion of the family. 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: NEW CERNOTINA CADDISFLIES FROM THE ECUADORIAN AMAZON (TRICHOPTERA: POLYCENTROPODIDAE) Review round: 1 Reviewer: 1

Basic reporting: I have made my comments regarding terminology, nomenclatural conventions, and typos or missing commas. Experimental design: The description of these new species is important, especially given the environmental threats and possible extinction of species and habitat. Validity of the findings: These are the first new species of this genus discovered from the region. 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: NEW CERNOTINA CADDISFLIES FROM THE ECUADORIAN AMAZON (TRICHOPTERA: POLYCENTROPODIDAE) Review round: 1 Reviewer: 2

Basic reporting: . Experimental design: . Validity of the findings: . Additional comments: Line 70; Dudgeon et al, 2005 is cited as 2006 in the literature, in this same line, Finlay 2010 has ae extra comma and a D before the ) Line 81; mentions a supplementary file 1 which I didn’t see Line 89; should be a comma after For examination and description Line 134; ..thumb-like, ca. as long as – what is ca., suggest authors write out word which ca. is describing, nearly?? Line 140, 165, 167; same comment about use of ca. Line 187; Cernotina hastilis Flint 1996, Flint 1996 is not in literature cited, nor is Botosaneanu 2002, in the next line (line 189) Line 201; the reference Paprocki et al. 2004 is not in the literature cited Line 212, 213; the reference Paprocki and Franca 2015 is not in the literature cited

You are one of the reviewers, your task is to write a review for the article. You will 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: SKIN AND FUR BACTERIAL DIVERSITY AND COMMUNITY STRUCTURE ON AMERICAN SOUTHWESTERN BATS: EFFECTS OF HABITAT, GEOGRAPHY AND BAT TRAITS Review round: 1 Reviewer: 1

Basic reporting: This paper describes the bacterial communities found in the fur and skin of 14 species of insectivorous bats (in a total of 186 animals) in 5 caves in the USA states of Arizona and New Mexico. Bats were caught either directly in the caves or outside of them, from 2011 to 2014. Using 454 massive sequencing, the authors analyzed 16S rRNA. The sampling appears to be good (but we are never informed of the total samples per bat species per cave, for instance), and in general, the molecular and statistical methods seem competent. The main result is that, according to statistical analyses, cave- caught vs. surface-netted was the most important factor in determining the differences in the bacterial communities, and the second more important factor was the eco-region were the animals were sampled. In relation to these two main findings, I have my most important concerns of the study. 1) Why do the bacterial communities associated to the bats change so much in a cave and outside? If they change so drastically, perhaps most of the bacteria the authors are finding are just transient, just acquired from the environment, but actually not living and interacting in the skin and hair of the bat, and perhaps they are not even active, since 16S rDNA does not distinguish if they are dormant or even dead. It would be critical to design a method to actually evaluate the active and resident bacteria in a bat: the bacteria that are alive, interacting with other microorganisms, both bacteria and fungi, and with the other organisms in the skin (ectoparasites) and also organisms conducting a functional role in the external biome of the bat. While we could devise molecular methods for disentangling this (to know which bacterial are the active vs. dormant or dead bacteria), such as analyzing the transcriptome of the bacterial RNA in the skins and hair of the bat (and should be discussed!), at least the authors could compare the bacterial communities that were found in a given bat species in a given time, both inside and outside the cave. These shared OTUs between the biomes inside and outside the cave may represent the true resident bacteria. 2) The sampling scheme is never clear. In particular, only 5 caves were analyzed, but in Figure 5 there are 8 environments. A table indicating caves, the external sites, sampling dates and bat species would help understand the final sampling design and to evaluate how well balanced is the study and which other possible analyses could be conducted with confidence. Also a clarification indicating if the bats in the caves are the same as the ones in the nets, or if the caves represent another population -- that may explain some of the results-- would be important. Experimental design: Collecting Methods: Lemieux-Labonté et al. 2016, used as a negative control a humidified sterile swab, page 4, that sounds like a good idea, as they detected evidences of contamination, page 5. Apparently in the manuscript there were not similar controls; this cannot be changed if it is the case, but this point at least should be discussed. Also, environmental samples of both the cave and the outside samples would have been interesting to check for the identity of the bacteria, instead of using the global or arbitrary data bases you used. Discuss. Validity of the findings: I think the main findings are adequate, but the presentation of them should be improved and better interpreted. See the other sections for comments on the validity. Additional comments: Abstract: Lines 28: Briefly, explain how and why the microbiomes from day to night can be so different! At first glance, this does not seem to make any sense. Line 31: Explain what is WNS. Introduction: In general, I feel that more emphasis should be given to the bacterial communities in the skin and hair in the Introduction. The skin and hair of the bats should represent complex environments, as the bats are notorious because the high number of ectoparasites found, including fleas, different mites, and other arthropods, and their immune system seems to be very developed. Clearly the environment also includes fungi, as the one responsible of the white-nose disease. The authors should make an effort to discuss better bacterial and the communities of other organisms from the skin (and hair in mammals or feathers in birds) in vertebrates. An important reference that should be reviewed in the Introduction and more carefully discussed is the study of Lemieux-Labonté et al. 2016. PeerJ. The introduction of that paper cites several relevant references to understand the skin bacterial ecology that should be explained in the ms. Also, apparently this is the only similar analysis of culture free bacterial communities in bats, and for this reason it should be carefully described in the Introduction, and compared in the Discussion. The paper is indeed mentioned in the Discussion in line 299, but is not mentioned in the Introduction and the paper is missing in the References section. Line 46: “in the Southwest”: Make it more general, so people outside North America will understand that you are talking about a specific part of the South of USA. Line 49: I am not sure what “syntopic” means, explain. Around line 66: Improve the review of the skin and hair communities in animals, in particular for mammals and bats. You mention the white-nose fungus, but as I explained above, many other organisms live there, and there are some interesting recent papers included and reviewed in Lemieux-Labonté et al. 2016. Also, a critical issue is the functional role of the bacteria in this environment, as it can include transient (that may be interesting but irrelevant), and resident bacteria, and methods to disentangle both. Lines 91 to 93: And if fungi communities are so important, Why not conduct a parallel study using ITS for the fungi? Not in this paper, but here you could discuss this (perhaps some bacteria defend from fungi in general) and you could use the DNA already extracted for this paper. Methods: As I mentioned above, the sampling is not clear, as there are 5 caves, but 8 eco-regions in Fig. 5. A table stating the sampled caves, eco-regions, dates, and number of bats per species will help the reader to understand the total sampling, and how unbalanced was the sampling, and if it is possible to compare microbiomes of a bat species and a given date with samples in and outside the cave, to explore for the existence of resident and transient species. Lines 111 to 122: As I mentioned above, some controls for contamination seem to be missing, and environmental samples would have been useful to interpret the data. Also, how do you decide the number and species of bats in each cave and sampling? How many animals were sampled at each sampling date? Lines 150 to 151 and 219 to 228: It is not clear if you rarefied or not the data, or rarefied in some analyses and not in other. I am not sure if McMurdie and Holmes 2014 is actually right, but I am sure that using different sample sizes would bias the estimates of diversity. Line 201: What is the meaning and interpretation of the VDS. I am not familiar at all with this statistic. Results and Discussion: In general, I do not like to make a joint section of Results and Discussion, as I feel the authors tend to confuse what they found and what the wanted to find. Separating both sections in clearer for the reader. I strongly recommend to separate both sections, and to carefully rewrite the Discussion, making clearer comparisons with other studies of external microbiome in animals, in particular to Lemieux-Labonté et al. 2016 paper, as I mentioned above. Fig. 1: It is very attractive, but make it more general: a) Mark the sampled caves. b) As these caves are more related to the southern populations, and as the national borders are meaningless for the biology, also include (North of?) Mexico (as Canada is included). This would make the map and the paper more useful and even more attractive. Line 247: The number of reads among samples is very variable, 843 to 20,515 and thus I would be very careful of comparisons between samples if they are not rarefied. What was the average number of reads? Lines 252: An easy to read and compare figure of the dominant bacterial groups in each sample, and perhaps in other categories, in particular of the bat species, would be important. For instance, you could show this data in a simple pie figure, as in Figure 1 of Lemieux-Labonté, et al. 2016. If you use the same order and colors, this figure could be directly compared and discussed. In contrast, your Figure S1 is completely useless. It is impossible to read or to make any sense of it. Lines 255 to 264: The Random Forest results apparently are the main finding of the paper. I am not familiar with the method, but I think that if it is so important, these results should be shown in a figure or table, to give more emphasis, but also include the associate statistics to evaluate the probability or reliability of this analysis. On the other hand, I am not so sure of the results of the Random Forest, given the values shown in Figure 7 for some other variables as soil PH, the values for some environments and some bat species, or sampling dates, that seem to be very important. I suggest to restructure the paper and give the Random Forest and the MDS analyses together, to try to disentangle what is happening and which are the more important variables that determinate the bacterial communities. Lines 265 to 278: Above, in lines 252 to 254, there is a brief description of the communities. Put both sections together, to support the suggested above pie figure. Figure 3 is interesting, but obscure, as the Y-axis is a complicated and non-intuitive variable (Log-relative normalized). Adding simple (additional?) pie figures or something like that may help understand the abundance of the bacterial groups. Also, explain better the estimate of the number in the Y axis, I guess a higher number means a higher number of that taxa, but it is not obvious. Line 270: Supplementary figure S5 is cited here, but apparently the figures (or tables) S2 to S4 have not been cited yet. S5 figure is very complex, and I am sure there should be an easier way to visualize this information. Improve, please. Lines 299: Extend and improve discussion of Lemieux-Labont et al. 2016. Also include reference. Fig. 5: I do not see any gray line. May be just a problem of my computer, but it is a good idea to make sure the figure can be opened OK in any software. Figure 6: As this analysis explains only 4% of the variance, I would send it to supplementary material, and just mention the statistic values in the text, lines. Line 317 and Figure 7: What is the meaning of the VDS corrected richness? How you interpret this statistic? Lines 342 to 359: While I think this analysis is interesting, I am not sure how reliable it is, as the comparison with the data sets are very heterogeneous: Barberán et al. 2015 seems to be for the complete continent, Newton et al. 2011 for all the available lakes in the world, and for the caves it seems very arbitrary, lines 213 to 218. I am not confident at all of this analysis. It would have been better to take as controls environmental samples of each sampling locality. Please, consider sending this to supplementary material and carefully discuss its limitations. Line 372: The “C” of cave is missing. Line 375: Indicate the dominant phyla. In general all supplementary materials will need a careful evaluation and edition, and a list of these materials was missing in the paper (and one of them I could not open). Some of the materials are very difficult to understand what they are or their meaning and the text is some case is too difficult or impossible to read, as was the case of S1.

You are one of the reviewers, your task is to write a review for the article. You will 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: SKIN AND FUR BACTERIAL DIVERSITY AND COMMUNITY STRUCTURE ON AMERICAN SOUTHWESTERN BATS: EFFECTS OF HABITAT, GEOGRAPHY AND BAT TRAITS Review round: 1 Reviewer: 2

Basic reporting: Winter et al. analyze the external surface microbiota of 14 species of bats across the Southwestern United States. This is a relevant study that addresses important questions about the factors influencing bat microbiomes. Unfortunately, I consider that the manuscript has several structural and technical flaws that prevent it from its publication in PeerJ the way it is. At the end of this report I describe general comments and suggestions to correct and improve the manuscript. Should the authors address the corrections and improvements suggested, I will be happy to review a new version of the manuscript. Specifically, the article fails in explaining in a clear way their main findings. The manuscript needs a better structuring of the information. The introduction requires a better background on skin microbiota and some of the figures can be improved. Experimental design: Overall, this study is correct in terms of the experimental design except for one major aspect that fails: my major concern is the decision of swabbing the entire skin and fur surfaces. Based on previous studies in other mammals, skin microbiota differs across skin regions so the decision of swabbing everything will obscure some important patterns. Also, the authors emphasize the importance of this study in the context of an emerging disease (WNS) however their swabbing technique neither replicates previous studies like Hoyt et al., 2015 nor it is focused on the regions infected by P. destructans (wings and uropatagium). Another important aspect, is that swabbing all external surfaces might also catch a high proportion of transient bacteria which are not specifically associated to bat skin, therefore making the interpretation of the results difficult. The authors need to clearly explain why they made this decision and how this would influence their results. Validity of the findings: Overall, this study is correct in terms of the validity of the findings. However at the end I have several suggestions that will improve this section. Additional comments: General comments Overall, I consider that the methods, results and discussion are confusing due to the way the manuscript is structured. It is not clear which are the main findings and it is hard to follow the main line of the manuscript. For example, in the abstract it is clear that ecoregion and location are the main factors influencing “external surface” microbiota, however in the results (using different methods) it is mentioned that species, sampling site, seasonality, NPP, soil pH, Conifer Woodlands and Savannas, and Chihuahuan Basins and Playas are also influencing bacterial diversity. The authors need to determine which are the main findings and streamline their results and discussion. This includes, evaluating which methods explain better their results. I have some concerns with some of the methods and analyses. Specifically, I don’t think the mantel test is strong enough to mention that geographic distance is playing a role. Even though the regression is significant the R value is extremely low. I would not include this test. Also, I am not convinced by the comparison of specific bacterial taxa with environmental sources. Bacterial communities in nature are highly diverse and therefore if the authors want to compare environmental microbes with the microbes obtained from bats they should have collected samples from the exact site where the bats were sampled. In my opinion, this is the correct way of comparing environmental microbes with potentially symbiotic ones and also to determine the proportion of transient bacteria present on the bat surfaces. Since the authors analyzed 14 different species it would be interesting to analyze if there were any patterns associated to the bats habits. Do these bats eat the same food? Do they have similar life histories? What about a phylogenetic component? Specific comments Introduction The authors need to expand the background on skin microbiota and their role to prevent emerging diseases in bats and in other non-human systems that have been studied with more detail. Line 42. Eliminate “second to rodents at 2277 species”. Lines 55-64. I am not sure how this information is relevant to this study, since all of these aspects are not analyzed later in the text. Lines 77-78. You need to be more specific here, since this is the only study about antifungal bacteria in bats. Also, I think this study needs to be discussed later in the text in the context of your results. Lines 79-85. Need more references of skin bacteria in bats such as Lemieux-Labonte et al. 2016. Methods The methods are a little confusing and they are disorganized. A better structure would help the reader follow the text better. For example, Sampling also includes molecular techniques like PCR. Normalization is mentioned twice. Diversity estimates are divided in different sections (alpha and beta diversity, NMDS plots etc) and they should be included in one. Line 118. Explain what is Ringer’s solution Line 151. Why did you use a depth of 1500? Please explain this sentence better. Line 191. Eliminate the last sentence that starts with “NMDS…” Line 203-209. There is no need to include the R script that you used here, unless you decide to include all the other scripts used for this study. If so, I would send all the scripts to supplementary figures. Lines 219-234. Explain with more detail how DESeq2 and CSS work. Also, explain better how the data obtained with these methods was correlated with rarefied data. If these were highly correlated with the rarefied data, why not use rarefaction? Maybe I am missing something here since I am not familiar with DESeq2 and CSS. Results and discussion The results in general are difficult to follow. The authors describe patterns of diversity associated to cave caught and surface netted and then explain pattern associated to ecoregions and then go back to describing cave caught etc. I would reorganize the results in subsections that reflect the main findings. Line 249: By looking at the supplementary figures, the authors need to explain why there was such a large proportion of unassigned reads. Line 252-254: This sentence seems out of place. I think it should go later in the results. Line 255: Change “The data were tested using a random forest model to see if the data…” to “A random forest model was used to see if the data…” Line 256-259: You mentioned in the methods that a reasonable ratio of the estimated generalization error compared to the baseline error should be two or greater, i.e. the random forests classifier does at least twice as well as random guessing for an unlabeled community. So why do you say the method was minimally successful if a ratio of 2 or greater seems enough? Please explain Line 274-277: To determine the effect of ecoregions and habitat with random forest the authors should only include regions where both bats from caves and from surfaces were collected. Otherwise it is not possible to distinguish the effect of the ecoregion versus location. Line 285: Why chloroplasts? Please explain. Line 285-288: This needs to be explained better. I would think the opposite, that cave caught bats would be dominated by bacteria adapted to cave environments and that surface netted would have a higher diversity of bacteria because they are in contact with many environments, soil, air, plants etc. Lines 289-299: There needs to be a stronger discussion section comparing with previous findings. The studies mentioned here were done in different species of bats with different habitats and the samples were obtained from different origins (guano, guts or ocular surfaces). There needs to be a more thorough explanation of why the authors find differences or similarities with these studies. Why did you not find differences among host species? were the samples taken differently that what Lemieux-Labonte et al. did? Lines 300-301: The authors need to discuss the fact that transient bacteria may be present in their samples. How do you take this factor into account? Lines 302-304: This was mentioned in the previous section too. Lines 337-338: What are the implications of these differences? Overall, throughout the text the presence of specific groups of bacteria need to be explained better: What are these bacteria characterized by? What does it mean if a specific group is enriched in cave caught or in surface-netted bats etc? Lines 339-341: I don’t think these sentences are relevant for the discussion Conclusion Lines 362: Why does comparing with one soil study (Ma et al., 2016) is relevant? Please explain. I would suggest to focus on specifying which are the factors that mostly contribute to explain your data instead of comparing with other unrelated studies. Lines 363: “Internal” means “gut”? Lines 372: What does “.Ave” mean? Lines 374-387: I think there needs to be a clearer conclusion. What are the main findings? Which factors determine the microbiome in bats? Which factors will require more studies in order to determine their relevance? Figures and tables Figure 1: I don’t think this figure is relevant in the context of this study. Figure 2: This maps need a scale. If you decide to keep the mantel test you could make a figure with the map and the graph together? This is just a suggestion. Figure 7: To make the interpretation easier I would highlight with some color the variables associated to each of the NMDS and richness.

You are one of the reviewers, your task is to write a review for the article. You will 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: SKIN AND FUR BACTERIAL DIVERSITY AND COMMUNITY STRUCTURE ON AMERICAN SOUTHWESTERN BATS: EFFECTS OF HABITAT, GEOGRAPHY AND BAT TRAITS Review round: 2 Reviewer: 1

Basic reporting: For this new version of the manuscript the authors changed the figures and answered some of my concerns. Nevertheless, there are still some issues, in particular about the figures that the authors will need to correct in the next version of the paper. Experimental design: See below. Validity of the findings: Somewhere in the Discussion or in the Conclusions, the limitation of the study should be clearly discussed in order to try to improve similar research in the future. From my original review I copy some of points that are still needed for this: 1) “It would be critical to design a method to actually evaluate the active and resident bacteria in a bat”, and explain how you could solve this. 2) “Lemieux-Labonté et al. 2016, used as a negative control a humidified sterile swab, page 4, that sounds like a good idea, as they detected evidences of contamination, page 5. Apparently in the manuscript there were not similar controls; this cannot be changed if it is the case, but this point at least should be discussed.” 3) “Also, environmental samples of both the cave and the outside samples would have been interesting to check for the identity of the bacteria, instead of using the global or arbitrary data bases you used”. Additional comments: Line 137: It should say “Figure 1”, as the figures were changed. Line 147: You should mention Figure 2a around here. Line 161: Figure 2b should be cited here. Line 197: Explain better. Apparently all the samples belonging to a bat species were removed. If this is the case, mention this bat species here. Lines 207 and 294: Explain what is BLM. Lines 276 to 286 need work. Move lines 276 and 277 to lines 195, were this explanation seems to belong. And move lines 279 to 286 to Sampling section, around line 162. Lines 286, remove an extra dot at the end. Figure 3: Include the units in the figure: 0 to 60 of what? And check the species of the bats and include the name of the “M” species that is missing in the figure caption. Also, put in italics the Latin names in the caption. Lines 360: Remove an extra dot at the end. Figure 5: Indicate what is the meaning of the colors of the dots in the graph. Change bat name to “Myotis velifer” Figure 6: Indicate the axis of the figure: latitude and richness, and also indicat the meaning of the two colors, red and blue of the dots. Figure 7: Indicate the meaning of the colors of the dots. Line 522: “likely behavior”? Maybe it is flying or feeding behavior, as in the graph. The figure should be 5, not 6. Correct.

