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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: MURINE PLURIPOTENT STEM CELLS THAT ESCAPE DIFFERENTIATION INSIDE TERATOMAS MAINTAIN PLURIPOTENCY Review round: 1 Reviewer: 2

Basic reporting: The English language should be improved. There are many typographical errors, including: s everal, detials, fonud, tarotoma… that could be corrected using Word. Several sentences start with ‘And’; Thus, your manuscript need to be reviewed/edited by a native English speaking colleague. Another important point is that all acronyms should be clearly defined as they first appear in the text. Some are not defined. For example, what are MES-FT? (I guess that it means Mouse Embryonic Stem- from First Teratoma?). I suggest that you change the title for: “Pluripotent mouse stem cells persist and can be isolated from in vivo teratoma”. Background section: To my knowledge, there is no clear demonstration that PSCs derivatives obtained in vivo are superior to derivatives obtained in vitro. So I would remove or at least temper this statement and I would rather write that “Teratoma is a widely used assay in the field of stem cells and regenerative medicine but the cell composition of teratoma is still elusive”. Experimental design: 1. You have used a GFP construct that reports Oct4 expression. First, it seems important to confirm that the GFP signal does report for Oct4 endogenous expression. To tackle this issue, I suggest that you perform immunostaining on your PSC cultures using an anti-OCT4 antibody and a secondary antibody coupled to Cy3 to determine whether or not GFP and Cy3 signals merge. Please note that the correct gene name is Pou5f1, not Oct4, even if Oct4 is widely used and accepted. 2. In your teratoma assays (Figs. 3, 5 and 6), immunostainings were performed using an OCT4 antibody, which could detect endogenous OCT4 that is expressed in adult tissues (see Bhartiya, Stem cells int., 2013). To rule out that you detected Oct4 expressed from the host and to show that your Oct4 signal came from the PSCs that you injected, I suggest that you perform additional stainings on teratoma using an anti-GFP antibody or that you show GFP fluorescence. 3. I suggest that you perform immunostainings on teratoma using antibodies against markers of the three germ layers, as this is the accepted standard in the field. 4. To confirm the pluripotency of your PSC cultures, in particular those issued from teratoma dissection (FT and ST), I suggest that you perform alkaline phosphatase staining. 5. It is not clear whether or not the green signal shown for Oct4 in Figs. 1, 5 and 6 is the fluorescence emitted by GFP driven by Oct4 promoter or whether it comes from an immunostaining? Please explain and mention it in the text. 6. The composition of the different culture media should be detailed in the material and methods part. 7. Scale bars should be included in photographs. 8. Please use an additional housekeeping gene in your PCR experiments (figure 2), as there is a great variability in your Hprt expression (use for example Gapdh, as in your qPCR). Please also provide a legend. 9. I thank you for providing raw qPCR data (Table S1). Your qPCR were performed in triplicates and were correctly analysed. But these triplicates are three measures performed on the same cDNA and therefore it only takes into account the technical variability. To take into account biological variability, RT-qPCR should be performed on at least two more biological samples for each line (so to have n=3 biological samples for each line).You might use Genorm to select three or 4 housekeeping genes to accurately compare expression in your different samples by qPCR (see Vandesompele et al., Genome Biology, 2002, 'Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes'). 10. It’s not clear if MEFs were removed so that RNA were extracted from pure PSCs samples? Please explain. Validity of the findings: Overall, the findings are valid and the conclusion are well stated and linked to the original research question. However, I have found that your conclusion on PGC markers is overstated.Indeed, your PGC markers are weakly expressed in your qPCR assay (levels that of course depend on the efficiency of your primers); Therefore I suggest that you mention that there is an increase in the levels of PGC markers but that they remain weakly expressed. Moreover, it is important to reproduce these findings on more biological samples. Additional comments: The study by Pei et al. reports the isolation and characterization of pluripotent stem cells (PSCs) from in vivo teratoma. As previously reported for cancer stem cells, the authors found that PSCs can be injected in immunocompromised mice, harvested from teratomas, expanded in vitro, and reinjected several times while keeping some molecular and functional characteristics of PSCs. This is an interesting study that requires a major revision, in particular the English language should be improved and additional experiments must be performed to reach higher standard.

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

Title: MURINE PLURIPOTENT STEM CELLS THAT ESCAPE DIFFERENTIATION INSIDE TERATOMAS MAINTAIN PLURIPOTENCY Review round: 2 Reviewer: 1

Basic reporting: The authors have made a substantial progress in the presentation of their study. The English is now understandable and the content is mostly clear. The authors should consider further clarification of the sentences in lines 14, 49-50, and 109-110. The figures have been significantly improved. Some abbreviations are not defined at their first appearance (MEF, line 83; DMEM, line 85; FBS, line 85; LIF line 86). The primer sequences should be better provided in a separate table than within the text. Experimental design: The description of the methods has been improved but some aspects need further attention: - the secondary antibodies, which have been used, are not identified (lines 98 and 119) - the description of the qPCR and statistics is mixed up - the injection site of the stem cells in the NOD/SCID mice is not mentioned Validity of the findings: The quality of the data has been improved but some aspects need further attention: - In Fig. 2 a negative control such as MEFs is missing. - The quality of the histology of the teratomas is much better now. However, the differentiations shown for endoderm in Fig. 3a and ectoderm in Fig. 5c are not completely undisputable in my view. Therefore, I would encourage the authors to do immunohistochemistry for germ layer-specific markers to further support their findings, although this might not be an absolute requirement since the reader can now decide whether she/he wants to follow the interpretation of the authors or not. - It is essential to reconsider the statistics for Fig. 7: The Wilcoxon test is suitable to compare two paired samples. One might discuss whether samples are paired in these experiments. However, it is clear that always three samples need to be compared. Therefore a Kruskal-Wallis test might be appropriate. 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: MURINE PLURIPOTENT STEM CELLS THAT ESCAPE DIFFERENTIATION INSIDE TERATOMAS MAINTAIN PLURIPOTENCY Review round: 2 Reviewer: 2