You are one of the reviewers, your task is to write a review for the article. You will 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: SKIN AND FUR BACTERIAL DIVERSITY AND COMMUNITY STRUCTURE ON AMERICAN SOUTHWESTERN BATS: EFFECTS OF HABITAT, GEOGRAPHY AND BAT TRAITS Review round: 2 Reviewer: 2

Basic reporting: I consider that the manuscript has improved from the last version. The introduction and the results are clearer now. However, I still consider the manuscript requires some improvements before being considered for publication. In particular, I consider that the authors didn´t properly address some of my concerns regarding the effect that transient bacteria may have on their results and also, they need to correct a few sections and figures. My comments are shown below (general comments to the author). Experimental design: I have no further comments regarding the experimental design. Validity of the findings: I have some comments here but I will present everything below(general comments to the author). Additional comments: I don’t think the term “external microbiota” is the most appropriate term: First I think it´s too vague, second, if external means skin and internal means gut I would argue that actually the gut is also external since it´s not INSIDE the host. I would modify these terms (external and internal) and be more specific: skin and gut microbiota maybe?. The authors expanded the methods section and explained why the sampled the fur in addition to the skin. However, I still think that this approach will be likely catching transient bacteria from the environment. In fact, there is a hole section in the discussion (lines 509-519) where the authors discuss how the differences they found between bats from different habitats may be related to changes in the environmental bacteria present in caves in contrast to surface environments. One interpretation of these results is that the sampling scheme is promoting the capture of transient bacteria and therefore the microbial communities on the skin are just a reflection of the environment. Therefore, I strongly think that the authors need to address the main caveat of the study, which is that they didn´t took environmental samples to compare with and therefore they can't discard the possibility of transient bacteria being sampled. Specific comments L42-44: I suggest adding more references on microbiomes in other animals (i.e amphibians hydra) in addition to the whale paper. L122-124: Maybe rephrase this first question. Something like: does the daily routine of bats (spending time outside or inside a cave) influences their skin bacterial composition? L197-200: This should be part of the section Sequence procesing. L236-237: Phylum? I guess you mean Class? Alpha, beta and gammaproteobacteria are classes. L262-265: I don’t understand the difference between the first and the second sentence. L276-277. This section should be included in the introduction. L279-286: This section should go in the “sampling section”. L299-306: I would add this paragraph with the next section and eliminate the first sentence which is unnecessary. L308: Change “Our study stands apart from culture-based studies and other next generation sequencing studies by focusing on the diversity of the external bacteria from...” to “Our study focused on describing the diversity of the external bacteria of 163 wild-caught bats from…” L314-316: Be careful here: you are confusing classes with phyla. Choose either one but fix the mistake here and throughout the manuscript and figures. L334. Change the title of this section to something like: The effect of habitat, ecoregion and species type on skin microbiota L357-360: I suggest eliminating this paragraph. I don’t think is necessary. L362: I don’t think you need so many subtitles for the results section. I suggest eliminating this one. L387-388: Eliminate this first sentence and add this paragraph as a continuation of the previous paragraph. You can start L379 with: “A bayesian hierarchical model was fit to explain the amount each predictor contributed to the community dissimilarity on the NMDS1 and NMDS 2 axis. In the case of NMDS1…. In the case of NMDS 2… L410: eliminate: (defined by QIIME’s alpha_diversity.py) Figures 6: This graph needs more work: What are the axis? Explain the top part of the figure, what are the colors of the dots? The same with figure 7. They both need to be clearer. L424: I am not familiar with these bayesian hierarchical models; thus, I really don't understand the values described below. I strongly suggest explaining this part more clearly. Specifically, the meaning of the values and the sign of the value need to be explained for a general audience: What does a negative or positive value mean? The larger the value the greater the effect? L436-440: This section to me is telling the readers that habitat and latitude are confounding factors and that maybe is not latitude but habitat what is explaining these patterns. I would reconsider presenting these data as it is. L487-498: I don’t think this paragraph of the discussion is relevant for this study. The comparison with other animal system at the bacterial phylum level is, in my opinion, not informative. The bacterial phyla that the authors describe here are pretty much present, not only on the skin of different organism but also on any environment. Correct all the “cofounding” by “confounding”.

You are one of the reviewers, your task is to write a review for the article. You will 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: INFERRING MICROEVOLUTION FROM MUSEUM COLLECTIONS AND RESAMPLING: LESSONS LEARNED FROM CEPAEA Review round: 1 Reviewer: 1

Basic reporting: This is a clearly presented and interesting account of changes in recorded morph frequencies in the snail Cepaea nemoralis over time. The animal is an important model for the study of changes, rapid or longer term, in response to changing environment. The evidence for rapid response and short-term fluctuation is an area of current interest and this paper is a worthy addition to it. I do, however, think that the authors need to think more fully about the conclusions from the data and make a much more forceful case for their interpretation if they wish to stand by it. Experimental design: Experimental design is clearly stated and appropriate to the objectives of the analysis. Validity of the findings: The data are robust and statistically sound and the analysis appropriate to meet the objectives set out. I have some issues with the interpretation, which will be set out in the next box. Additional comments: Comments from Laurence Cook. From my own experience of the subject of your paper I have the following comments to make on the conclusions. Line 55. ff. The authors will know that I have reservations about the treatment of eU as a genetic category. Although Cain and Sheppard (line 206. particularly Cain, who emphasised the fact that this was a visual phenotype) noted the predictive value of eU in relation to habitat, analysis of further data suggests that it is no improvement of the category unbanded itself (Cameron & Cook. 2012. Folia Malacologia 20, 255-263). It would be safer to refer to the change as adaptive rather than genetic. Line 61. ff. Another important long-term study, which appears to show stability and also considers movement and one of the selective agents, is by Charles Goodhart ( 1956 Proc. Linnean Society of London 167, 50-67; 1956 Proc. Linnean Society of London 169, 163-167. Line 127. ff. The sample sizes for Lobith are colossal, surely much larger than a possible effective population? I have not gone back to Wolda’s paper, but is there a possibility that some frequencies quoted are ensemble values whereas with the later smaller densities you would have been examining sections? I think a note on this possibility should be added. Line 153. Does that imply that some morph or morphs entered the samples after the first sampling? Line 218. ff. Surely the caution should be applied to the short-term states not the long-term states (see below)? Line 250. ff. But patterns can be perceived, and are very much dependent on one’s confidence that the appropriate populations are being sampled at the different times (Cameron & Cook. 2012. Folia Malacologia 20, 255-263; Cook. 2013. J. Moll. Stud. 80, 43-46). General. I think it is important to investigate and interpret the results you describe. Nevertheless, the problem I have with the interpretation is in your sentence beginning line 202. If short term fluctuations characteristically exceed longer term change, apparently sometimes by large amounts, the implication must be that some kind of stabilizing force also operates. Otherwise fluctuations would accumulate and lead to monomorphism or extinction. Why does this not happen? We carried out a very detailed analysis of the data looked at by Bell (Cain et al., 1990) and in the end concluded that there was no evidence of forces shifting frequencies on a yearly basis. The estimations of ingress and egress did not show up age classes suffering from high mortality or radical difference in contribution of morph types to a particular recruitment. The alternative would have to be that, for whatever reason, the methods or circumstances of sampling sometimes introduce biases. At Point of Air (Cook & Pettitt, 1998. Biol. J. Linn. Soc. 64, 137-150) there is no evidence of selection introduced by climate etc. but plenty of reason to suppose that extensive movement of individuals took place that could account for what would look like a selective change if sampling was restricted to a limited part. In another sampling (Cook 2003. J. Conchology 38, 73-78) I also noted a change in frequency in the middle of a three-sample (in fact, a four-sample as one of yours) sequence which later appeared to be cancelled. A possible reason discussed could have been that when density was low relative visibility of morphs changed. I think that at least you need to discuss these problems more fully before concluding that frequencies do in fact change radically on a very short-term (sometimes apparently within-generation) basis.

You are one of the reviewers, your task is to write a review for the article. You will 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: INFERRING MICROEVOLUTION FROM MUSEUM COLLECTIONS AND RESAMPLING: LESSONS LEARNED FROM CEPAEA Review round: 1 Reviewer: 2

Basic reporting: This short manuscript is right to the point with an accurate and relevant message. However, I found the message so trivial that it should not be necessary to point out these things. So, what I should like of the authors is that they give more meat on the bones when referring to the older literature and give very detailed examples of when authors has drawn conclusions of long-term trends from just two data points (one historical and one contemporary). Moreover, it would be interesting to learn if this is the case for colour-variation in Cepaea snails, usually, or if similar inferences have been drawn for other traits that are tentatively under selection. Examples would be welcome. Finally, I would suggest yet another possible way of assessing interannual variation in order to separate this from long-term trends if only one historical sample is available: Instead of using only one year of contemporary sampling, repeat sampling over 2-3 contemporary years to assess interannual variation. Experimental design: The experimental design is straight forward and only basic statistics is used. Validity of the findings: I wish I could conclude that this information is of very little value because conclusions of long-term trends are never drawn from only two data points. But, if this is so (and as suggested above, I think the authors must do more to show that this is actually the case) then this study has some value. In particular, if the authors (as is partly done) suggest approaches to solve the problem. However, if the authors fail to find good example of studies in which conclusions are based on only one historical and one contemporary sample, then I think this study has only very minor value, as this should be rather obvious to anyone. Additional comments: My main point is that the authors should put more emphasize on examples of studies in which this has been a problem. It would also be good to focus the study more on how the problem can be solved. There is some discussion about this but this part can be more of a focus in both Abstract, Introduction and aim of the study. How do you deal with a situation when there is one historical data point available.... Such a focus would be much more of a contribution to the scientific society than only pointing to an obvious problem.

You are one of the reviewers, your task is to write a review for the article. You will 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: INFERRING MICROEVOLUTION FROM MUSEUM COLLECTIONS AND RESAMPLING: LESSONS LEARNED FROM CEPAEA Review round: 1 Reviewer: 3

Basic reporting: Please see general comments. Experimental design: Please see general comments. Validity of the findings: Please see general comments. Additional comments: The authors examine how the frequencies of genetically-based color morphs of Cepaea change from historical samples to the present, paying special attention to how conclusions can change depending on which specific year from a given time period is used for a comparison between time periods. In essence, conclusions as to how frequencies change from the past the present depend on which specific year from the past is used for comparison. The implication is that studies need to have replicate samples from each time period if they are to generate reliable estimates of change BETWEEN time periods. This is all true but not especially surprising, as long term data sets (e.g., Darwin’s finches) show tons of year-to-year variation despite the lack of long term trends. I suppose there isn’t any harm in restating the point for another study system but the insight isn’t particularly novel (see also fig 2 of Hendry and Kinnison 1999). I do object to how the implication is sometimes stated as the lack of replicate sampling leading to incorrect or “misleading” inferences. Instead (as is implied at other parts of the MS), the robust conclusion is that short term evolutionary changes can be greater than long term changes. I would prefer that to be the point of motivation for the paper rather than the more obvious and less interesting point that somehow “incorrect” conclusions can result from incomplete sampling. Instead, the observed changes are “correct” but they don’t necessarily reveal long term average trends. Thus, the problem isn’t in how the comparison is done but rather in how an investigator might attempt to extend a particular temporal comparison to general statements about long-term trends. This is perhaps a subtle point but the wording in the MS could be much improved in this regard. Technical comments: 1. Line 38: cite general meta-analyses and quantitative reviews rather than a few arbitrary examples out of hundreds that could be cited. 2. Line 45: Of course, this point is obvious from essentially any long-term study. 3. The emphasis seems to be on the fact that SIGNIFICANCE of temporal changes depends on year of sampling. However, significance is not what matters here (given that significance is also dependent on sample size). Instead, it is effect size that matters – that is, the actual frequency change. So the real question is how much allele frequency change differs depending on the year of sampling. Of course, these data are in the figure but the point is not clear in the text, nor is the figure optimal. For best inference, maybe prepare a graph of “change using one historical year” on the x-axis and “change using another historical year” on the y-axis. Deviations from the 1:1 line then reveal the extent to which the chosen year matters. 4. Also, the allele frequency change that can occur is dependent on the starting allele frequency, so it would be good to examine if the two are correlated. 5. As the lead author has shown in other work, the multivariate phenotypes of snails can vary among similar habitats. I would like to see a graph something like Fig 3 of Ozgo et al. (2011 – BJLS 102:251-262). 6. The starting part of the Discussion essentially just lists the results again. I think the discussion could be much more interesting by focusing on the processes that shape short-term versus long-term change and how these process influence expectations under climate change (or something like 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: AUGMENTED CARTILAGE REGENERATION BY IMPLANTATION OF CELLULAR VERSUS ACELLULAR IMPLANTS AFTER BONE MARROW STIMULATION: A SYSTEMATIC REVIEW AND META-ANALYSIS OF ANIMAL STUDIES Review round: 1 Reviewer: 1

Basic reporting: 'no comment' Experimental design: 'no comment' Validity of the findings: 'no comment' Additional comments: Manuscript entitled “Augmented cartilage regeneration by implantation of cellular versus acellular implants after bone marrow stimulation: a systematic review and meta-analysis of animal studies (#18289)” is well written and discussed. However, following comments should take care during the revision. 1. Manuscript lacking the future implication of this kind of analysis in cartilage tissue engineering. Please discuss the importance of the study in discussion and conclusion parts of the manuscript. 2. Growth factors are the one of the important components of tissue regeneration along with scaffold and cells. It would be nice to include the growth factors description in the manuscript. 3. Stimulation in form of bioreactor is again one of the crucial factors for differentiation of stem cells into chondrocyte lineage. Please also include the bioreactor in search, and discussion in the manuscript. 4. Recently, cryogels special kind of the hydrogel which synthesized at sub-zero temperature to achieve good porosity and mechanical strength in the scaffold, gained significant attention in cartilage tissue regeneration. Please also include cryogels (PMID:28230077, 27886065, 27185069, 25498693 etc.) in the search study. 5. In line 185, what’s I2? Please define and discuss the importance of I2. 6. Line 132, what’s Safranin-O-staining does in cartilage research? Please discuss briefly. 7. Please briefly discuss “Cochrance approach” in line 153. 8. Conclusion lacking the importance of the study. 9. Please include growth factors and bioreactor in figure 1 illustration of cartilage tissue engineering.

You are one of the reviewers, your task is to write a review for the article. You will 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: AUGMENTED CARTILAGE REGENERATION BY IMPLANTATION OF CELLULAR VERSUS ACELLULAR IMPLANTS AFTER BONE MARROW STIMULATION: A SYSTEMATIC REVIEW AND META-ANALYSIS OF ANIMAL STUDIES Review round: 1 Reviewer: 2

Basic reporting: The authors have used clear language throughout and present a well defined research question with good background information. I recommend removing the word "unbiased" from line 80 and line 312. A systematic review is not an unbiased overview as it is subjected to biases at all stages of the review process. For example, the authors make decisions on types of study to include, which databases to search and the methods to use. Line 284-287. Please include effect size data for adipose-derived stem cells (mean and confidence intervals). Experimental design: This is a primary research article with a well defined research question- to present a overview of all studies reporting regeneration of articular cartilage by implantation of cell-laden versus cell-free biomaterials in the knee and ankle joint after bone marrow simulation in animals comparing different cell types and methods. Did the authors publish a study protocol before carrying out their review? Can you provide a link to this protocol or a copy of the protocol? I would recommend that the authors include the specific search terms used in their search strategy instead of referencing other publications. This could be included in the methods or as supplementary information. I would question whether the use of semi-quantitative histological scoring systems (which are observer-dependent and thus subjective (Ref. Rutgers et al. 2010) is the best method to determine the success of regeneration? I suggest discussing the limitations of this as outcome measure in the review. I do not understand the meta-analysis methods used specifically, line 162-163. "modeled with a compound symmetry covariance matrix, as this resulted in the lowest Akaike Information Criterion value." Can you give more details? I would recommend statistically testing for asymmetry in the funnel plot. The reviewers do not report data extraction being carried out by two independent reviewers. I suggest discussing the limitations of this. Validity of the findings: The included data has appropriate controls. As requested above, I would like a bit more information on the meta-analysis methods before assessing if the results are statistically sound. The conclusions based on the results appear to be justified and linked to the original research question. Additional comments: This is a well written review. I would recommend that the authors revise the manuscript bu clarifying some methods.

You are one of the reviewers, your task is to write a review for the article. You will 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: AUGMENTED CARTILAGE REGENERATION BY IMPLANTATION OF CELLULAR VERSUS ACELLULAR IMPLANTS AFTER BONE MARROW STIMULATION: A SYSTEMATIC REVIEW AND META-ANALYSIS OF ANIMAL STUDIES Review round: 1 Reviewer: 3

Basic reporting: The authors of this manuscript have presented a summarized overview and meta-analyses on the previously published research articles on the augmented cartilage regeneration after bone marrow stimulation. This study utilized various tools for screening the suitable reports on this topic with full details of experiments to compare the results for concluding remarks. This review suggests that cellular biomaterials/implants have higher cartilage regeneration efficacy compared to acellular biomaterials/implants. In my understanding, this study of authors is the continuation of their previous finding (Ref. 8 of the manuscript), which demonstrated the role of acellular biomaterials in cartilage regeneration. The manuscript is written well, however, few grammatical errors have been noticed, that can be rectified in revision. Experimental design: This is a systematic review and meta-analysis of the already published research. The findings are relevant to understand the suitable combination of biomaterials, biologics, and cells for the augmented cartilage regeneration. The study was started with search finding of 11,248 reference articles on this topic, which were further screened rigorously to obtain 146 full-text studies for further analysis. Validity of the findings: The provided methods of research data screening and meta-analyses are suitable to demonstrate the unbiased conclusion on the role of cellular/acellular biomaterials in cartilage regeneration after bone marrow stimulation. Authors of this study concluded that cellular biomaterials have shown 18.6% higher regeneration capacity compared to acellular biomaterials. I believe that the provided hypothesis on augmented cartilage regeneration using cell seeded biomaterials after bone marrow stimulation is not significant to understand the basics behind higher regeneration ability. I may suggest discussing more in this aspect in the discussion section using suitable reference articles will enhance the understanding and readership quality. Additional comments: I have found few points that need to take care by authors of this manuscript; 1. Section 2.1_the provided ref. (like 32, 33..) are not clear. Can authors justify the relevance of these references? If these references are used in support of methods used, then they need to be cited with proper elaboration. 2. section 2.2, 2.4 etc., Authors have used the term 'reviewer' at few places to demonstrate their role, I believe that the correct terminology to indicate their role by the name of 'authors' of the present study. 3. I have found the repetition of many sentences in little-modulated form, for example; line 228-230 'cells were either.....' is providing the same meaning as provided in the next sentence, line 230-233, 'in some studies....'. Authors should revise the manuscript in this aspect to make more crisp and attractive text content.

You are one of the reviewers, your task is to write a review for the article. You will 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: AUGMENTED CARTILAGE REGENERATION BY IMPLANTATION OF CELLULAR VERSUS ACELLULAR IMPLANTS AFTER BONE MARROW STIMULATION: A SYSTEMATIC REVIEW AND META-ANALYSIS OF ANIMAL STUDIES Review round: 2 Reviewer: 1

Basic reporting: Well-defined question, clearly written Experimental design: Methods and analysis described clearly and included sufficient details. Validity of the findings: The conclusion is well stated and directional for preclinical research. Additional comments: Thanks to the authors for addressing my comments. The manuscript is significantly improved and readable well. Now, I am recommending the manuscript for publication in PeerJ journal.

You are one of the reviewers, your task is to write a review for the article. You will 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: AUGMENTED CARTILAGE REGENERATION BY IMPLANTATION OF CELLULAR VERSUS ACELLULAR IMPLANTS AFTER BONE MARROW STIMULATION: A SYSTEMATIC REVIEW AND META-ANALYSIS OF ANIMAL STUDIES Review round: 2 Reviewer: 2

Basic reporting: NA Experimental design: NA Validity of the findings: NA Additional comments: The modified manuscript is showing improvement in its technical information and better readership quality. The revised manuscript is now recommended for consideration for publication in Peer J.

You are one of the reviewers, your task is to write a review for the article. You will 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: MULTIVARIATE ORDINATION IDENTIFIES VEGETATION TYPES ASSOCIATED WITH SPIDER CONSERVATION IN BRASSICA CROPS Review round: 1 Reviewer: 1

Basic reporting: Clear, unambigous, professional English language used throughout. Intro and background were used to show content. Structure conforms to Peerj standards, discipline norm, or improved for clarity. Figures are relevant, high quality, well labelled and described. Raw data supplied. Experimental design: Original primary research within Scope of the journal. Research question well defined, relevant and meaningful. It is stated how the research fills an identified knowledge gap. Rigorous investigation performed to a high technical and ethical standard. Methods described with sufficient detail and information to replicate. Validity of the findings: Data is robust, statistically sound and controlled. Conclusions are well stated, linked to original research question and limited to supporting results. Speculation is welcome, but should be identified as such. Additional comments: The authors present an interesting research that confirms the importance of environmental heterogeneity when establishing populations of generalist predators useful for agricultural crops. In addition, the manuscript presents statistical tools that can be useful in research on agricultural crops that are conditioned by seasonality and extreme weather phenomena. If there is a weakness, it is in the way that some of the results are presented (as I have noted above) which should be improved upon before Acceptance. Various references to supplementary material are made in the results section. It becomes difficult to follow the text with so many references to different figures that do not appear in the main manuscript that are mentioned profusely in the results. Please clarify it. Specific comments: Line 71: The reference to spiders studied as belonging to 'Araneida' is risky because the taxonomic organization of spiders (in terms of order and suborder) is still discussed. In any case if the authors wish to maintain their denomination they should explain which criterion they follow to define the spiders like 'Araneida' and not 'Araneae'. Line 108: Indicate the bibliographic sources used to identify the spiders.

You are one of the reviewers, your task is to write a review for the article. You will 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: MULTIVARIATE ORDINATION IDENTIFIES VEGETATION TYPES ASSOCIATED WITH SPIDER CONSERVATION IN BRASSICA CROPS Review round: 1 Reviewer: 2

Basic reporting: English should be carefully checked throughout. I pointed out a few issues and some paragraphs that need re-writing. More background literature should be added to hypotheses and discussion. The discussion feels incomplete. With all the analyses done a lot more can be discussed. Experimental design: The methods have some issues pointed out in the PDF. Also, the methods are not clearly described and make it confusing to understand. Validity of the findings: The data would me more robust by adding suggested analyses (see PDF). Discussion and conclusion need to be re-written and or expanded. Additional comments: This is a great and valid contribution, however the paper has several flaws all pointed out in the PDF. Mainly the written english, some of the methods and the discussion.

You are one of the reviewers, your task is to write a review for the article. You will 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: MULTIVARIATE ORDINATION IDENTIFIES VEGETATION TYPES ASSOCIATED WITH SPIDER CONSERVATION IN BRASSICA CROPS Review round: 1 Reviewer: 3

Basic reporting: The manuscript is globally understandable. However, there are numerous grammatical and spelling mistakes, as well as syntax errors that need to be corrected. The introduction and particularly the discussion are often too general and miss concrete examples, especially the discussions about the pests/natural enemies/vegetation types. In addition, some parts of the discussion (quite general) would best fit in the introduction. Some information is missing in the figure captions. Figure 6 is illegible. Some figures included in the Supplemental material would be best in the MS (e.g., RDA graphs). Also, one table could be added to summarize some pertinent information about the methodology. Main results needed to answer the research question are provided. However, numerous figures are part of the Supplemental material (only figure about site 1 is provided in the manuscript). Experimental design: The manuscript entitled "Multivariate ordination identifies vegetation types associated with spider conservation in brassica crops" meets the aims and scope of the PeerJ journal. The purpose of the study is well stated and relevant, and the justification of the research is provided. However, the hypothesis is too general and could to be more precise. It would be interesting to make hypotheses about which habitat types would provide the highest abundance and diversity of spiders in brassica fields and why. The sampling design is acceptable to answer the research question. However, a more rigorous work could have been done by identifying the spiders to the species level (instead of the morphospecies) or by conducting the experiment over several years. The most important issue with this manuscript is the methodology section (experimental design and statistical analyses) that needs to be further explained. There is a lack of details at several places in the manuscripts that limits the comprehension of the work done. For instance, a figure of the sampling design or a Table that summarizes all the essential information may help to understand how the samplings was performed (e.g., number of sampling points per site, number of sampling points per habitat types and per site). The statistical analyses section also needs further details to completly understand what was done and why. Validity of the findings: The authors succeed to identify some habitat types that seem important for the conservation of spider abundance and diversity. However, the results greatly differ between the 3 sites and discussion remains often too general. The discussion may be improved by being more specific and, for instance, by giving specific recommendations (overall, which habitat types exactly should be present near brassica crops to provide a high abundance and diversity of spiders?). The data consisted in only one year of sampling and, due to an exceptional event (typhoon), not all the sampling dates were taken into account in the analyses. However, the data seem correctly analyzed. Because of the numerous analyses done, the "take-home message" is not always clear and needs to be better identified. The discussion may also be improved by adding critics of the methodology/results found. For instance, results and discussion should be moderated since 1) the study was performed in only one year and that an exceptional event happened during this year (typhoon), and 2) identification of the spiders were not performed at the species level (but morphospecies, so with possible bias). Additional comments: General comments The manuscript is globally understandable; however there are numerous grammatical and spelling mistakes, as well as syntax errors that need to be corrected. The major issue of this article is the lack of important information, especially in the methodology and result sections, which limits the comprehension of the experimental design, the statistical analyses performed and the results obtained. In addition, the introduction and particularly the discussion are often too general and miss concrete examples. The discussion could also be improved by adding critics of the methodology used and results found, and comparisons with other articles observing effects of habitats type on spider abundance and diversity (not only natural enemies in general). Abstract L16: “the importance of vegetation…”: You may precise “Natural vegetation”. L17: Replace “disturbance” by “disturbances” L18: Add “the” before “types of vegetation” L19: Remove “into” L19-20: “Here we explore… and non-crop vegetation”. You may consider rewriting this sentence, it sounds odd. Keywords I am not convinced about the term “Ecological Engineering” Introduction General comments The introduction is overall well written but often lacks of details. It is often too general and may be improved by given some precise examples (see specific comments below). Specific comments L38-45: This part could be improved by adding an idea of the period of time you refer to. “Anthropogenic activities” since when (from few years ago, decades ago…)? L71-74: More information should be given regarding the spider communities found in brassica crops. You mentioned it in the discussion but it can also be described in the introduction. Actually, some information given in the discussion should actually be part of the introduction (e.g. L236-241). It is also important to precise in the introduction (not only in the discussion) that spiders are generalist predators. Furthermore, essential information is missing: Which brassica pests can be controlled by spider in brassica fields? Please provide more details. L71-84: The last paragraph could be divided into 2 paragraphs: 1) One paragraph about spider communities found in brassica crops and their importance to control pests; so you can add more details about their biology and ecology (e.g., do they overwintered in non-crop habitats adjacent to crop fields?) and 2) second paragraph about the purpose of the study and hypotheses. L81-84: The hypothesis remains very general. “different vegetation communities would share the spider fauna of brassica fields to varying degrees…”. Did you have an idea of which habitat types would share more species with brassica crops? Material and methods General comments The most important issue of this manuscript is the lack of important information in the methodology. A lot of details are missing and prevent the complete comprehension of the study. The methodology section could be improved by adding a table that compiled all relevant information for the comprehension of the sampling design: sites, total number of sampling points per site, number of each vegetation type sampled per site, details about the sampling period (exact dates of sampling)... Also, a figure of the sampling design may help understanding how the sampling where performed (e.g., how many sampling points in each habitat type?). Finally, I wonder whether one year of data is relevant enough to make conclusions, as effects of landscape on insect communities can greatly vary from one year to another, and considering that one event had perturbed the samplings. Specific comments L90: Which brassica species? Were there different species? If so, it would be important to list the different species. L91: “50 x 50 m grid”. Why this distance? You need to justify the choice of this specific scale. Is that because of the dispersal ability of spiders? L95: “not uncommon”. So it is common? L98-99: First, it is not clear in the MS whether you have 25-29 sampling points for each one of the 5 sampling periods or for the entire sampling period. Also, when looking at your dataset, it is not clear how many sampling grids you had per site. Did you initially have 30 sampling points for each date and site? In Minquing for instance, in the data file, the sampling points are numbered from 1 to 30, but 19 is missing. Why? So there are a total of 29 points, but only 28 in the other data file (Data_set_Sp). How can you explain that? All these details are essential to understand the experimental design. If there were 30 sampling points at the beginning you should say so. Same problems appeared with the other sites (Nantong 1: 25 or 26 grid points? Nantong 2: 27 or 28 grid points?) Second, how many samplings were performed in each vegetation types? And how many vegetation types did you consider? These details could be summarized in a Table. L99: “all vegetation types”: I do not understand the classification of the different vegetation types. This must be clarified in the methodology section. What does “non-crop vegetation” refer to? It is mentioned in the methodology (L92-94) that “non-crop vegetation consisted of adjacent field margins and fallow fields… as well as uncultivated areas…”. But in the results, non-crop vegetation, fallow and field margin are separated. So I wonder what were included in the “non-crop vegetation” category. L100: “five occasions”. You need to precise the different sampling dates (could be included in a Table, as suggested before). Was all the vegetation types sampled during the 5 sampling dates? It is not clear. L103-104: So how many sampling dates do you have overall? L108-109 “(genus in some cases) and assigned to morphospecies”. It is not clear whether the analyses take into account the genus or morphospecies level. You need to be more precise. I assume that the morphospecies was used to calculate the diversity index. If so, it should be mentioned. You don’t need to say that some species were identified to the genus level if you don’t do analyses at this level. Statistical analyses General comments The statistical analyses seem ok to me there are a lack of details and missing important information at numerous places, which limits the comprehension of the analyses done. I am familiar with RDA and variation partitioning analyses, but less with cluster analyses so I gave fewer comments on this part. In addition, the authors need to verify that the correct references are cited for all R packages mentioned. L114 “spider abundance and diversity data”. What was the unit of replication in the analyses? Did you use the total abundance or mean abundance? As I understand when looking at your dataset is that you keep the data per sampling point. But, did you pool all the data from the 5 sampling periods? Did you use the genus or morphospecies level to calculate the Shannon index of diversity? All of this is not clear and need to be mentioned. L117: Hellinger transformation: Also used when a lot of zeros in the matrix, like in your dataset. L121: “differing vegetation types”. “different” instead of “differing”. What vegetation types did you include in the variation partitioning? L121 “principal” instead of “principle” L121-122. You may better explain why you decide to perform a variation partitioning between the vegetation type and the principal coordinates of neighbor matrices (PCNM). You may better explain what the PCNM is about. L124: “effects of distance”: the distance between what and what? Is that the distance between the different sampling points within each site? L128: It is said that the “adjacent land cover” is different between sites. It would be good to have a more precise idea of these differences. Can you add a characterization of the 3 different sites? For instance in a Table (e.g., including the proportion of the different land covers)? L129-140. I am less familiar with this sort of analyses, so I am less able to criticize thoroughly this part. However, I have one comment about the dissimilarity index. L145: “occurrence of spiders”. Occurrence per site? Per sampling point? For the entire season or per sampling dates? L147: “spatial correlation”. Did you mean “Spatial autocorrelation”? L148-156. The term “spatial variation map” is not clear. In addition, this part looks more like results than methodology. L150-151. I am familiar with multivariate analyses and forward selection but I don’t understand what you did here. Why the “spatial variation maps” were included in a forward selection. L155; “Whist”. ? Verify English. L159: “p-value<0.00”. Replace by “p-value < 0.001”). L159-161: You need to cite the references for all the R packages used. Results L163-164: It is important also to have the details of the captures per site (abundance and diversity). Moreover, why didn’t you identify to the species level? Did you confirm the different morphospecies with an expert? If not, you should discuss about it in the discussion (you may consider having an over/under-estimation of the diversity). L164-171: “Variance partitioning showed that abundance of spider families was vegetation-type dependant…”. Important information is missing here. First, Fig 1a misses the total value of the effect of the variable X1=Vegetation and X2 = PCNM (by the way, you must change the second X1 in Figure 1 to X2, because you have 2 variables called X1). The value 0.13 means that the vegetation type alone explains 13% of the variation in spider abundance, which is not very high. What is the total effect of the variables X1 and X2 (unique contribution of each variable + share effect)? If the residual effect is 0.94, than the total effect of X1 and X2 is 6%, which is quite low. Second, you precise in the following sentence that you have tested “the significance of each variance fraction”, but you precise only one p-value (p=0.07). Is that the p-value for the global model (including effect of X1 and X2)? Also, you mentioned that there are “strong effects” but I disagree with that. All the p-values you mentioned are > 0.05. I disagree also with the term “strong effect”. Actually, you have a strong effect when the R2 of your model has a high value, and the model is highly significant when p-value is very low. The description of the results needs to be rewritten. Third, you mentioned only the results of the variation partitioning for spider abundance in Minqing, and spider diversity in Nantong 1 and Minquing. What are the results of the other variation partitioning? Even if you found no effect, you have to say it. It is not clear. L174-176: I am not totally in agreement with you observations. According to me, when looking to Fig 3, Non-crop vegetation are associated with Thomisidae (maybe Oxyopidae), but Salticidae and Lycosidae are more associated with Fallow land than Non-crop vegetation. L177 “pumpkin showed a strong association with Oxyopidae”. I disagree. The association is not so strong. L177-183 It would be interesting to have all the RDA graphs in the MS. L180: “Oxyopidae showed …” Oxyopidae abundance or diversity? Please, be consistent in the structure of the sentence. Put the species family first (e.g., Oxyopidae showed strong association with….) or the habitat first (e.g., Non crop vegetation are associated with…) L181: Replace “Fig 2a” by “Fig S2a” L184-185: Quite general. “most of the spider families” “Other surroundings vegetation types”. Can you be more precise. It is difficult to understand the results for people that have never done such analyses. Please explain in more details what do you mean by “same coulour in heatmap”. You should explain more precisely, at least in the figure caption, what does the color key correspond to (what is the unit of the value 0-10?). L186-187: “Lycosidae showed strong differences in abundance between…”. You can add “and diversity” so that you don’t have to repeat Lycosidae in the next sentence. L188-189: “Oxyopidae depicted strong differences in diversity among different vegetation types…(different colour)”. I disagree for Nantong 1 and Nantong 2 because it is actually the same colour for all the vegetation types (Fig S3b and S4b). L189: Remove one “Fig” L192-193: What does it mean concretely to have such AU-value and BP values? Can you explain briefly? It is important to give some precisions if you want people that are not familiar with such analyses to understand your results. Here, it looks like only a series of values, without explanations. L196: “Spatial autocorrelation”: between sampling points? L197: Spatial correlation or autocorrelation? Do you refer to the same thing? Please explain what you mean. Be consistent in the writing and correct when needed Figure 1: What is the meaning of the intercept? Figure 2: Which site? Figure 4-5: Did you sampled in brassica fields? It is not clear n the methodology. Why Brassica appears in Figures 4 and 5 and not in the RDA graphs? In the figure caption, Figure 6 is very small and illegible. Discussion General comments First, several part of the discussion are too general and need some precisions. Some hypotheses could also be added to explain some of the results found. The discussion may also be improved by adding critics of the study (e.g., over/underestimation of the spider diversity because no identification to the species level…). What could have been done or analyzed (e.g., analyze of the effect of the vegetation type on the distribution of spider depending on the sampling periods…). Results would have been different if more than one year studied? Specific comments L208: “and non-cropped and non-sprayed zones”: You may rephrase (the two “and” are a bit redundant). L210: “various pests”: can you give examples of pests controlled by spiders? L214-216: “Requires….to require”: Replace one of the two “require” L218-220: What did you mean by “conventional statistical approaches” and “advanced statistical approaches”? I am not sure that the pertinence of this section. L224: 2a function of spatial distance”: Distance between what and what? L224: “its intercept”: What does it mean exactly in an ecology point of view (not only statistics)? L225-228: So it means that “cursorial families” are more widely distributed than “web-builders”? Can you explain why or at least give some hypotheses? L227-228: “as observed (Blitzer et al. 2012…”. Correct the end of the sentence: “as observed by Blitzer et al. (2012) and Schmidt et al. (2003). L230: “spatial distance”. It is not clear (between what and what?). L230: “cabbage”. In the rest of the MS, the word “brassica” was used. Please, be consistent in the terminology. L231: “meso-scale”. Is 50m really a meso-scale for spiders? I would have used the qualification “local scale” instead. Unless you can have justifications. L232: “alternative food”: Can you be more precise (e.g., examples of preys)? L236-214: This part should be part of the introduction. 241-244: I do not understand. Are you referring to your results or to other authors (Schmidt et al 2003…)? L244-246: Too general. “alternative food”, “place where spider have time and space…” Can you be more precise? What kind of food and what kind of habitat? L252: Replace “this” by “which” L253-255: “may be because of the availability…”. You may replace by “may be due to the availability”. Remove “may also be”. L267 “vary in space”: Did you mean between habitats? L269: “certain adjacent crop”: Can you be more precise and give some examples? Can you give some recommendations? Which habitat type should be present near brassica crops to provide a high abundance and diversity of spiders? L278-280: Spatial correlation or autocorrelation? L280: Replace “with” by “and”. “various vegetation types”: Can you precise? L282: I don’t see the need here to emphasis again about the typhoon. L283-284: I am not totally convinced. L286: Replace “is” by “has been” L287: Replace “likely” by “great” L288: Replace “nearby to crop fields” by “nearby crop fields”. Replace “conservational” by “conservation” L293: Replace “Further” by another word (In addition, furthermore…) L294 “temporal effect”. As you have several sampling periods, it would have been very interesting whether the effect of vegetation type on the distribution of spider differ over time.

You are one of the reviewers, your task is to write a review for the article. You will 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: ANCIENT DIVERGENCE TIME ESTIMATES IN EUTROPIS RUGIFERA SUPPORT THE EXISTENCE OF PLEISTOCENE BARRIERS ON THE EXPOSED SUNDA SHELF Review round: 1 Reviewer: 1

Basic reporting: The quality of written communication is excellent. There is a wide literature base employed, although literature from other homologous study systems (land faunas that were periodically connected via Pleistocene sea level fluctuations) was not employed. Many examples exist (Taiwan, Japan, Tasmania, New Zealand, Sri Lanka, England). Tables and Figures were all of a high standard, although Fig1 needed the locality names on the figure for each sample, to avoid the need of cross-referencing with other sources. Likewise, Figure S1 only had codes for samples, and hence was uninformative without consulting other information. These figures need to be "stand alone" with respect to interpretation. One of the main deficiencies was that the Introduction started straight with the study species. The question is very interesting and broadly relevant, so the paper should start with the over-arching issue, that is of broadest interest and significance (genetic history of taxa on landbridge islands, and predicting their past connectivity using SDMs as verified by genetics). Experimental design: It was unfortunate that the previously studied specimens and DNA sequences could not be integrated into this study. As such, it comes across as the third study to make a "first attempt" at the phylogeography of this species. In terms of phylogenetic reconstruction, concatenation of genes is only one option. The alternative, that is more realistic, is to build population trees that allow each gene to have evolved along an independent gene tree. There are methods out there that do this (including BEAST). That said, it seems most of your variation is harboured in mtDNA, and the nDNA are not adding much. I was not sure about your models employed during tree building. I cannot see why the BEAST strategies needed to be different from MrBayes and RAXml, and vice versa (particularly with respect to codon models). Even if you need to fragment your datafile, you can make datasets of 1st codons all together, all 2nd codons together, and so forth, and then give them separate models in MrBayes or RAXml Given the depth of divergences at play, is a coalescent tree prior likely to be the most appropriate for BEAST analysis, versus a Yule tree prior? I am not very familiar with MaxEnt, but I am familiar enough to know that a basic acceptance of default parameters is inappropriate, and you should find good, more rigorous examples of its implementation to follow as a guide. This will indicate the degree of sensitivity to parameter selection. No outgroup in the analyses? Although the Philippine split looks obvious, without an outgroup, you actually cannot say where the deepest split (phylogentically) occurs. Validity of the findings: The findings are probably valid, but rely heavily on the mtDNA variation, which should be more adequately explored. Otherwise, the results may be given the impression that they are more reliable (strong multilocus support) than reality. One of the big issues I have is an apparent contradiction. The species could not move across the Sunda shelf, to mix with other populations. However, the SDMs should that they also did not persist at most of the contemporary sample sites during the Pleistocene glaciations. So, if they couldn't move widely, and they could not persist where they are today, how did they come to have their present distribution, and indeed, deep genetic divergences? I would be interested to know whether there are any modern day homologs of the glacial Sunda shelf habitat, and whether the species occurs there today or not. Essentially, is the Sunda shelf glacial habitat novel, relative to today's environment? If so, we have no evidence (from present species distributions) that they could not occupy this historical habitat, because there are no modern homologs. Additional comments: The paper seems to end strongly on the point "we need more sampling". This weakens the study, because it points out your main deficiency, and leads to the question, why didn't you do more sampling?

You are one of the reviewers, your task is to write a review for the article. You will 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: ANCIENT DIVERGENCE TIME ESTIMATES IN EUTROPIS RUGIFERA SUPPORT THE EXISTENCE OF PLEISTOCENE BARRIERS ON THE EXPOSED SUNDA SHELF Review round: 1 Reviewer: 2

Basic reporting: My main suggestions for revision are in "General Comments for the Author" below. Included in these comments are suggestions for improving the focus of the text on the major discoveries made possible by the new data. Much of what exists in this draft seems largely extraneous to this central purpose. Experimental design: I have no major changes to suggest on this topic. Validity of the findings: Validity seems fine to me. Additional comments: This manuscript reports new comparative DNA sequence data and phylogenetic analyses for skink populations of the species Eutropis rugifera. Geographic sampling is broad but sporadic, providing a historical biogeographic overview for this species. The results are worth publishing, but the manuscript needs a reasonable amount of revision to be effective in highlighting the new findings. The major problems with the presentation are evident from the abstract, which should describe the new data (genes, number of populations sampled) and analyses, and the major conclusions. For the latter, a verbal account of the contents of Figure 3 would be a good strategy: “The earliest phylogenetic divergence within E. rugifera separates Philippine populations from those of the Sunda Shelf with an estimated divergence of 16 million years ago; the Philippine populations thus cannot be considered conspecific with their Sunda congeners. Within the Sunda Shelf, the oldest divergence separated Borneo populations from a common ancestor of populations in peninsular Malaysia and the Mentawai Archipelago approximately 6 million years ago. Our data suggest that within Borneo, populations from Sabah and Sarawak separated approximately 4-5 million years ago in the early Pliocene, followed by further cladogenesis in Sarawak through the Pleistocene. Separation of continental Malaysian populations from those in the Mentawai Archipelago occurred approximately 5 million years ago. Divergence among samples from the Mentawai Archipelago likely dates to the Pliocene/Pleistocene boundary approximately 3.5 million years ago, and our samples from continental Malaysia appear to coalesce in the middle Pleistocene, about 1.5 million years ago.” With this temporal and spatial framework, focus the historical biogeographic analysis and discussion specifically on these events. Given the full temporal scope of the divergences revealed within E. rugifera, the emphasis on the Pleistocene in the abstract and elsewhere is misplaced; only the most recent divergences within Sarawak and coalescence of the continental Malaysian samples date to the Pleistocene, so these are the only events for which Pleistocene climatic history is particularly relevant. Limit your discussion of the historical climate of the Sunda Shelf to what should be your main point: the most parsimonious explanation of the historical events revealed by your data analysis. Your bar on Figure 3 showing episodic land connections probably suffices for this purpose. Extensive discussion of grasslands and rivers in the past history of the Sunda Shelf seems unwarranted by the data; introduce these considerations only if necessary to explain a specific case of cladogenesis on Figure 3. Some additional problems in the abstract also recur throughout the manuscript. “Last Glacial Maxima” is a misnomer. The correct phrase is “Last Glacial Maximum,” the single most recent time that glaciation reached a maximum southern extension from the northern pole. The LGM occurred about 10,000 years ago, much too recent to be of explanatory value for the divergence events revealed by your data. Statements such as “unexpectedly ancient divergence” in line 26 are too vague to be informative; substitute the specific numerical estimates from your data and analyses for “ancient,” “recent,” “close” or “distant” in describing divergences, as illustrated in my suggested rewording of your main discoveries. In describing recurring cycles of inundation versus exposure of Sunda land areas, I favor “episodic” rather than “periodic,” the latter being a special case of “episodic” in which successive episodes have a constant temporal spacing. Figure 3 indicates that perhaps the Pleistocene cycles approach periodicity, but “episodic” is the more conservative claim. One important concept is missing from your discussion of historical biogeography: “incumbency” of populations in their interaction upon secondary contact. Vicariant populations of E. rugifera that have evolved as distinct lineages over many millions of years likely have made secondary geographic contact episodically following their initial divergence as climatic conditions temporarily removed a geographic barrier (for example, a drop in sea level permitting formerly separated populations to make contact by expanding into a newly formed land bridge between them). Because of their ecological similarities, each population lineage likely blocked the spreading of its geographic neighbor into its own range at these times of transitory secondary contact. The geographic pattern of vicariance thus persisted over many millions of years despite episodic removal of the physical barrier that initially led to allopatric speciation. Many authors have discussed this phenomenon, perhaps most notably John Wiens and Kenneth Kozak, although my recollection is that the term “incumbency” traces to writings by Elizabeth Jockusch and David Wake on Batrachoseps salamanders. Given your sparse geographic sampling, detailed discussion of incumbency is not advised here; the phenomenon nonetheless allows you to focus your explanations on what caused the initial cladogenesis of evolutionary lineages without having to explain why they subsequently maintained their patterns of vicariance despite episodic geographic contact during the Pleistocene. I focus next on the figures, which mostly succeed in conveying the main new discoveries, but which could be improved. Figure 1 – Label all of the main areas discussed in your text. The Mentawai Archipelago is the most obvious omission; move “Eutropis rugifera Range” to the top of the circled area to make room to label the Mentawai Archipelago where “Eutropis rugifera Range” now appears. Within Borneo, distinguish Sabah and Sarawak. Because you mention them in lines 235-236, label Nicobar Islands, Bali and Bawean Island. Also, number the localities and use the same numbers to cross-reference taxa in Figures 2 and 3. Figure 2 – After numbering the geographic localities shown on Figure 1, include the numbers in the tip taxa for cross-reference. Figure 3 – Again, add numbers to the tip taxa to reference Figure 1 as requested also for Figure 2. Because the main purpose of this figure is to convey the temporal information, remove Bayesian support from the nodes and replace it with the estimated time of divergence. If you want to convey branch support, then perhaps put a small black circle at each node whose Bayesian posterior probability is 0.95 or greater. Then your numbers and confidence intervals on the figure will be synchronized. Because only one event of cladogenesis coincides with the transition from mesic to xeric climate at the late Pliocene, this point is perhaps overemphasized. The overall importance of this transition is to explain why episodes of land connection are much more frequent following this point, although your most important events of cladogenesis precede it. Figure 5 – Perhaps cut this analysis entirely. My earlier comments explain why I consider it largely irrelevant to your data. You want to have some hypotheses of the geographic barriers that explain the initial cladogenesis of your main lineages. It is potentially of interest to identify what areas of refuge allowed these lineages to persist when unfavorable climate restricted their distributions; nonetheless, your geographic sampling is too sparse for giving too much emphasis to this point. Perhaps show one map that depicts the areas most likely to have been continuously occupied by lineages of E. rugifera through the cycles of fluctuating climate. This is the main point that your readers will want to know. The current presentation is too complicated to convey this point effectively. From my preceding comments, it is evident that I would greatly condense and rewrite the introduction and discussion sections to focus on explaining the main new results. Material on lines 210-224 of the Discussion should be consolidated with the introduction. Include at the start of the Results section a formal description of your new data before covering results of the phylogenetic analyses of those data. Organize your discussion section chronologically by summarizing the main divergence events and their best historical geographic explanations from the deepest to most recent cladogenetic events. As you reach the tips of the tree, identify the minimum number of separate geographic lineages that your data have revealed. Lines 267-271 are mostly a distraction. Where internal branches on a phylogenetic tree are small enough that topology cannot be resolved as successive dichotomies, emphasize instead the approximate time at which the various branches coalesce. Especially for cases of vicariance caused by interaction of a global climatic feature with details of topography, it is likely that some phylogenetic splits will be true polytomies rather than successive dichotomies. In such cases, the relevant phylogenetic resolution consists in putting a precise time estimate on the polytomy, not in trying to force it into a dichotomous scheme. Your main message then should be that the various lineages arose approximately simultaneously at the estimated divergence time that best fits your 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: CONSTRUCTING STAGE-STRUCTURED MATRIX POPULATION MODELS FROM LIFE TABLES: COMPARISON OF METHODS Review round: 1 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: Review of ``Constructing stage-structured matrix population models from life tables: Comparison of method" by Masami Fujiwara and Jasmin Diaz-Lopez Stage based demographic models are useful when life stages other than age of an individual better capture survival and reproductive rates. Such models have been discussed in ecological and demographic literature for some time though issues related to construction and interpretation have persisted. This ms is a useful contribution that addresses a major issue in linking age-based models with stage-based models. It provides some general clues about when stage-based models would work and provide guidelines for construction of such models. Here are a few suggestions that I hope would help in improving the readability of the ms: 1) I would like to see a bit more clarity in the Discussion section about the discrepancy between age- and stage-based models with regard to estimates of $\lambda$ and generation time. It is shown that generation time as opposed to $\lambda$, is largely way off when derived from stage-based models. Can authors provide some intuitive reasoning into why this is so? Is this a numerical issue or is there anything problematic when using equations 13-14 for stage-based models? Since only in the case of constant hazards (and constant fertility) stage-base models do well I am wondering which, the aggregation of age-groups to make stages, or individual heterogeneity contribute more to the error in estimates. Can this information be dished out from the results? 2) The ms is generally well written though it was hard for me to go back and forth between the text and the 16 tables at the end of the ms. I would suggest converting the tables into figures if possible. You could plot deviations between estimates and true values, for instance. I would also think of moving some of the model details (equations 1-20) to an appendix leaving only qualitative information in the text. Please give citations to the different survival/mortality and fertility functions if they have been used previously. I would also suggest providing the codes as some of the methods used to compare different approaches can be used as tools to make models when data is available. 3) Line 177: why is $l(x) = n_1(x)+ n_2(x)$? If so, is it guaranteed that $l(x) \le 1$ for all $x$, as $n_1$ and $n_2$ are population densities? 4) Line 232: should be $l(z)l^{-1}(x-0.5)$? 5) Line 237: what is $l(0.5)$? 6) Line 269 (and 278): what are the symbols L, M and O etc. inside the matrix? Never explained, I think. 7) Line 384: I am not sure if anything is said about the convergence; may be good to provide some details about how fast the methods converged etc. 8) Line 395: check sentence: ``higher threat than" 9) Line 403: ``age-range" 10) Line 453: check sentence 11) Lines 461-467: this paragraph is not very informative and seems redundant. 12) Line 481: ``We" recommend..(unless both the authors listed are same!) 13) The title should be "Comparison of methods" I guess (not "method")

You are one of the reviewers, your task is to write a review for the article. You will 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: CONSTRUCTING STAGE-STRUCTURED MATRIX POPULATION MODELS FROM LIFE TABLES: COMPARISON OF METHODS Review round: 1 Reviewer: 2

Basic reporting: The article is mostly clear, properly referenced, and put in adequate context. Presenting the main results in a long series of tables is not the most effective strategy for communicating the results. I think you could use histograms to summarize results for lambda and generation time, perhaps also split by life-history type. Color coding could be used to embed more information into such histograms, e.g differentiate different types of averaging, or whatever you deem worth highlighting. The language is a bit imprecise in some places. This issue is most serious for the terminology related to rates, ratios, and probabilities. To me a "rate" is something that has dimension 1/time. This manuscript starts with introducing "rates" that are not "true rates" thus defined. In a discrete time model, survival is not a rate but a probability. Reproduction and lambda are ratios. Of course, I readily admit that the use of term "rate" is often quite liberal, and people have tendency to include even when it is not needed or is "wrong" (I have also done so in the past). This is not necessarily a big issue - when the context is clear, they may not be a risk of confusion. But in this article some "rates" are "true rates" whereas others are ratios or probabilities. The text would greatly benefit if you could clean up this terminology. Experimental design: This manuscript presents a useful comparison of different ways of aggregating data for stage-structured population models. The design of the analyses is adequate for the task. Validity of the findings: No comments Additional comments: Specific comments: L19, abstract: I would shorten this, even though it probably is within the allowed limits. You would want to provide a concise overview of your work L33: discretization ... introduce*s* L44: "life history types" (simpler and less clumsy) L47: "I recommend" should be "we recommend", assuming that both authors stand behind this recommendation. L74: remove "However" that hints to a non-existent contrast L90: usually simply "Euler-Lotka equation". In any case, no caps for "Renewal Equation" (also later) L94 and below: why "a renewal equation" when you specifically have defined it as referring to the Euler-Lotka equation? L105: remove the decimal from "1.0" because the value is exactly "1" L110: here you should write "*the* renewal equation" L123: maybe you should define what "the second stage" is? L132: I would call this "mortality rate" (this is a true rate because its unit is reciprocal of [x]) L134: why alpha_3 and beta_3 here, and alpha_2 and beta_2 in eq. 6 introduced later? If the notation has no mnemonic basis, at least the ordering should be logical. L141: this equation is missing a scaling parameter (fecundity per length^3), otherwise R in this equation is completely different from R in equations 4 and 5. I would subscript the R's differently anyway because the exact interpretation is different even after standardizing the units. L206: as L105 L269,278: in my pdf, I see L, M and O where there probably should be ellipses L343: Should this be "similar"? They are the same under the chosen precision. Either say that or use "similar". Also further below - "same" literally means identical and should not be used loosely. L380: you need to add "with the true values", otherwise "consistent" is ambiguous (or better "similar to the true values ") L387: I don't understand what "and they performed poorly" refers to L390: pompous, "two key parameters describing population demography" or something would be better L390: insert "expected to be" before "declining" L392: of population, not species L414: there is a fundamental difference in that adults reproduce and survive while juveniles only survive, so the situation is unlikely to be symmetric L429: only when growth is indeterminate, which is typically true for fish, but also for many other cold-blooded vertebrates as well as many invertebrates

You are one of the reviewers, your task is to write a review for the article. You will 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: CONSTRUCTING STAGE-STRUCTURED MATRIX POPULATION MODELS FROM LIFE TABLES: COMPARISON OF METHODS Review round: 1 Reviewer: 3

Basic reporting: The authors studied the convertibility between age-structured and stage-structured models with a life table and examined the convertibility using two basic demographic statistics. i.e., population growth rate and generation time. The description of the introduction is easy to understand the background of the issue they want to solve. It is written thoroughly citing many related references. The purpose is clear, that is, to investigate the performance of methods for such conversions. The manuscript is organized well according to PeerJ standards. The figures and tables are well-organized and well-labeled such that the readers can understand what they meant. Experimental design: The original question is clear and well-defined, i.e., how to extent age-structured model can be converted to stage-structured model in obtaining the population growth rate and generation time. Most researchers, who have used population matrix models and the matrix database, would be interested in it and be eager to know the answer. It is definitely within the scope of the journal. Most of the assumptions made in the calculation are good and sufficiently described, but I have several complaints and concerns on a part of the assumptions (see comment 2 and 3). Validity of the findings: Their findings, namely the conversion performed well in identifying the population growth rate for many life history strategies of organisms and performed poorly in estimating generation time, is astonishing and interesting. The recommendation in Conclusion is rationale and includes meaningful implication. However, the result on the discrepancy of generation time among the methods are not fully explained and compared (see Comment 1). Additional comments: (1) The most important point to be revised is on the description in the result of generation time (377l. to 382l.). The result showed that the discrepancy is substantial for most scenarios. However, the explanation is very short and insufficient to understand the discrepancy. Many questions arose when I read the result. Which scenario is the worst (or medium)? Does it depend on the maturation timing (early- and delayed-maturation)? Why does the discrepancy arise? I need more explanation about them in Results and Discussion including your speculation. (2) My second concern is the different assumptions of survivorship between early maturation type and delayed maturation type. The authors assumed two types of survivorship both for juveniles and adults (Eqs. 1 and 2) in early maturation, and assumed Eqs. 1 and 6 in juveniles and Eq. 1 in adults. I do not understand why the authors change the assumption because I think it is not necessary in this study. The difference leads to the difficulty in comparing the results between early- and delayed- maturations. The comparison must be very valuable. (3) The authors used 2 by 2 matrix in the construction of stage-structured matrix for early-maturation type. Several researchers cautioned that population growth rates strongly depend on matrix size. The variation in matrix dimensions complicates the comparison among the statistics of the matrices (see Salguero-Gomez 2010). Therefore, I expected that the results in tables 4 to 6 are very different from those in tables 7 to 9. Though they seem to be similar and not to affect the difference so much in your manuscript, it’s better to note lightly in Discussion. (4) This is just a comment. You explained the result of the estimation of population growth rate on lines 349 to 376. I recommend you to make a summary table in order to advise in which case we have to be careful when we used the converted stage-structured matrices. It would be useful for readers to understand the results at a glance. Miscellaneous points: (i) On line 134: The parameter alpha_3 and beta_3 should be alpha_2 and beta_2. The subscripts should be numbered in order of their appearance. (ii) On line 173: “m_1 = 0.5 for x > x_2” There must be a mathematical contradiction here because x1=x2 in the first type of maturation. The equality symbol is necessary? (iii) On line 197: the stage -→ stage i (iv) On lines 231 and 233: l^-1(x-0.5) appears the product of l^-1 and (x=0.5). It’s better to use the fractional form here. Is the b(x)in the equation b(z)? (v) On line 260: Equation 13 is a typical formula of generation time. Please cite a good textbook on demography. Eqs.13, 14 and 18 are denoted as G using the same symbol. It’s better to denote them as G_1, G_2 and G_3. You should specify which generation time was used in the tables. (vi) On line 266: at age x, respectively. --→ at age x, respectively (in Eq. (7)). (vii) On line 269: f_2 -→ f_1.5? (viii) On line 324: priori? Is it “a priori”?

You are one of the reviewers, your task is to write a review for the article. You will 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: CONSTRUCTING STAGE-STRUCTURED MATRIX POPULATION MODELS FROM LIFE TABLES: COMPARISON OF METHODS Review round: 2 Reviewer: 1

Basic reporting: The manuscript is reasonably well written but the results are still rather heavy to follow. This is because one has to refer to several figures at the same time, the figures are not made as accessible as they could, and the way of referring to their elements in the text is different from the figures itself. See also my "general comments". I do not like calling everything as "rates", especially probabilities, but I acknowledge that there is no universal standard for this. Experimental design: No new comments, this is fine Validity of the findings: No new comments, conclusions reflect the results without over interpretations Additional comments: Specific comments: L41 "stage-structured" (hyphen, for consistency) L52 Split this sentence after "and" --- the statement being made is not related to the first clause. L79 It is never "necessary", so I would say something like "little is gained by converting..." L180 I wonder if there is some reasoning behind using different means, or is it just ad hoc choice? L322 Tell the reader which columns shoes this L329 "(Models D-F in panels a-c of Figures 5-7" - the use of lower/upper case letters here is the opposite to their usage in the figures. Check this throughout, very confusing! L339 The figure ref should be moved to the end of the sentence L359 Figs. 11-13 do not include model B L365 This should probably read "are generally not accurate", because they can be accurate in special cases L391 "enable the estimation of"->estimate L404 "it is possible that in reality" sounds odd and should be deleted, given that there are lots of insects that do exactly this, periodic cicadas being an extreme example. L407 I would add here "In our model, the heterogeneity ..." because there are many other reasons, more in general L478 Here you could advise against estimating generation time from stage-structured population models L492 Remove the hyphen from "Orcinus-orca" Table 1 None of the parameters are dimensionless. It would be good to make some statement about the measurement units or dimensions. Figures: * Each figure results from a 2x3 factorial design, but the panel order does not reflect this. With three rows and two columns, it would be logical and reader-friendly to let "H" with two levels to vary by column, and "F" with three levels by row. It looks like that you should fill the figures column-wise. * The figures would probably benefit from marking the "a" column differently from the others, perhaps also adding a horizontal reference line * More in general, indicating certain types of models with coloring or patterning would probably be helpful. Of course, this should not be overdone. * Given that the figures 5-7 are structurally identical, you could probably merge them into one, with e.g. grouped bars or whiskers to show results for the three levels of lambda. The same applies to the other figure triplets.

You are one of the reviewers, your task is to write a review for the article. You will 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: CONSTRUCTING STAGE-STRUCTURED MATRIX POPULATION MODELS FROM LIFE TABLES: COMPARISON OF METHODS Review round: 2 Reviewer: 2

Basic reporting: no additional comment. Experimental design: I suggested two points to be revised (Comments 2 & 3). It's now appropriately revised. Validity of the findings: I commented "the result on the discrepancy of generation time among the methods are not fully explained and compared". It's now appropriately revised. Additional comments: Most of my comments are appropriately responded. However, there are still several typos and points to be revised. Reply to 1st comment: The expansion in discussion is good. It's now appropriately revised, I think. Reply to 2nd comment: The author probably misunderstood what I meant in the comment. However, clarifying the reason or the mechanism of the difference between early-maturation and delayed-maturation is not a central theme in this paper. I agree with the author’s opinion, i.e. their recommendation is to use age-structured model to calculate lambda and generation time when age-specific vital rates are available. Reply to 3rd comment: The revised discussion on 399l. to 406l. is enough to answer my comment. Miscellaneous points: (i) On line 177: the stage begins at age x_i. I understand “the stage” in the senetnce means stage i in stage-structured model, I think. Otherwise, there is no definition of i. (ii) In Eqs. (15) and (16): There are many 2s in matrix-element subscript and some of the subscripts are described as 1.5. I do not understand the inconsistency. Which is correct, 2 or 1.5? (iii) On line 362: They gave similar to the true values -→ They gave similar result to the true values? (iv) Table 4: You defined three types of generation time, G_1 to G_3. Please describe which generation time is use in which model type. (v) Figures to 16: Why do you use A to F in the panels. The capital A to U are used to identify the models you used in Table 4. It’s very confusing. The horizontal axes should be described using upper cases?

You are one of the reviewers, your task is to write a review for the article. You will 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: CONSTRUCTING STAGE-STRUCTURED MATRIX POPULATION MODELS FROM LIFE TABLES: COMPARISON OF METHODS Review round: 3 Reviewer: 1

Basic reporting: The authors have adequately addressed my residual comments. I have no further comments. Experimental design: No new comments Validity of the findings: No new 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: CONSTRUCTING STAGE-STRUCTURED MATRIX POPULATION MODELS FROM LIFE TABLES: COMPARISON OF METHODS Review round: 3 Reviewer: 2

Basic reporting: No additional comments. Experimental design: No additional comments. 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: SPECIES–SPECIFIC CRAB PREDATION ON THE HYDROZOAN CLINGING JELLYFISH GONIONEMUS SP. (CNIDARIA, HYDROZOA), SUBSEQUENT CRAB MORTALITY, AND POSSIBLE ECOLOGICAL CONSEQUENCES Review round: 1 Reviewer: 1

Basic reporting: This MS by Carman, Grunden and Govindarajan is excellent, providing new data and observations on predation on the hydrozoan jellyfish Gonionemus by crab species both living in eelgrass habitats in Massachusetts, USA. The MS is written clearly, with experiments, results and implications expressed in sufficient detail and fairly. Main conclusions include that the native majoid crabs Libinia dubia readily consume Gonionemus and that mortality of L. dubia increases in experiments when more medusae are consumed. In contrast, the introduced crab Carcinus maenus avoids consuming the jellyfish. Anecdotal evidence is presented that Callinectes sapidus may consume Gonionemus, but C. sapidus was not common enough to assess this relationship in any detail. All told, these results are relevant because L. dubia and C. maenus live in the same habitat and consume many of the same prey items, suggesting that the differential feeding bahaviors on Gonionemus benefits the non-native C. maenus. The complicating detail in this paper is that recent work has shown that the Gonionemus jellyfish likely in this region may include lineages that are particularly noxious to humans and that have recently been introduced. Some care should be taken to make clear that toxicity to humans is not necessarily a good proxy for toxicity to crabs, or evidence to the contrary should be presented. Also, it may be helpful to explicitly lay out potential future work investigating whether there is differential impact when Libinia feed on the different lineages of Gonionemus. Experimental design: The experiments are fairly straight forward. Validity of the findings: The experiments are fairly straight forward, consistently showing that: 1) more medusae are consumed by L. dubia over time and in higher densities, and; 2) mortality increases as more medusae are consumed. The apparent inverse relationship between crab size and mortality (with smaller crabs being less likely to suffer lethal effects) is difficult to understand and the authors correctly assert that more evidence is needed to understand whether this relationship is real or not. Additional comments: A few notes/suggestions, all minor. 1) I do not necessarily agree that "jellyfish" refers to gelatinous zooplankton. Perhaps this is true of the word jelly or jellies, but I tend to think of jellyfish referring to only the medusa phase of medusozoan species that have a medusa phase. In any event, it is a common name and it is unclear how that introductory sentence helps the overall narrative of this paper. It is not a big deal, but the authors could use the term "gelata" , as introduced by Haddock (Haddock, 2004 in Hydrobiologia) in order to move the focus to the medusa-bearing groups. 2) Line 223: "Even if the medusae were cling. . ." should have "were" deleted. 3) Lines 246-250. It might be worth providing a bit of caution into this discussion. Crabs and humans are quite different organisms and there is no evidence that the toxins behave similarly on the two groups. I think a caveat to this effect might be warranted. It is interesting to note the individual variation in human response to Gonionemus venom, and the fact that a single sting can elicit symptoms in a human, but this is merely suggestive of possibilities in regards to the crabs. 4) The discussion of jellyfish predators starts (lines 257-258) with a partial list (sea turtles, fish, molluscs, chaetognaths, ctenophores, and other cnidarians) and that through me off a bit. But it goes on to become more complete. Phyllosoma larvae should be added to the list (Biol Bull. 2012. 222(1):1-5. Predation by the phyllosoma larva of Ibacus novemdentatus on various kinds of venomous jellyfish.) and I have also seen images of galatheid crabs feeding on medusae in deep reef settings. I was unable to find anything in a quick literature search, but the authors might consider checking that. 5) Lines 288-290 states "The hard shells of the crabs probably provided protection from Gonionemus stings upon initial contact with the jellyfish. However, the soft interior tissues appear to be vulnerable." Is there really any evidence that the stings do not occur through the handling and manipulation of the jellies, rather than the consumption? I suspect that the authors have this correct, but it might be more fair to state " However, the soft interior tissues are more likely to be vulnerable." 6) Lines 308-312. Here again the authors suggest that what we know about Gonionemus toxicity to humans may be directly relevant to toxicity in crabs (when discussing Carcinus avoidance of Gonionemus). It could also be the case that Gonionemus in Europe is toxic to Carcinus even if the Gonionemus in Europe isn't particularly noxious to humans. 7) The authors may want to lay how to test (and the importance of testing) whether the less toxic lineage of Gonionemus (to humans) is also less toxic to Libinia.

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

Title: SPECIES–SPECIFIC CRAB PREDATION ON THE HYDROZOAN CLINGING JELLYFISH GONIONEMUS SP. (CNIDARIA, HYDROZOA), SUBSEQUENT CRAB MORTALITY, AND POSSIBLE ECOLOGICAL CONSEQUENCES Review round: 1 Reviewer: 2

Basic reporting: This is a straightforward experimental paper on the effect of Gonionemus cf. murbachii on Libinia dubia and other crab species. Experimental design: Very clear. Validity of the findings: The results are sound and worth publishing. 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: SPECIES–SPECIFIC CRAB PREDATION ON THE HYDROZOAN CLINGING JELLYFISH GONIONEMUS SP. (CNIDARIA, HYDROZOA), SUBSEQUENT CRAB MORTALITY, AND POSSIBLE ECOLOGICAL CONSEQUENCES Review round: 2 Reviewer: 1

Basic reporting: All clear, and reviewer comments addressed satisfactorily. Experimental design: Well done. Validity of the findings: Conclusions are well founded. Additional comments: All reviewer comments have been addressed and the MS is ready to be 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: INDIGENOUS AUSTRALIAN HOUSEHOLD STRUCTURE: A SIMPLE DATA COLLECTION TOOL AND IMPLICATIONS FOR CLOSE CONTACT TRANSMISSION OF COMMUNICABLE DISEASES Review round: 1 Reviewer: 1

Basic reporting: Basic reporting is good, with clear presentation of the work. No concerns about this. There is one typo in equation 2 (page 4): the minus sign in front of the lambda should not be there. Perhaps there could have been a bit more of a discussion of the literature relating to measuring contact patterns relevant to infectious diseases and the potential limitations of relying on the number of household contacts alone. Some possible reference are Ogunjimi B et al Mathematical Biosciences 2009 Xiao X et al Epidemics 2016 Melegaro et al Epidemics 2011 Zagheni et al AJE 2008 Stehle et al PLoS One 2011 Fournet et al PLoS One 2014 One limitation of relying on contact data alone is children have consistently been found to more infectious than others (for example, see Ghani et al 2010 paper cited, but several others as well such as Viboud et al Br J Gen Prac 2004). Would be nice for the reader if Fig 3 could use colours corresponding to those chosen in Fig 2. Minor point: "data are" is usually preferred to "data is" (though I note the Peerj review website uses "data is" so I'm probably fighting a losing battle on this one). Experimental design: I have no concerns about the experimental design. I believe this is relevant, original, meaningful research with appropriate methods that clearly fills a knowledge gap. The outbreak simulations are probably the weakest part of this (it seems a bit odd to collect good household data but then not look at a household model). However, this is not an essential part of the paper - this is more just to illustrate the point that contact patterns can be important. These simulations do not represent a novel finding and could easily be dropped without affecting the quality of the paper. If these stay in I think at least worth discussing limitations of this model (which can be contrasted with a household model), + also reporting R0 values for the three populations considered. Perhaps also worth mentioning that different methodology was used to collect data from the Melbourne site, so contact matrices are not necessarily comparing exactly like with like. Validity of the findings: I have no reason to doubt the validity of the findings and the conclusions, though I think there could be more of a discussion of the relevance of the household data alone for informing transmission models. First because the number of effective contacts made by a single individual with household members may not necessarily scale linearly with the number of other household members, and secondly because many other factors (other than numbers of people per household and per room) may be important determinants for household transmission risk. Most importantly age, but other factors such as ventilation/humidity + hygiene practices may also be important. 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: INDIGENOUS AUSTRALIAN HOUSEHOLD STRUCTURE: A SIMPLE DATA COLLECTION TOOL AND IMPLICATIONS FOR CLOSE CONTACT TRANSMISSION OF COMMUNICABLE DISEASES Review round: 1 Reviewer: 2

Basic reporting: The manuscript is well-written (structure and language). From my point of view, the analysis of the data should be deepened and better embedded in discussions around validity / reliability of measurements (see section 3) and relevant literature should be cited. I will suggest literature in section 3. Experimental design: I have a few methodological questions: 1) How comes that the authors had more respondents for the magnetic board than for the questionnaire? I thought that questionnaire participation was mandatory and magentic board was not; hence the magnetic board respondents should be a subset of the questionnaire respondents? (ll. 94/95) 2) The authors state that the magnetic board was separated into four rooms. What happened if the respondent's house had more or less than four rooms? (l. 108) 3) How exactly was the weighting done (ll. 150/151)? Maybe an equation would help? Validity of the findings: 1) The authors introduced a new method to measure contacts within a household. At least some data were measured with two methods (questionnaire and magnetic board). From my point of view, a new methods should be thoroughly assessed re how reliably data are collected. What are potential biases? Is the underlying contact definition the same as in other methods? If not, what are the potential implications of that? There is a huge body of literature on contact measurement methodology. Some paper that might be helpful to deepen this analyses are the following (in reverse-chronological order): BMC Infect Dis 2016, 16: 341 PLOS ONE 2015, 10: e136497 BMC Infect Dis 2014, 14: 136 Epidemiol Infect 2012, 140: 744-52 PLOS ONE 2012, 7: e37893 Proc Biol Sci 2011, 278: 1467-75 2) Since the numbers of respondents are at the lower end, it would be advisable to calculate confidence intervals when creating contact matrices for specific strata to assess whether apparent differences are statistically significant. This could be done with bootstrapping. 3) Since the authors have introduced weights for being in the same room, they also might want to discuss this in light of the literature around contact duration / intensity, e.g. Biostatistics 2014, 15: 470-83 Epidemics 2011, 3: 143-51 R Stat Soc C 2010, 59: 255-77 Theor Biol Med Model 2009, 6:25 Epidemiol Infect 2007, 135: 914-21 Additional comments: - ll. 55/56: what kind of skin infections? Impetigo? - l. 81: what is meant with "Top End"? The northern part? - l. 153 what is a "shire council"?

You are one of the reviewers, your task is to write a review for the article. You will 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: INDIGENOUS AUSTRALIAN HOUSEHOLD STRUCTURE: A SIMPLE DATA COLLECTION TOOL AND IMPLICATIONS FOR CLOSE CONTACT TRANSMISSION OF COMMUNICABLE DISEASES Review round: 2 Reviewer: 1

Basic reporting: Changes are good. Just a few minor typos to be fixed (line numbers are from marked up version): line 99 punctuation: ”. The questionnaire asked the question ?Who slept in the house last night?” line 221 + 260 "NT" used inconsistently. I think better to spell out Northern Territory each time as abbreviation isn't used often. line 252 "proportion of contact" ->"proportion of contacts" line 276 "population" -> "populations" line 277 "household" ->"households" line 278 remove comma line 290 "contribute" - >"contributes" line 309 & 332 comma needed: ...., however, ... Also, no mention of methodology used to calculate the R0s which have been added in the results section in this revision. Presumably dominant eigen-value of next generation matrix, but worth saying and including the appropriate reference (Diekmann + Heesterbeek). 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: KNOWLEDGE AND BELIEFS REGARDING CERVICAL CANCER SCREENING AND HPV VACCINATION AMONG URBAN AND RURAL WOMEN IN LEÓN, NICARAGUA Review round: 1 Reviewer: 1

Basic reporting: Line 7: Please define VIA. Lines 7-10: The authors should clarify that the type of work described in lines 7-10 has not previously been conducted in Nicaragua specifically because it has been conducted elsewhere. Line 16: Unadjusted linear regression is typically referred to as simple linear regression, “single variable regression” should be replaced with “simple linear regression.” Lines 18-19: What was the overall goal of in-depth, qualitative interviews? Line 27: The 3rd theme does not sound like a barrier. Please revise to more clearly convey how this statement represents a potential barrier to screening. Line 31-32: This sentence refers to an important finding (i.e., barriers to screening did not differ for urban and rural women); however, it is not mentioned in the Results section. This sentence would be more appropriately placed in the Results section. Line 47-48: The source of the data reported in the following sentence should be referenced: “Vaccination against HPV-16 and -18 can prevent nearly 70% of cases of cervical cancer.” Line 54: What is the cervical cancer mortality rate in Nicaragua? Line 59-60: Please provide information on HPV and cervical cancer screening recommendations in Nicaragua. For whom (certain age groups?) is screening recommended in Nicaragua? With what frequency is screening recommended? Does the recommended frequency vary by age and risk? Lines 73-74: Given that numerous studies have examined the acceptability of HPV vaccination programs, the authors should clarify that their statement in lines 73-74 refers to prior work in Nicaragua. Line 91: When (i.e., months and year) was the study conducted? Line 127: Does “diseases” refer to HPV and cervical cancer? Line 133: Five weeks or six weeks as stated on line 92? Line 141: Why were 92 surveys incomplete? Line 188-189: The belief score clearly measures knowledge about and attitudes towards screening, but it is not defined in that way in the outcome variable section (lines 144-156), so it is unclear why the authors state that they examined correlates of knowledge and attitude (lines 188-189) in the quantitative data analysis section. Line 189: Unadjusted linear regression is typically referred to as simple linear regression, “single variable regression” should be replaced with “simple linear regression”. Line 190: What “variables of interest” were considered? Please clarify. Line 247: According to Table 1, 52% of women were “just getting by” and 53% of rural women were “just getting by.” Please correct line 247 or Table 1 depending on which is currently incorrect. Line 255: If aged 18-49 was an eligibility criterion, how were data on age missing? All statements made about qualitative findings in the Results section should be limited to what women actually said. For example, it’s unclear whether the following statements reflect what women actually said during in-depth interviews or simply reflect the authors’ hypotheses about how women’s views about or attitudes toward screening might impact their willingness to undergo screening. If they reflect the latter, then they belong in the Conclusions section: • Lines 287-289: “As these services are not affordable for many women in the population, the uncertainty of the gender of the provider may dissuade women from receiving Pap smears.” • Lines 289-291: “The embarrassment felt by some women receiving a Pap smear may be exacerbated by a male Pap provider – effectively discouraging some women from regularly seeking cervical cancer screening.” • Lines 334-335: “In addition, the perceived link between cervical cancer screening and sexuality could discourage women from seeking regular Pap smears.” Line 337-338: The 3rd theme does not sound like a barrier. It should be revised to more clearly convey how this statement represents a potential barrier to screening. Lines 367-375: Everything other than the following sentence between lines 367 and 375 seems to reflect the authors interpretation of their findings and belongs in the Conclusions section: “Furthermore, as shown in Table 2, 26% of women who were surveyed at 372 health centers were unsure of the health benefits or purpose of a Pap smear.” Line 403: Delete “of” between “against” and “cancer-causing.” Line 788: In question 37, change “you” to “your.” Line 806: Should question 40 be changed to: You would consult with YOUR partner before receiving a Pap test? Line 984: In question 67, change the final word from “method” to “cancer”. Line 1159: In the table of survey items included in the belief score, in item 6, change “you” to “your.” Line 1160: In the table of survey items included in knowledge score, in item 9, change the final word from “method” to “cancer”. Experimental design: The research question is well-defined and the authors state how this study fills an identified gap in knowledge. However, the title of the manuscript, the first aim (lines 76-78), and the authors interpretation of findings from analyses addressing the first aim do not accurately reflect the quantitative data collected. Barriers to screening were measured via the belief score, which the authors state “measures a woman’s likelihood of being screened based on her responses to 22 belief questions” (lines 348-349). Although the beliefs measured likely have an effect on women’s decisions to undergo screening, the survey did not measure whether these beliefs actually serve as barriers to screening. For example, women were asked whether they agree or disagree with the statement that “Pap tests are painful.” Women were not asked whether they are unwilling to undergo screening because Pap tests are painful. Thus, it is unclear how the belief questions measure women’s likelihood of being screened. As such, the authors statement in line 348-349 requires justification. Furthermore, the authors should either (1) address the discrepancy between the first aim of the study and justify their use of belief scores as the outcome of interest in quantitative analyses or (2) revise the Title, first aim of the study, and their interpretation of findings from analyses addressing the first aim to reflect the fact that barriers to screening were not actually measured in the survey. Validity of the findings: Line 419-420: This statement is somewhat misleading. While the identified themes may pose barriers to screening, most of the quotes provided from in-depth interviews suggest that the identified themes reflect women’s views about or attitudes toward screening. Few of the quotes provided illustrate how a woman’s views/attitudes actually served as a barrier to screening. This statement should be modified to better reflect the findings presented. Line 439-440: The following is not reported in the Results section: “Our results indicate that there was no significant difference in barriers to cervical cancer screening for urban and rural women.” All findings discussed in the Conclusions section should be reported first in the Results section. Additional comments: I was able to open the raw data in Excel, but not in STATA. I may have done something wrong, but when I tried to open it, I got the following error: . use C:\Users\Desktop\peerj-18174-Rees_STATAdata2.dta file C:\Users\Desktop\peerj-18174-Rees_STATAdata2.dta not Stata format

You are one of the reviewers, your task is to write a review for the article. You will 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: KNOWLEDGE AND BELIEFS REGARDING CERVICAL CANCER SCREENING AND HPV VACCINATION AMONG URBAN AND RURAL WOMEN IN LEÓN, NICARAGUA Review round: 1 Reviewer: 2

Basic reporting: This is a paper sharing results from a study in Nicaragua to understand knowledge, attitudes, beliefs about cervical cancer screening and HPV vaccination. Participants included a convenience sample of women recruited at 6 clinics (3 rural and 3 urban) in León, Nicaragua. Experimental design: -This is a paper about beliefs and knowledge rather than “barriers” to screening. Please review and revise the aims, research question and focus throughout to reflect this. - The methods section is very long, while it is clarify all methods there is some repetition. For example, it is stated several times that interviews were conducted in waiting areas. Validity of the findings: no comment Additional comments: Review of: Barriers to screening for HPV and cervical cancer among urban and rural women in León, Nicaragua (#18174) This is a paper sharing results from a study in Nicaragua to understand knowledge, attitudes, beliefs about cervical cancer screening and HPV vaccination. Participants included a convenience sample of women recruited at 6 clinics (3 rural and 3 urban) in León, Nicaragua. Major comments: -This is a paper about beliefs and knowledge rather than “barriers” to screening. Please review and revise the focus throughout to reflect this. -There is a lot of discussion in the results section, this should be moved to the discussion section. Abstract: -define the acronym VIA Introduction: -Line 59-61: You state that only 31.5% of women who are recommended to have a Pap test do have one each year. What are the screening recommendation for Paps in Nicaragua? This may be important to include. In the U.S. PAP screening is recommended every 3 years (not every year) for women age 21 to 65 years. On the PAHO Nicaragua cancer profile page (available here: http://www2.paho.org/hq/index.php?option=com_docman&task=doc_download&gid=23004&Itemid=270&lang=en) it states that screening is recommended every 3 years, so it wouldn’t be surprising that few women report having a Pap test in the past year. -Line 77: No need to define the acronym HPV more than once in the manuscript. Methods: The methods section is very long, while it is clarify all methods there is some repetition. For example, it is stated several times that interviews were conducted in waiting areas. -Line 189: Single variable regression, please be specific and specify that this is linear regression. -Please provide an explanation as to why so many surveys were incomplete and as to why those surveys were completely excluded. Results: -Line 346: “a multiple linear regression results” should be reworded. -Most women report that that they have ever had a Pap test. How recent were those tests? This would be interesting to see since even though some women report that there are barriers to screening, they are still being tested. -Move the discussion to the discussion section. Questionnaires: -Question 19 asks about how long ago participants had a Pap test. These results would be interesting to include in the paper. -Question 23 doesn’t make sense as written. Pap tests do not test for HPV. -Question 24 asks for number of children, it would be interesting to know more about how likely parents would be to vaccinate their children for HPV among those women that have children. -Question 58 is not clear. Is this a result of the translation or is this exactly how the question was asked? Please provide some explanation for this question. -There seems to be an error in question 67. -What was the reason that you only asked about if people would be willing to vaccinate their daughters and not their sons (Questions 80+)? How many of the participants actually had daughters? Tables: Add more descriptive title to tables and figure. -Table 3, add footnote describing the statistics used so that results can be interpreted without going back to the methods section.

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

Title: KNOWLEDGE AND BELIEFS REGARDING CERVICAL CANCER SCREENING AND HPV VACCINATION AMONG URBAN AND RURAL WOMEN IN LEÓN, NICARAGUA Review round: 2 Reviewer: 1

Basic reporting: 1. I thank the authors for re-framing their manuscript from barriers to beliefs regarding cervical cancer screening so that their focus is more aligned with what was actually measured. However, the rationale for measuring beliefs is under-developed. One way the authors could strengthen their rationale is by discussing how women's beliefs regarding cervical cancer screening and HPV vaccination might influence (i.e., promote or inhibit) their uptake of these HPV and cervical cancer prevention strategies. This would make most sense prior to line 30 in the Abstract and line 103 in the Introduction. 2. It is unclear what the authors mean by “beliefs to promote or limit” in the Abstract (lines 34-45) and Introduction (line 107). If the rationale is further developed as suggested above, the authors could replace “to promote or limit” with “regarding.” 3. Line 40: Since the outcome was women’s belief scores, "and attitudes" should be deleted from line 40. 4. Line 41-42: This sentence should be deleted (“The outcome variable was the screening-promoting belief score.”). It would make more sense to define the outcome before (i.e., in line 39) describing the analysis. For example, “We assessed women’s beliefs regarding cervical cancer screening via a 22-item scale, with higher scores indicating screening-promoting beliefs.” 5. The authors’ suggested revision to lines 42-43 should be incorporated into the abstract: Twenty qualitative interviews, which sought to explain sociocultural dimensions of knowledge and attitudes indicated by our quantitative findings, were conducted with a sample of 13 urban and 7 rural women (aged 19-46). 6. I think the manuscript is greatly improved by shifting the focus of the manuscript to beliefs regarding cervical cancer screening. However, I think the manuscript requires a careful review to ensure that all aspects of the manuscript are up-to-date with respect to this shift in focus. For example, themes (1) and (2) sounds more like barriers than “knowledge and beliefs” regarding cervical cancer screening. Theme (5) also seems out of place with this description. Please revise (Abstract lines 46-55, each theme in the Results section, and lines 454-463 in the Conclusions section) to more clearly convey how these themes represent knowledge and beliefs. Perhaps attitudes towards cervical cancer screening would be an appropriate addition to the description of the themes identified? 7. Lines 55-56: Because this result is from the multiple linear regression analysis, this sentence should be presented with the quantitative findings above (i.e., before the presentation of qualitative findings). 8. Line 126: What is "reported acceptance"? The authors should consider deleting the word "reported" here. 9. Line 177-178: The latter half of this sentence describes how the belief score was analyzed (i.e., with respect to demographic characteristics and participant knowledge) and should be deleted as that information belongs in the Quantitative Data Analysis section. 10. Lines 183-187: While some items listed in Appendix B reflect positive statements (i.e., Pap tests are safe), most of the items listed in Appendix B reflect negative statements. Thus, it’s unclear how assigning a score of 1 if participants agreed with a negative statement would yield an overall score where higher values indicate screening-promoting beliefs. Please verify that the scoring is reported accurately and clarify if necessary. 11. Line 228: Knowledge should be deleted here since the women’s belief scores were used as the outcome, while knowledge of HPV and cervical cancer were explanatory variables of interest. 12. Lines 403-406: It’s great to see that these data have been added to the manuscript; however, these data would be more appropriately reported with the quantitative data at the beginning of the Results section. Experimental design: The authors acknowledge that their sample is biased due to the fact that women were recruited from health centers, but their data suggesting that the majority of women in their sample are in compliance with national guidelines (lines 403-406: “80% of women at the health centers had received a Pap smear within the past year, with 37% of women screened within the past 3 months [Table 4]”) are in stark contrast to the estimate provided in the Abstract (line 30) and Introduction (lines 86-87): “only 10% of women receive Pap smears at the nationally recommended frequency.” This large discrepancy should be addressed in the manuscript. Validity of the findings: Some of the authors qualitative findings directly highlight the mismatch between beliefs and actions (e.g., the quote from Participant 16 on lines 301-306). Because this study measured beliefs regarding cervical cancer screening rather than actual barriers to screening, it would be helpful if the authors further developed their discussion of how their findings could be used to develop interventions to promote cervical cancer screening and HPV vaccination. 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: KNOWLEDGE AND BELIEFS REGARDING CERVICAL CANCER SCREENING AND HPV VACCINATION AMONG URBAN AND RURAL WOMEN IN LEÓN, NICARAGUA Review round: 2 Reviewer: 2

Basic reporting: This is a revised paper sharing results from a study in Nicaragua to understand knowledge, attitudes, beliefs about cervical cancer screening and HPV vaccination. This manuscript has improved since my prior reading, however there are still some issues that should be addressed prior to acceptance. The language is clear. I suggest adding to your methods that the interviews and data collection occurred in Spanish and was translated after (or before) data analysis. I also suggest adding URLs to your references to allow readers to access your referenced documents. Lastly, the conclusion needs some editing to be more specific to the results from this particular study rather than sweeping statements- I have given some examples below. Experimental design: The research question for phase 2 (qualitative) is not clearly articulated and different language is used to explain the purpose of this phase in different parts of the manuscript. Please see below. Validity of the findings: Please see comments regarding the conclusion below. Additional comments: -Abstract: It is redundant to say your simple linear regression analyses were unadjusted and multiple linear regression was adjusted. Introduction: -Line 70: Please provide a reference for “The vast majority of cases of cervical cancer are caused by persistent infection with specific strains of human papilloma virus (HPV).” -Line 72: Please provide a reference for “In addition, screening through the use of cervical cytology is an extremely effective method to identify pre-cancerous lesions and prevent the development of cancer.” Methods: -Line 127-130: “The preliminary quantitative results influenced the creation of the open-ended discussion guide, which sought to elucidate sociocultural dimensions of knowledge and attitudes about HPV, cervical cancer, screening practices, and acceptance of HPV immunization that might further explain our quantitative findings. “ Is this sociocultural dimensions of knowledge, attitudes and acceptance, or personal/individual level dimensions? Later in Line 141-142 you state “The interviews thus placed a greater focus on understanding attitudes and beliefs towards HPV and cervical cancer.” This seems different than the aims previously stated. Then again later in Line 234-236 you state: “The purpose of Phase Two was to expound upon quantitative findings regarding gaps in knowledge and past experiences with Pap smears, HPV and cervical cancer. In addition, the interviews enabled a conversation regarding the views of women on a potential HPV vaccination program in Nicaragua.” These should be consistent and there is no reason for repetition. I suggest providing the aim/purpose clearly and concisely for phase 2 one time. -Line 207-211: What was the reference level for your dummy variables? Results: -Line 274-276: “Fewer than half of the participants completed secondary school (46%) in both the urban (48%) and rural (44%) populations.” Looking at your table, that category is not completion of secondary school, but any secondary school. In addition, this doesn’t look like the accurate percentages – you can add categories together to stratify in this way. -Table 3: Define “belief score” in a footnote. -You present some data on health insurance. Please introduce the reader to the health insurance system in Nicaragua. Is any healthcare provided by the government? Are preventive health services like Pap Testing provided at a charge? Conclusions: -Thank you for the addition of Table 4. Table 4 demonstrates that almost all participants had had a Pap test within the past 3 years - just as the guidelines recommend. Therefore this sentence from line 455 (and other sentences from the conclusion) may not be relevant: “Our results suggest that women may be dissuaded from seeking regular screening due to embarrassment associated with the intimate nature of the exam as well as the association of Pap smears and cervical cancer with sexual promiscuity.” These contrasting results should be discussed. In addition did women specifically say that they were “dissuaded” from seeking regular screening? Or did they say that this was an issue they faced? -Line 458: Did you measure health center attendance? -Note: HPV vaccine is important for both males and females and may prevent cancer in both men and women. I suggest mentioning this in your discussion and providing an explanation as to why you focused on daughters rather than adolescents in general.

You are one of the reviewers, your task is to write a review for the article. You will 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: DIVERSITY OF FISH SOUND TYPES IN THE PEARL RIVER ESTUARY, CHINA Review round: 1 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: General suggestions: 1.In Result section, the authors mentioned 23 sciaenid species had been recorded in Hong Kong waters by citing a 1999 paper. Information about the sciaenid species occurring in the survey area in more recent years, if available, is useful to help confirm the caller’s identity. The voluntary calls of J. macrorhynchus and J. belangerii match some of the call types, it is expected that these two species should present in the study site. 2.The authors should give the relative abundance of each call type occurring in each recording day (or in each of the two continuous recording session) if possible. This information is helpful to interpret number of soniferous species in the area that produced these 66 call types. 3.Please aware that no vertical lines should be used in tables. 4.All characteristics of 3-8 section calls are presented in the supplementary section. It is suggested to add one figure following Fig. 7 to illustrate three of these call types. 5.Comparisons were made among the call types recognized in this study with the hand-held disturbance sounds of several sciaenid species published in the literature and no match was noticed except for Johnius macrorhynchus. Despite of the similarity among the disturbance and voluntary call types in J. macrorhynchus (Lin et al., 2007), this condition may not be a general rule for other sciaenid species. Hand-held disturbance call might be a vocal response toward the highest stress to a fish and this high stressful condition may not be the one causing the voluntary calls heard in the survey site. The purrs emitted when the fish was confined in a small tank, the repeating cycle reduced (with a longer pulse period). Johnius macrorhynchus emits two call types (Lin et al., 2007), the dual knocks and the purr. It is possible that J. macrorhynchus emit these two types in series and creates a multiple section call type. 6.The spotted seatrout (Cynoscion nebulosus) is one of the few sciaenid species that produces as many as four types of call (Mok, H.k. and R.G. Gilmore. 1983. Analysis of sound production in estuarine aggregations of Pogonias cromis, Bairdiella chrysoura, and Cynoscion nebulosus (Sciaenidae). Bulletin of the Institute of Zoology, Academia Sinica. 22(2): 157-186.). It is likely that most sciaenid species have fewer call types. Because there are 66 call types recognized in the survey sites, one can guess some of the call types must come from a single species. As most of the call types were identified based on the number of sections and the repetition of the anterior section, it is likely that a species might be able to produce several call types by varying the anterior sections of the call as a response to the variable external stimuli. According to the result of cluster analysis, five clades were revealed. Can the authors hypothesize that these groups belong to the call repertoire of five species? 7.Spiny croaker is a small croaker species, its call type could be distinct to other related species due to it small sonic muscles. 8.Huang (Huang Po-Wei. 2016. Sound characteristics and spatiotemporal variability of Scienidae in the coastal water of northwestern Taiwan. Master Thesis, National Dong Hwa University, 109pp.) recognized only four call types from three recording sites on the northwestern coast of Taiwan where 9 sciaenid species have been recorded (Johnius amblycephalus, J. distinctus, Lamichthys crocea, Nibea althiflora, Pennahia anea, P. argentata, P. microcephalus, P. pawak, and a undescribed Johnius sp. which has been has been misidentified as J. macrorhynchus. The call type is identical to the purr call of ‘J. macrorhynchus’ was the most abundance type at these sites. The soundscape was dominated by only one sound type. 9.Please explain how the order of the call types in Fig. 8 was decided? Specific suggestions: 1.Line 22, ‘species-specific sound’ should be use cautiously because sounds of some soniferous fishes might to so similar that they don’t have a species-specific call type. 2.Line 70, : ‘sp.’ should be changed into ‘sp.’ (in italics) 3.Line 71: : ‘spp.’ should be changed into ‘spp.’(in italics) 4.Line 88: ‘Nibea diacanthus’ should be changed to ‘Protonibea diacanthus’. 5.Line 101:’ the spatial and temporal patterns of soniferous fishes, should consider to be changed into ‘the spatial and temporal distribution patterns of soniferous fishes in the estuary’. 6.Line 102: ‘of the damage to aquatic environments’ may consider to be changed into ‘of the damage to aquatic environments (e.g., spawning grounds of the sciaenids)’. 7.Lines 202-205”… and the call duration was dependent on the number of pulses in a call these parameters were not included in the PCA, Canonical discriminant analysis and hierarchical cluster analyses.” However, pulse number in a call was a parameter valuable in matching call type 1+N19 with the known voluntary call of Belanger’s croaker. As such call duration remains a useful taxonomic character. 8.In line 261-262, ‘Hierarchical clustering using a between group method that measure the squared Euclidean distance automatically group the 31 extracted call types…’. Please distinguish ‘the 31 extracted call types’ with the 66 call types mentioned elsewhere in the text. 9.Line 352: ’underhand held condition’ should be changed into ‘under hand-held condition’. 10.Line 501: ‘chinese ‘ should be changed into ‘Chinese’. 11.Line 504 : ‘spp.’ should be changed into ‘spp.’ (in italics) 12.Line 364: ‘sp.’ should be changed into ‘sp.’. *(in italics) 13.Line 12: ‘Soundfrom’ should be changed into ‘Sound from’. 14.Line 362: ‘Sound from other soniferous families, including cutlassfish (Trichiurus haumela, family Trichiuridae)(Ren et al., 2007)’. Because the subject of Ren et al. (2007) paper is about the sound of large yellow croaker (not about the cutlassfish sound), I suggest the authors should recheck if cutlassfish is actually soniferous. 15.Species names in the references should be printed in italics. 16. Lines 681, 692, 701, 702, 704, a full stop should be put behind the table number.

You are one of the reviewers, your task is to write a review for the article. You will 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: DIVERSITY OF FISH SOUND TYPES IN THE PEARL RIVER ESTUARY, CHINA Review round: 1 Reviewer: 2

Basic reporting: see below Experimental design: see below Validity of the findings: see below Additional comments: The sheer number of figures (often multiple panel) and tables as well as non standard nomenclature to describe the sounds, prevents me from investing the time to adequately review this manuscript. I suggest the authors give serious thought about what and is not necessary as most of the figures are unneeded even as supplemental files and revise the manuscript to length that is more fitting its overall content

You are one of the reviewers, your task is to write a review for the article. You will 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: DIVERSITY OF FISH SOUND TYPES IN THE PEARL RIVER ESTUARY, CHINA Review round: 2 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: 1. Line 49: I suggest that the authors should consider changing the word ‘Most’ into ‘Some’. 2. Line 60, ‘Pseudoscianea crocea’ should be changed to ‘Larimichthys crocea’. 3. Line 71: ‘(signature)’ is suggested to be deleted. 4. Line 87: ‘water proof’ changed to ‘waterproof’. 5. Line 115: ‘Total call duration’ changed to ‘Call duration’. 6. Line 150: ‘Fig. 4-5’ should be changed to ‘Figs. 4-6’. 7. It is suggested that (1) at least one representative call type of each call category differing in number of sections should be included in figures 4-6 so that they can be cited in the text (i.e., putting at least one representative call type for 1-8 section calls in these three figures); (2) some redundancy or highly resembling call types original placed in these three figures should be reduced; (3) figures and tables in the supplementary section should not be cited in the text. If these suggestions are accepted, then (Fig. S1-S26) in line 150 should be deleted. 8. Line 178: ‘Ethical statement’, I don’t think this study involves ethical issue with the animals surveyed as only the biological sounds in the field were recorded by an noninvasive method. 9. Line 230-231: “Call types with an analyzed number greater than five were extracted for further discriminant and cluster analyses and 31 call types meet the requirement.” Does it mean that these 31 types were more common than the other 35 call types in this study site? If this is true, then it should be stated clearly in the Occurrence pattern of call types in Discussion (line 353 or other more appropriately part of the text) to give an idea about the relative abundance of the 66 types. Also see comment 17. 10. Line 268: ‘with Sciaenidae sounds’ should be changed to ‘with sciaenid sounds’ 11. Line 327: ‘of the species’ should be changed to ‘of the sciaenid species’ and Line 329: ‘Sciaenidae’ should be deleted. 12. Line 335: ‘Arius sp.’ should be changed into ‘ Arius sp.’ (‘sp.’ following a generic name should not be in italic type.) 13. Line 341: ‘Comparison with other passive acoustic monitoring sounds’ is suggested to be changed to ‘Comparison with biological sounds from other passive acoustic monitoring sites’ 14. Line 360: ‘come from a single species’ changed to ‘come from the same species’. 15. The problem with peak frequency was discussed between Line 375-378, as length of the sonic muscle fibers with also related to body size, this factor should be mentioned as well. The book chapter by Parmentier, E and M.L. Fine., entitled, ‘Fish sound production: insights.’ (In R.A. Suthers et al (eds.). 2016. Vertebrate Sound Production and Acoustic Communication, Springer Handbook of Auditory Research 53, Springer International Publishing Switzerland, DOI 10.1007/978-3-319-27721-9_2) should be cited. 16. Line 637: “inter-pulsepeak interval’ differs from ‘inter pulse peak interval’ used in labelling the X-axis of Fig.4 B. Please check. 17. There are too many insets within Figures 4-6. For example (1) Characteristics of the (A) N9, (B) N10, (C) N13, (D) N17 call types were given. The authors should consider leaving just N9 and N17 in this figure. (2) The insets on regression of call duration and number of pulses in a call for each call types are not necessary. 18. In Table 1, ‘Typ and e’ in the upper left corner of the table are separated ; they should be together.

You are one of the reviewers, your task is to write a review for the article. You will 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: STATINS: ANTIMICROBIAL RESISTANCE BREAKERS OR MAKERS? Review round: 1 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: I consider that the review is well-written and well-structured. It provides new information related to the antibacterial effects of statins, which results useful for the control of bacterial infections that can become in resistant strains. The review gives information about the evidences that deal with the development of bacterial resistance in response to statins, and those that agree with the opposite effect (“AMR breakers”) of these molecules. In my opinion, this review is worth to be published in PeerJ. I only have minor concerns that should be corrected in the manuscript: 1. In line 284, authors describe that the mechanism for the antibacterial effects of statins could be apoptosis. Please, eliminate this sentence, since apoptosis is a cell death mechanism restricted to eukaryotic cells. The term “apoptosis” to explain regulated cell death in bacteria is under discussion, and its molecular basis are very different from that detected in eukaryotic cells. For your reference, Microbes Infect. 2013 Jul-Aug;15(8-9):640-4. doi: 10.1016/j.micinf.2013.05.005. 2. The period (.) before each reference is wrong. It should be located after. 3. The sentence in Lines 285-288 is confusing, and it appears contradictory: How the reduction in a metabolite in the mevalonate pathway, essential for bacterial membrane stability, can protect bacteria from statins’ cytotoxic effects? It should be the opposite effect. Please, clarify. 4. Is there any study related to the analysis of microbiota in people under prolonged statin treatment? This kind of information is missing in the review, and will strength the discussion related to the negative effects of statin against microbiota.

You are one of the reviewers, your task is to write a review for the article. You will 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: STATINS: ANTIMICROBIAL RESISTANCE BREAKERS OR MAKERS? Review round: 1 Reviewer: 2

Basic reporting: This work presents a compilation of MIC data for Statins against Gram-positive and Gram-negative bacteria (Results and Tables 1 and 2), proposed mechanism(s) of action of Statins, and knowledge gaps in the field. The manuscript is well written. References, background and tables are provided in sufficient detail. Line 235 – Title needs editing. (suggestion: Fungal origin does not correlate with antibacterial activity/efficacy.) Experimental design: Although a summary of the current status of research on Statins may be useful for researchers in the field, the specific aims and scope of this review are unclear. In the Introduction (Conclusion), it is claimed that this review 'provides evidence' of a link between statins and antimicrobial resistance. This is inaccurate, as the only data presented in this work are MIC values from previous studies and molecular structures of Statins. At best, this manuscript proposes how synergistic use of statins with other antibiotics may affect microbial outcomes. As detailed in section 3 below (validity of the findings), the utility of summarizing the MICs is not obvious, especially in the light of significant variation in MIC data and the limited scope of MIC assays as tools for understanding mechanism of antibacterial action. It is suggested that whether statins support or oppose primary drugs in their killing action is contextual. Several examples are presented in the discussion section, based on previously published works. Although this is a significantly important part of the review, it seems disconnected from the previous analysis of MICs and plausible mechanism. Validity of the findings: The Results section summarizes MIC values of various statins reported in the previously published studies. The authors then go on to list the potential pitfalls of MIC analysis, including differences in assay times, growth media and other experimental conditions (e.g. with or without shaking). Given the large variation (~5-fold) in MIC values reported for the same statin with the same bacterium, the utility of the MIC comparisons made by the authors seems limited. It is important to note that MIC assays have a limited scope as far as elucidating the mechanism of antimicrobial action is concerned. Within these limitations, the authors have analyzed the structures of Statins and proposed a plausible mechanism of action, namely binding to the bacterial outer membrane proteins, wall teichoic acids and lipoteichoic acids. It is not clear how the authors arrived at this based on their analysis of MICs and/or the structure of Statin molecules. I urge the authors to clarify how the quarternary chiral carbon, and attached methyl groups, might affect the mechanism of action? Suggestion of a possible interaction between the methyl groups and alanine groups of teichoic acids is speculative. Is there data on Statin-bacteria interactions to support this statement? For e.g. are there any docking studies of the di-methyl moiety to components of LPS, lipid bilayer, outer membrane proteins, etc? Lines 55-59: Difference in the MIC and sub-MIC regimes is not clear. Both seem to involve growth of resistant bacteria and inhibition of growth of susceptible bacteria. Lines 103 -106 are identical to lines 15-18. Line 113 – ‘relative’ and ‘comparison’ are redundant. ‘quantitative’ comparison might be more appropriate. If the authors agree, this change should be made for all instances in the text. Line 251 – Absence of antimicrobial activity of PRV could be due to other factors (for e.g. absence of quarternary chiral carbon, which the authors have suggested might be important for binding to outer membrane components). These may be essential or preceding steps in the action of statins, prior to involvement of the dihydroxy acid moiety. Therefore, inhibition of HMG-CoA could still be one of the mechanisms of action of statins. Line 321 – Binding to statins to proteins is suggested as one of the mechanisms of the inhibitory action of Statins. The readers might benefit from a table summarizing the % binding for various Statins. Lines 326-336: The hypothesis that more membrane area per volume requires more statin molecules seems an oversimplification. There could be other differences between MRSA and MSSA that may explain the difference in MIC values. For e.g. It is unclear how the virulence of both strains compares. If MRSA were more virulent than MSSA, that would significantly affect the MIC values. Line 304 – Please clarify what is meant by ‘bacterial adhesion’. Line 309 – Suggestion of a possible interaction between the methyl groups on Statins and alanine groups of teichoic acids is speculative. Is there data on Statin-bacteria interactions to support this statement? Line 321 – Binding to statins to proteins is suggested as one of the mechanisms of the inhibitory action of Statins. The readers might benefit from a table summarizing the % binding for various Statins. Line 340-347 – Statins are electrically neutral. How do they interact with the bacterial LPS (hydrophilic) which is charged, and the phospholipid bilayer (anionic)? Line 547-550 – It is not clear what the authors are trying to convey here. Additional comments: I suggest the authors re-analyze the purpose and scope of this manuscript and revise this draft prior to resubmission. The purpose of the MIC analysis needs to be considered and clarified in any revisions the authors might make. A possible route to redrafting this manuscript would be to focus solely on the synergistic action of Statins with other antibiotics. This might help provide a clear framework for the review, and serve as an important resource for researchers interested in the effects of synergistic drug treatment on antimicrobial resistance.

You are one of the reviewers, your task is to write a review for the article. You will 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: STATINS: ANTIMICROBIAL RESISTANCE BREAKERS OR MAKERS? Review round: 2 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: I consider that the manuscript has been significantly improved including all the suggestions that I made at the first round of the peer review process.

You are one of the reviewers, your task is to write a review for the article. You will 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: MEASURING LANGUAGE LATERALISATION WITH DIFFERENT LANGUAGE TASKS: A SYSTEMATIC REVIEW Review round: 1 Reviewer: 1

Basic reporting: Overall, this is a clearly-written manuscript that thoroughly describes the process of conducting a systematic review. The authors' literature search is thorough and appropriately documented with a Prisma diagram. The parameters for the review are described on osf.io. The authors note making the R code for the forest plots available at https://osf.io/t24rv/, but when I access this link I get an error - does this need to be made public? Experimental design: The research question - namely, how different experimental tasks affect language localization - is timely and important. I am not aware of other reviews (certainly not systematic ones) that address this topic. The methods are described in enough detail that the searches could be replicated. Validity of the findings: The conclusions are well stated and relate to the authors' findings. Additional comments: Overall I very much enjoyed reading this manuscript, and my lack of substantive comments is due to the excellent job you have done with the manuscript. I did think that it would be nice to more thoroughly address the various approaches to quantifying lateralization, but i understand the rationale for putting these in a separate paper. Other minor comments: -It would be worth having a careful proofread of the references. In my quick glance, I noticed: some journals underlined and italicized (most just italicized); some unusual capitalization (Bmc instead of BMC, Neuroimage rather than NeuroImage); for Hickok & Poeppel (2007) I don't generally see "Opinion" included in the article title; Peelle (2012) is incorrect in the main text (Peele) but correct in the references; some citations have month included (e.g. Warrington & Shallice 1984) but most don't. -For the Prisma diagram (Figure 1) it would be nice to include reasons for the 8 excluded articles. -For the figshare link (https://figshare.com/articles/Forrest_Plots_of_LI_values_for_different_language_tasks/4977950), I believe "Forest" plots are mis-spelled, which might lead some people to have difficulty accessing the link. Also, if you want to share the graphs, I would suggest a smaller file size: PNG for raster-based, or perhaps a vector-based format such as .eps.

You are one of the reviewers, your task is to write a review for the article. You will 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: MEASURING LANGUAGE LATERALISATION WITH DIFFERENT LANGUAGE TASKS: A SYSTEMATIC REVIEW Review round: 1 Reviewer: 2

Basic reporting: Well written and generally clear. See comments under "General comments for the authors". Experimental design: NA Validity of the findings: NA Additional comments: Abstract Line 32: What does “closely matched tasks” mean? I think the authors mean tasks with baselines that actively control for as many factors as possible, isolating the linguistic process in question. This could be stated more clearly in the abstract (at this first mention of ‘closely matched’). Introduction Lines 83-85: “Answering such questions requires optimisation of protocols for LI measurement, to ensure that variability in LIs measured for different language functions cannot be attributed to differences in task sensitivity or measurement error.” I understand what the authors are arguing for here, but is this really even possible? How does one equate apples (e.g. language production) and oranges (e.g. language comprehension)? For instance, how does one define sensitivity without becoming circular (depending upon the LI)? Results Line 382: “This subtracts out non-linguistic work memory processes (see Table 3).” Probably worth noting that it subtracts out basic visual processing as well. Again, this highlights the futility of the idea of pure insertion though, as what is essentially a phonemic task, mediated via visual means (orthography), is contrasted with what is essentially a visuospatial form task. Lines 392-397: “…a recent meta-analysis of functional imaging studies by Rice, Ralph and Hoffman (2015) suggested that while conceptual knowledge does appear to be represented bilaterally in the anterior temporal lobes, left lateralised activity was more likely when semantic content was accessed linguistically. This is in contrast to the predictions of Hickok and Poeppel’s (2007) model of language in which mapping of sound to meaning is considered as a bilateral process.” Is this really a problem for the Hickock and Poeppel model? Doesn’t it just suggest that linguistic information is left lateralised. Line 467: A carriage return is missing before, “Effect of ROI” Line 545: “be” appears to be missing between “may” and “influencing” Lines 545-546: “In this way, CTA represents a theoretical alternative to baseline tasks to subtract domain-general activity.” This is not really true as the activation analysis for any given task in a CTA still depends on the definition of a baseline. Lines 557-559: in the references cited here (dissociated language functions within healthy individuals) I would suggest adding (though in the interests of transparency I note that I am an author on one of the papers cited below): • Tailby, C., Abbott, D. F., & Jackson, G. D. (2017). The diminishing dominance of the dominant hemisphere: Language fMRI in focal epilepsy. NeuroImage: Clinical, 14, 141-150. • Berl, M. M., Zimmaro, L. A., Khan, O. I., Dustin, I., Ritzl, E., Duke, E. S., ... & Gaillard, W. D. (2014). Characterization of atypical language activation patterns in focal epilepsy. Annals of neurology, 75(1), 33-42. Line 566: Why would CTA be inappropriate for investigating dissociated language functions within individuals? I don’t follow the logic here – if there really is dissociation (say left Wernicke’s and right Broca’s) why should that not come up across tasks (any less so than the localisation of Wernicke’s and Broca’s in non-dissociated individuals)? Summary and Conclusions Line 583: the phrase “active baselines” should be qualified, along the lines of say, “carefully selected active baseline aimed at controlling for all but the language related process of interest.” I don’t think an active baseline per se is the answer the authors are proposing, but rather one that is crafted according to the language laterality question that is being addressed. I think this point is worth belabouring at first appearance in the Discussion. Lines 588-590: “Future studies should try to more closely match task demands of word generation and sentence generation tasks.” Some further exploration of what kind of form such closer matching might take is called for here, as in some sense this is an (to use fruit as a reference again) apples and oranges question – how does one objectively identify that one has matched word and sentence generation tasks, especially once one has taken into consideration the influence of baseline too? Lines 611-614: I still do not understand the argument that CTA is somehow inappropriate for dissociated dominance cases, any more so than for non-dissociated individuals (as per comment above). I agree that CTA collapses across inter-task processing particulars, but why this affects dissociated individuals more than non-dissociated individuals eludes me? Lines 614-616: I agree with the sentiment that “research should focus on developing batteries of closely matched task that tap a variety of language functions to allow systematic comparisons in within-subject studies.” I think, however, that in a manuscript such as this – aimed at providing guidance to future “users” of language lateralisation approaches – that some text should be allocated to sketching out what such an approach might look like. Otherwise statements like the one quoted above just sound like empty rhetoric, rather than being instructive and useful to the reader. Inclusion of a sketch of what such a battery might look like would greatly enhance the utility of the article. As a key take home message from this review article is the importance of ‘closely matched tasks’ I think the authors are beholden to offer some more concrete suggestions here. Tables Table 2: ‘Verbal fluency’ should probably have a parenthetic tick in the ‘Orthography’ column, as in the case of letter based fluency (knowing, for instance, that “phlegm” begins with ‘p’ and not ‘f’). Indeed, an alternative name for letter based fluency is Orthographic Lexical Retrieval. Similarly, ‘Phonemic judgement’ should probably have a tick in the ‘Speech motor planning’ column, given that these kinds of tasks almost invariable require some kind of subvocal rehearsal (arguably also ‘Sentence comprehension’, depending on the length of the sentence). I happily acknowledge the subjectivity of the table (making categorical judgments about the presence/absence of a given cognition process during a given task), but these seem important constituent elements of these tasks. More generally, I don’t think that splitting into Table 2 (functions involved in various language tasks) and Table 3 (functions involved in various baseline tasks) is necessarily the most helpful organisation in the context of the goals of the paper. The authors seem to be arguing for the use of better controlled tasks, including better control over differences between active and baseline states in fMRI (e.g. lines 175-176: “Tables 2 and 3 highlight the difficulty in designing a task which isolates a single language function in order to study its laterality”). For the ‘wishing to be informed’ reader to make best use of this article it would seem ideal to have the same ‘cognitive function’ headings in both Table 2 and Table 3, or even better to have everything combined into one ‘mega-table’ where column one is task (be it active or baseline) and columns 2-n are the purported cognitive functions involved; then one can just scan pairs of rows to find active and baseline states that appear to isolate a given linguistic operation. For instance, Working Memory is listed as a cognitive function in Table 3 but not in Table 2. I realise I am being a bit literal here but if I use a baseline task for which there is a tick under Working Memory in Table 3, but there is no corresponding Working Memory column in Table 2, what am I as the reader supposed to take from this? Could any deactivation that I see for the language task be due to no working memory load in the active state I have used? A mega-table would be nice in that it would enable the reader to choose a pair of rows, one active and one baseline, and compare where they did or did not have ticks and use this to identify the cognitive operation they are attempting to isolate. Another option that could prove useful would be to have an example table that goes through the exercise of selecting active and baseline tasks. For instance, using one example language task and a range of different baseline tasks, to illustrate clearly the implications of the decisions made in task design for the observed patterns of activity (e.g. using an example from the text, an active task of sentence generation compared with either (1) a baseline that is passive fixation or (2) a baseline that consists of covert recitation of over-learned sequences like the months of the year). I realise that these ideas are all said in text in the body of the document, but a graphical representation of the idea has the potential to render this clearly, succinctly and compellingly and I think is worth exploring. (I realise I am treading a fine line here as a reviewer, potentially straying into editorial comments, but as the tone of the article is didactic as a reviewer I would like to see a clear visual representation of the information conveyed within the body text.) The authors seem to imply that there is a ‘true’ measure of laterality that the fMRI paradigms are trying to reveal. If laterality does vary as a function of the baseline task, which is the ‘true’ laterality, or which is the ‘clinically relevant’ laterality? Can we even know this? Implicit in some of the discussion in the paper is the idea that strong laterality is a marker of a good task, but is this really the case? If we are seeking the ‘truth’ of laterality for a given region or task, then selecting the task that has the strongest laterality might be biasing the results. This really comes down to the question of what are we hoping to obtain through our laterality measurements? Is reliability/stability more important than strength of laterality? Or is it important, for instance, to identify what degree of laterality is indicative of redundancy of function, such that unilateral resection would be safe? I think the Discussion would benefit from some consideration of “how do we know that we have accurately captured laterality of a region/task?” At the least this would make overt the idea that the strongest lateralisation is the best. I admit though that this is a difficult question to answer. I just worry that the ‘strongest lateralisation is best’ approach runs the risk of relying on the metric do dictate meaning, rather than having the underlying functionality of an area dictate meaning. Followed to the extreme, we may continue refining tasks for a given region until we come up with one that yields very strong lateralisation, ignoring those with weaker lateralisation that we tested in earlier iterations. If followed to this extreme, have we then potentially lost important information about the non-dominant hemisphere (that resecting it might cause a deterioration in function; or that resecting the dominant hemisphere might not be as disastrous as feared because the non-dominant hemisphere could support residual function)? These are difficult questions to answer – I don’t pretend to have the answers to them. I am just urging caution with the ‘strongest LI is best’ approach, and consideration of the question regarding functional consequence of laterality measures (the difficulty of evaluating the relative utility of different LIs obtained via different tasks). It seems to me that one of the best ways to answer these questions is with surgical outcome studies. And once viewed from this light, the relevant metric would be the task for which the LI is best predictor of outcome, regardless of whether this is also the task with the strongest LIs. =============================== In summary, I found this manuscript to be a useful summary of the literature, raising some important issues in the field. If some of the above comments could be addressed I believe this will increase the utility of 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: MEASURING LANGUAGE LATERALISATION WITH DIFFERENT LANGUAGE TASKS: A SYSTEMATIC REVIEW Review round: 1 Reviewer: 3

Basic reporting: The manuscript is very well written in Standard English and there are no issues with the basic reporting. Literature references and context seem to be in order. The standard article structure for systematic reviews is followed. The article does not seem to have a clear hypothesis, though, that is something the authors could potentially work on. Experimental design: As far as I can judge it, the authors followed all standards for such studies. I do not see any issues with the design of this systematic review. The research question is well defined and meaningful. Technical standards are fulfilled and methods description is sufficient enough for potential replication. Validity of the findings: This seems to be a very robust and statistically sound finding, and conclusions are in line with the data. Additional comments: This is an interesting systematic reviews that covers an important question in the literature. The authors should work on clearly stating a hypothesis, but apart from that I see no problems with accepting this work.

You are one of the reviewers, your task is to write a review for the article. You will 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: MEASURING LANGUAGE LATERALISATION WITH DIFFERENT LANGUAGE TASKS: A SYSTEMATIC REVIEW Review round: 2 Reviewer: 1

Basic reporting: ok Experimental design: ok Validity of the findings: ok Additional comments: I don't see any remaining issue with the paper and think it could be recommended for acceptance now.

You are one of the reviewers, your task is to write a review for the article. You will 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 HEAD TURN PARADIGM TO ASSESS AUDITORY LATERALITY IN CATS: INFLUENCE OF EAR POSITION AND REPEATED SOUND PRESENTATION Review round: 1 Reviewer: 1

Basic reporting: 109: Something is wrong with the grammar in this line/sentence. Otherwise, the manuscript is written clearly and well. The references cited are adequate. However, see my request to add more details on the method used, rather than slavishly referring the reader to previously published methods. Experimental design: The authors rely too frequently on citation of previous papers that have used this method to score head turning. Instead, all of the details of the method should be given in this paper. 150-151: Say what is the evidence that the calls played back are of high versus low arousal since this is critical to interpreting the results. The sentence “The stimuli were distinct with regard to the arousal state of the sender….” does not make sense. Please clarify. Also, it is not clear what data have been tested statistically. 172: Here it is stated that the head had to be held “approximately straight”. This is too vague, please give the acceptable angles of the head. 174: Say precisely what position of the ears was considered to be “straight”. Validity of the findings: 231 and 232: What do the values n=7 and n=9 refer to here? 253 to 264: There was a significant group level bias to the left for the first 7 presentations but later it is said that the left turn bias did not differ significantly from chance. Does the latter statement refer only to the first head turn and, if so, how was a HTI value calculated? Figure 3: Given that 7 of the 15 subjects shifted to an HTI of 1.0 in the second 7 presentations, it would be interesting to draw lines linking the score obtained in the first 7 presentations for each individual with the score for that individual in the following 7 presentations. These could then be tested using the paired Wilcoxon tests to see whether there has been a significant change at group level. Furthermore, since the data for the “following 7” look skewed, it would be better to show a median rather than a mean. Then a shift from left to right may be apparent and, at least, considered. Additional comments: 161: Correct ‘in details’ to ‘in detail’. 310-314: This interpretation of the results of Siniscalchi et al. (2008) is most unlikely to be the case because these authors looked at habituation and chose to analyse responses to the first 7 presentations since no significant habituation of responding occurred over the first 7 presentations (see their Figure 2). I am positively inclined towards your examination of problems with the head turning paradigm but the manuscript would have beeen improved if you had included other calls that might have given different results, especially using a wider selection of calls that might elicit low versus high arousal responses.

You are one of the reviewers, your task is to write a review for the article. You will 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 HEAD TURN PARADIGM TO ASSESS AUDITORY LATERALITY IN CATS: INFLUENCE OF EAR POSITION AND REPEATED SOUND PRESENTATION Review round: 1 Reviewer: 2

Basic reporting: The paper is reasonably well written, although spelling and grammar require attention in quite a few places Experimental design: No comment Validity of the findings: No comment Additional comments: This paper explores head turning in response to auditory stimulation at the individual level. The paper addresses an interesting and understudied area and draws attention to some important potential confounders in studies on this nature. I only have some small issues, highlighted below. Abstract This is well written, although inclusion of more methodological detail in places would certainly strengthen it. Introduction This is largely well written until the final few paragraphs, when the rationale for the study seems to become less clear. The authors start to talk about female cats (implying a sex effect?), but it is not quite clear whether they are referring to a sex effect or the study they mention only used female animals as its subjects. The last paragraph of the Introduction needs further expansion as it is confusing in nature and does not really set the reader up for the purpose of the investigation. Spelling and Grammar Line 30. Add ‘the’ before ‘individual’ Line 31. Add ‘the’ before ‘group’ Line 33. Change ‘influences’ to ‘influenced’ Line 43. Add ‘the’ before ‘behavioural’ Line 60. Change ‘over’ to ‘to’ Line 107. Change ‘will be….investigated’ to ‘would be…investigate’ Line 109. Remoe ‘these’ and change ‘shows’ to ‘show’ Line 161. Change ‘details’ to ‘detail’ Line 169. Change ‘maximal’ to ‘maximum’ Line 171 and 184. Change ‘to’ to ‘of’ Line 231. Remove ‘only’ Line 270. Change ‘on’ to ‘an’ Line 353. Change ‘indicators’ to indicator’ Line 358. Change ‘more a’ to ‘a more’

You are one of the reviewers, your task is to write a review for the article. You will 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 HEAD TURN PARADIGM TO ASSESS AUDITORY LATERALITY IN CATS: INFLUENCE OF EAR POSITION AND REPEATED SOUND PRESENTATION Review round: 2 Reviewer: 1

Basic reporting: This has been improved. Experimental design: No change but some extra details added. The authors' response included a photograph of the testing apparatus and that should be added to the paper itself to assist the reader in understanding the paper. Validity of the findings: The findings are valid. 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 COMPARISON OF MOLECULAR AND MORPHOLOGY-BASED PHYLOGENIES OF TRICHALINE NET-WINGED BEETLES (COLEOPTERA: LYCIDAE: METRIORRHYNCHINI) WITH DESCRIPTION OF A NEW SUBGENUS Review round: 1 Reviewer: 1

Basic reporting: The authors present a comprehensive molecular phylogeny of trichaline net-winged beetles whose taxonomic rank remains a subject of debate. The MS is written in clear English overall with minor editing (mostly grammatical) required to improve the coherence and sentence fluency (e.g., in line 131: “maximum parsimony (MP) analyses,” not “phylogenetic parsimony (MP) analyses;” and in line 136: “morphology-based phylogenetic relationships” rather than “phylogenetic morphology-based relationships”). Please also check for typos; there are still quite many, though I did not explicitly check for them. Sufficient review of relevant literature is provided and all figures and tables are prepared professionally. Experimental design: The MS represents an original research that fits the aims and scope of the journal. The authors attempt to propose a natural classification system of trichaline net-winged beetles based on their comprehensive molecular phylogeny, together with morphological cladistic analyses at the genus level. The authors have employed a wide range of the state of the art methods with extensive sampling of molecular data. However, more detailed information and/or further elaboration on some of their analyses should be provided in Material and Methods. - Lines 111–112: “The concatenated supermatrix was partitioned for all fragments and codon positions when appropriate.” Please provide how the final supermatrix was partitioned for each analysis. Was any software, such as PartitionFinder, used to determine the best partitioning scheme? - Lines 113–116: Please explain why different models of nucleotide substitutions were used for ML tree search (i.e., GTRCAT) and its BS calculation (i.e., GTRGAMMA). In most cases, same model should be be used to calculate BS values. - Lines 116–117: “The BI analysis was run in MrBayes . . . under the GTR+I+G model identified as above . . .” I highly recommend using softwares like PartitionFinder, which not only determines the best partition scheme, but also the best fitting models for each partition. The assignment of proper substitution model for each partition can significantly improve the phylogenetic reconstruction results. - Explain how all the trees were rooted. This is essential for making assessment on monophyly of the ingroup, as well as its systematic position within the larger group (i.e., Metriorrhynchini). Validity of the findings: The molecular phylogeny reported in the MS shows relationships among 143 samples representing 86 species of trichaline beetles, whereas the morphology-based phylogeny only include 11 terminals, each of which represents different genus within the so-called “trichaline clade.” The title of the MS, “The congruence of molecular and morphology-based phylogenies of trichaline net-winged beetles . . .,” can therefore be misleading since it sounds like the morphological analyses were conducted on the same set of samples included in molecular analyses. Furthermore, the authors do not seem to have found strong evidence for the congruence between their DNA-based phylogeny and morphology-based phylogeny. In lines 266–271, the authors write: “The morphological analyses did not support the monophyly of the DNA-based trichaline clade. . . The deeper relationships were poorly supported,” and again in line 645: “The morphological analyses indicate different relationships,” which essentially refutes the main argument made on the congruence of molecular and morphology-based phylogenies. Therefore, a different title that more accurately describes the main finding of the MS seems necessary. In lines 161–164: “The phylogenetic trees inferred . . . using the ML criterion and Bayesian inference were well-resolved and suggested similar relationships . . .” Please provide the BI topology as a supplementary figure and briefly discuss any discrepancy observed between the ML and BI trees, if there is any. In lines 630–634: “The terminal position of the trichaline clade in Metriorrhynchina has already been demonstrated in the molecular analyses of Metriorrhynchini . . . Our analyses of the current more extensive dataset confirm the terminal placement of the trichaline clade (Fig. 1A).” This statement depends largely on how the trees were rooted in the present study. As I pointed out earlier, please explain in Material and Methods how your trees were rooted. In the section Taxonomy, the diagnosis and redescription of “trichaline clade” is provided, but it is unclear which taxonomic rank this proposed clade represents. A clade can be a group of organisms at any taxonomic level, so long as the constituents are believed to be the decedents of a common ancestor. Therefore, the designation of trichaline as a “clade” does not address the main problem with alpha-taxonomy discussed in the introduction of the MS (see lines 42–43). In Figure 1, note that there are two (B)’s in the legend and be mindful of their order (i.e., Fig.1B is the BI tree, and 1C the MP tree). Also, please clearly state that both 1B and 1C are morphology-based trees. I highly recommend collapsing all the nodes with support values below 50% in 1B and 1C trees and present them as polytomies, as already done for one of the nodes in Fig. 1C. You may provide all of the three original equally parsimonious MP trees as supplementary figures. 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 COMPARISON OF MOLECULAR AND MORPHOLOGY-BASED PHYLOGENIES OF TRICHALINE NET-WINGED BEETLES (COLEOPTERA: LYCIDAE: METRIORRHYNCHINI) WITH DESCRIPTION OF A NEW SUBGENUS Review round: 1 Reviewer: 2

Basic reporting: Manuscript is written in clear and concise English. References are ample, treated in concise format, sufficient background is provided in the intro part. Structure of the paper corresponds with usual arrangement of phylogenetic papers. Rich figures illustrates crucial structures of the morphological data, phylogenetic analyses are presented in clear and visually attractive form. Molecular data are added as supplementary file in FASTA format. Morphological data are added as a table in the text. Probably it would be good to add it also as supplementary file, maybe in NEXUS file, to be quickly available for the readers? Phylogenetic hypothesis is clearly formulated and correspond with text in results and discussion part of the ms. Experimental design: The manuscript presents original biological research. Research questions are clearly formulated, corresponding with improvement in classification of the group, but also trying to link morphological and molecular data. This is highly interesting in a group developing mimetic rings, where many morphological characters have homoplastic distribution within different genera and classification based on morphology only can be highly misleading. This concept is clearly formulated in the text. The research is rigorous, corresponds with high standards in phylogenetics, use advanced methods for evaluation of molecular data. In my opinion, it is in no conflicts with ethical standards. Most of the methods are described in sufficient details. I have only two comments which can improve the clarity of the text: 1. Twenty species are treated as outgroup in the phylogenetic analysis. I will appreciate comments on logic how these taxa were selected – how closely related they are to the ingroup. Should be inserted in a paragraph on lines 107-122. 2. In morphological data matrix, the first taxon (Metriorrhynchus) presents probably outgroup and is used to root the resulting trees. This should be added to the text. Also, explicit information how character states were polarized should be mentioned in the text (paragraph on lines 124-142, 174-177). List of morphological characters, included in Results (starting on line 174) should be moved in M&M section in my opinion. Some characters have detailed discussion about distribution of character states in individual genera or lineages, some have no comments. This should be unified. Several characters included presents only autapomorphies of a single genus, and bring no information to the phylogeny of genera (which is main goal of the paper). Validity of the findings: I have no important comments to Results and Discussion parts, which are generally very well written. The following details can be however improved in the text: Diagnosis of trichaline clade (lines 280-294). Here, on lines 286-294, you listed five very different states how male genitalia look like – this makes no diagnosis for the group! Maybe should be commented, or this part should be deleted? Spinotrichalus, lines 427-428: “[these characters …] are the autapomorphy of S. telnovi and Spinotrichalis may be trated as synonym, when its position renders Lobatang paraphyletic.” But if you combine Spinotrichalus as subgenus of Lobatang, the same apply for the nominotypical subgenus! The newly described subgenus, Maibrius, is characterized on lines 503-505 only as “differs from the nominotypical subgenus only in the serrate male antennae …” – but serrate/flabellate antennomeres are homoplasious within the group. You contradict this in the Discussion, lines 696-700), where you stated that “…serrate … male antennae … their value is questioned by variable morphology in related species”. Would it be possible to characterize Maibrius with some other morphological apomorphies as well? Further, in a Remark on lines 507-513, you states that “the molecular phylogeny identified F. (Maibrius) horaki sp. n. as a genetically distant sister-lineage to other Flabellotrichalus”. But, as seen from Table 1 (lines 65-80), your sampling of this genus for molecular analysis is based exclusively on nine unidentified species (sp. A to sp. I in Table 1) from New Guinea. There is no info in this ms how many species of this genus is described, what is their geographical distribution etc. You should comment on this – why you are not able to identify your samples to species? Is your sampling covering the general distribution of known species of Flabellotrichalus – this means, is the nominotypical subgenus as proposed distributed in PNG and the newly described species from West Papua? In the identification key, you are using exclusively male characters in some couples, but exclusively female ones in others (see lines 618-625). This is not very “user friendly”. Can this be improved? Also, info that female sex is not known for Maibrius should be inserted on lines 618-619. Additional comments: Minor comments and typos: line 50: “which is now a part” – it means a junior synonym or subgenus? line 200: “1,” should be “(1)” line 270: “Their stict consensus and one of the most parsimonious trees were unresolved” – I understand how consensus tree can have unresolved some nodes. But can be this applied also for a single parsimony three? Possible typo? Effect of missing data? This is not clear to me, please explain. line 405: add comma line 979: second “(B)” should be in fact “(C)”

You are one of the reviewers, your task is to write a review for the article. You will 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: SELF-EFFICACY, RELATIONSHIP SATISFACTION, AND SOCIAL SUPPORT: THE QUALITY OF LIFE OF MATERNAL CAREGIVERS OF CHILDREN WITH TYPE 1 DIABETES Review round: 1 Reviewer: 1

Basic reporting: The article is written in English. The authors have used clear text. The introduction should include more information, changes in the daily life of caregivers due to the specific characteristics of type 1 diabetes, such as glycemic control, dietary restrictions, physical exercise, and mothers' fear of The disease causes irreparable damage, hypoglycemia, etc. It should also include a brief description of the disease (type 1 diabetes) and its psychosocial characteristics. Therefore, the introduction is incomplete Experimental design: It is not clear in the text the purpose of the study I consider it important to include in the text the average age of the participants, as well as the standard deviation. Text include age of the child but no of the mothers. Used instruments are correct, and have high levels of reliability Methods is described with sufficient information to be repproducible by another investigator. The time of diagnosis of diabetes in childs has not been taken into account. This variable will affect the results, It would be important to include it in the text. Validity of the findings: The results are novel and easily replicable by other authors. Additional comments: The study brings knowledge to science. However, it would be interesting to carry out comparisons between mothers of children with type 1 diabetes, and mothers of children with other chronic diseases, such as asthma. Studies indicate differences in quality of life between the two.

You are one of the reviewers, your task is to write a review for the article. You will 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: SELF-EFFICACY, RELATIONSHIP SATISFACTION, AND SOCIAL SUPPORT: THE QUALITY OF LIFE OF MATERNAL CAREGIVERS OF CHILDREN WITH TYPE 1 DIABETES Review round: 1 Reviewer: 2

Basic reporting: The article is written in English. Literature is well referenced and relevant. The authors have used a clear text and an adequate terminology. In my opinion, the introduction of the document should raise more accurate information regarding the different typologies that exist within diabetes. Likewise, it is important to indicate in a more detailed way sociodemographic aspects that may be directly influencing the variables of self-efficacy, social support and satisfaction in relationships. Experimental design: Research question well defined, relevant and meaningful. Used instruments are correct, and have high levels of reliability. Methods are described with sufficient information to be reproducible by another investigator. I believe that one should, on the one hand, delve more precisely the aspects related to sociodemographic variables and, on the other, make clear the reasons why the application of model 1 and model 2 offer different results (lines 210-215). It is necessary to explain what rights are taken to choose one model instead of another. If we add other measures, we must explain the reason. Validity of the findings: Results are fascinating and easily replicable by other authors. Additional comments: This work contains very relevant aspects that confirm the tendency or relationship between levels of self-efficacy, satisfaction relations and social support of mothers caring for children with type 1 diabetes. I suggest paying more attention the psychosocial and environmental aspects that affect the variables of Study, as well as the comparison with other groups of mothers with children with chronic diseases of particular attention.

You are one of the reviewers, your task is to write a review for the article. You will 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: SELF-EFFICACY, RELATIONSHIP SATISFACTION, AND SOCIAL SUPPORT: THE QUALITY OF LIFE OF MATERNAL CAREGIVERS OF CHILDREN WITH TYPE 1 DIABETES Review round: 1 Reviewer: 3

Basic reporting: See below Experimental design: See Below Validity of the findings: See below Additional comments: Thank you for the opportunity to review “Quality of life of maternal caregivers of children with type 1 diabetes and the role of self-efficacy, relationship satisfaction, and social support.” This manuscript examined differences in quality of life between mothers of children with and without type 1 diabetes, as well as the role of self-efficacy, relationship satisfaction, and social support in predicting quality of life. A strength of the manuscript include the inclusion of a comparison group. A major weakness of the manuscript is the lack of inclusion of child variables, including child age. The parents’ role in management of T1D differs greatly among very young children, school age children, pre-adolescents, and adolescents. There is a developmental transition of responsibility that occurs over time, which would certainly impact parent quality of life. Inclusion of the child’s age is needed to assist with interpretation. Other child variables such as duration of T1D diagnosis would be helpful to include as well. There are a number of additional concerns, which are described below. Introduction 1) Many of the references cited are over 10 years old - I recommend reviewing the introduction and discussion to ensure that this paper is placed in the most current context, and to highlight how this paper adds to the contemporary knowledge base. 2) Line 76. It is unclear whether the caregiver or child experiences difficulty in emotional functioning as a result of caregiver diabetes stress. 3) Line 80. Not all readers will be familiar with the transactional stress and coping model, so the authors should either briefly review the model or more clearly specify which elements provide a theoretical basis for the investigation and why. 4) Line 95. It is not clear what type of relationships the authors are referring to until later in the paragraph Given that there is a body of literature on parent-child relationships/conflict in T1D, it should be made clear that the authors are referring to marital relationships earlier on in the paragraph. 5) Line 104. It is not clear whether the authors are referring to the parent or child’s level of social support. Additionally, there is some research on social support in parents of children with T1D. The authors should review and cite this literature to expand this paragraph, which is only two sentences. See Wysocki T, Greco P. Social support and diabetes management in childhood and adolescence: influence of parents and friends. Curr Diab Rep 2006: 6: 117–122.; Lewandowski A, Drotar D. The relationship between parent-reported social support and adherence tomedical treatment in families of adolescents with type 1 diabetes. J Pediatr Psychol 2007: 32: 427–436; Carcone AI, Ellis DA, Weisz A, Naar-King S. Social support for diabetes illness management: supporting adolescents and caregivers. J Dev Behav Pediatr 2011: 32: 581. 6) Please explain why only the quality of life mental health domain was used as the criterion variable as opposed to the total score or other subscales. 7) In hypothesis b, it seems as though the authors are only going to examine the regression in the diabetes group and it is unclear how the comparison group fits into this hypothesis. Materials & Methods 8) In accordance with people first language (see APA Manual), the term “diabetic child” should be changed to “child with T1D” throughout the manuscript. Additionally, “diabetic group” should be modified. 9) Please specify whether there is a total score for the SF-36. Please also indicate the response format for the SF-36 and the DAS (i.e., responses range from X to Y). 10) In the description of the SF-36, it is unclear what the domain “role-physical” measures and how this differs from physical functioning. A brief explanation is needed to assist with interpretation of findings. 11) Line 151-153. It is unclear what “termed the reported health transition” means. Also, a citation noting similar research that excludes the single item is warranted. 12) The Statistical Analyses section should specific which analyses were run (e.g., t tests, regression) and why. Results 13) The explanation of results needs further detail. It is unclear why the mother group was entered into the model when the hypothesis only specifies “in mothers of children with diabetes.” Discussion 14) There is a growing body of literature on sleep disruption in parents of children with T1D, which could indeed be construed as having an impact on physical functioning. This issue warrants mention and interpretation. 15) The authors should offer an interpretation for why demographic factors were found to negatively impact certain variables.

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

Title: SELF-EFFICACY, RELATIONSHIP SATISFACTION, AND SOCIAL SUPPORT: THE QUALITY OF LIFE OF MATERNAL CAREGIVERS OF CHILDREN WITH TYPE 1 DIABETES Review round: 2 Reviewer: 1

Basic reporting: Suitable Experimental design: Suitable Validity of the findings: Suitable Additional comments: I have read the changes made, and I can say that these reflect the suggestions made by me in the evaluation process.

You are one of the reviewers, your task is to write a review for the article. You will 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: SELF-EFFICACY, RELATIONSHIP SATISFACTION, AND SOCIAL SUPPORT: THE QUALITY OF LIFE OF MATERNAL CAREGIVERS OF CHILDREN WITH TYPE 1 DIABETES Review round: 2 Reviewer: 2

Basic reporting: . Experimental design: . Validity of the findings: . Additional comments: I have read the changes made, and I can say that these reflect the suggestions made by me in the evaluation process.

You are one of the reviewers, your task is to write a review for the article. You will 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: SELF-EFFICACY, RELATIONSHIP SATISFACTION, AND SOCIAL SUPPORT: THE QUALITY OF LIFE OF MATERNAL CAREGIVERS OF CHILDREN WITH TYPE 1 DIABETES Review round: 2 Reviewer: 3

Basic reporting: See below Experimental design: See below Validity of the findings: See below Additional comments: The authors did a nice job addressing reviewer feedback. I have several additional suggestions for improving the manuscript. The additional details provided in the first paragraph are helpful; however, the types of oversight and degree to which caregivers of children and adolescents with T1D provide this oversight varies with the age of the child and the duration of diagnosis. This warrants mention. Additionally several details added are already discussed further below in the introduction and warrants them unnecessary. For example, the second paragraph is now almost entirely covered in the new details added to the first paragraph. Likewise, so is the last sentence in the second paragraph on page 4. Additional comments regarding authors' reply to initial review points. 6) Please explain why only the quality of life mental health domain was used as the criterion variable as opposed to the total score or other subscales. REPLY: We have added an explanation at the end of the introduction. Reviewer response to 6: This explanation is circular. The authors are hypothesizing that reduced QOL impacts caregiver ability to maintain good mental health. However, they are not measuring these things separately. The difference between quality of life – mental health and mental health in general warrants explanation as this seems to be overlooked entirely in the manuscript. 7) In hypothesis b, it seems as though the authors are only going to examine the regression in the diabetes group and it is unclear how the comparison group fits into this hypothesis. REPLY: This mistake in the hypothesis has now been corrected; it now reads “… (b) lower levels of general self-efficacy, less relationship satisfaction, less satisfaction with social support, and fewer social support providers would be significant predictors of poorer maternal mental health in mothers based on the quality of life mental health domain.” 13) The explanation of results needs further detail. It is unclear why the mother group was entered into the model when the hypothesis only specifies “in mothers of children with diabetes.” REPLY: Again, this mistake in the hypothesis has now been corrected; it now reads “… (b) lower levels of general self-efficacy, less relationship satisfaction, less satisfaction with social support, and fewer social support providers would be significant predictors of poorer maternal mental health in mothers based on the quality of life mental health domain.” Reviewer response to 7 and 13: It is still unclear how the comparison group fits into the hypothesis. It is not clear if both groups are being combined to examine the impact of the stated variables on mothers as a whole regardless of whether their child has T1D. The manuscript continues to lack explanation as to why certain variables were chosen as control variables in the hierarchical regression. For example, if maternal education differed by group, then why was it not entered into the model as a control variable. Moreover, it is unclear why mother group (T1D vs. comparison) was entered as a control variable. This indicates that worse mental health was predicted by low social support, low relationship satisfaction, and low general self-efficacy regardless of mother group. However, in the discussion, the authors interpretation is unclear implies that these findings only apply to mothers of children with T1D.