Basic reporting: The English language is now correct. But there are still some mistakes that need to be corrected, including: Takahashiand Yamanaka (a space is missing after the name of the first author) Teratoma and not Tarotoma as in the panel A of figure 4 and of figure 6. Nanog and not Naong as written (last page of results) Mean and not MS in methods MES-FT and iPS-FT are not defined when they first appear in the text. SAS program (SAS stands for what?, please define this term) Experimental design: The anti-GFP antibody that was used is not indicated in the mat and methods I should have noticed this before but when I read again your manuscript I realize that a negative control line is missing for you PCR and qPCR data. So please add a negative control, such as fibroblasts, which do not express pluripotency/germ cell markers for PCR in figure 2 and qPCR in figure 7. . Validity of the findings: Overall it is good. However, I would temper and remove ‘highly’ from the sentence “OCT4-positive cells … highly express germ cell markers”. To affirm this, you should have compared the expression levels of these markers in your cells to those in primordial germ cells (positive control) and to a negative control line (such as fibroblasts). So, in your situation, you can conclude that they express germ cell markers, but not that they highly express them. Additional comments: Your manuscript is now much better. I will add a negative control for your PCR and qPCRs and pay attention to details in your 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: DOMESTIC CARNIVORE INTERACTIONS WITH WILDLIFE IN THE CAPE HORN BIOSPHERE RESERVE, CHILE: HUSBANDRY AND PERCEPTIONS OF IMPACT FROM A COMMUNITY PERSPECTIVE Review round: 1 Reviewer: 1

Basic reporting: The paper shows a clear language throughout. However, I’m not a native English speaker and can’t not assess if it can be improved. Context and background in intro are well raised and most references are adequate. Structure fits the journal standards. Figures and tables are adequate. Experimental design: Research and experiment are well designed and performed to ethical standards. Methods are described in detail and data allow replications. Statistical procedures are adequate and especially GLMs well applied, as well as model selection based on AICc ranking. Validity of the findings: My main concern about the paper is the application and weakness of the results. Sample size is relatively small and geographic ambit restricted. Although data seems robust and statistically significant results seem of limited application. I suggest dealing with this discussing what happens in the rest of the area (Biosphere Reserve). Is the problem with dogs similar in rest of the reserve? Do property and households follow the same pattern than in the island? The conclusion of the paper is “free-roaming dogs are perceived to interact in an anthropogenic context” and the recommendation is to develop environmental education actions. In most of the places where free-roaming dogs cause problems the solution comes from control actions or culling and always in the origin of the problem is the man hand. What kind of education actions does propose the authors? More details are needed here. Environmental education can be a proposal for the future but does not solve actual problems. Therefore, this kind of guidelines should be complementary of other actions. Which other solutions can be applied there? 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: DOMESTIC CARNIVORE INTERACTIONS WITH WILDLIFE IN THE CAPE HORN BIOSPHERE RESERVE, CHILE: HUSBANDRY AND PERCEPTIONS OF IMPACT FROM A COMMUNITY PERSPECTIVE Review round: 1 Reviewer: 2

Basic reporting: Introcution lack of relevant information that help to define the necesity of this research Experimental design: - Research questions are difficult to understand - Methods are not well described Validity of the findings: - Although data obtained from questionaires are used to obtain information regarding impact of domestic animals upon wildlife, this data must be used with caution and be very autocritic with the data obtained if no other confirmation method is used. If valid, data provided in this MS would fit better in a local journal. - It is not clear enough how model building was carried out biological and stasticaly. Additional comments: General comments Ms aimed to understand perceptions of impacts of free-roaming dogs and cats to wildlife. Although the objectives of this MS are of interest in the area, there are several aspects that difficult fulfilling the objectives proposed in this study. Overall, it’s not clear for this reviewer what authors wanted to test with the questionnaire and if data they could get with this could be used for what they wanted to predict, which difficult interpretation. In introduction it’s is not clear what was the aim of the study. 1) it’s about how free-ranging dogs and cats can get access to a conservation area? Can that be assessed through questionnaires??, The questionnaire can give perceptions more that access to a conservation area. Also, questions are applied to determine predictor of dog’s access to wildlife, which is not clear how questions asked can help to explain this. Methods are not clear and result section is not well written. The MS is about domestic carnivores and in some paragraph cats are included, models run, but in others only dogs are mentioned. Specific comments Introduction L68-69: Dogs and cats hybridization...this has proved with some species only. Clarify or remove sentence. L82-88: access to wildlife? Or allowing dogs roaming freely? Methods L92-99: Ethics should go at the end of methods L116-126: This paragraph would fit better at the introduction where could add more emphasis on the need for conducting this study. L135: CI of 95%? L145-146: how degree of confinement was defined? L166: what’s the aim of include rural and urban dogs as factors? Not clear the response variables on this analysis. Definitions: How free-ranging dogs were defined?? How feral dogs are defined and how authors are sure they are not owned dogs? Just by interviews? Statistic: aims is about dog and cats but the statistic is just regarding dogs, please clarify. Data on the questionnaire: it is not clear enough what authors wanted to test with the questionnaire. In methods it is not clear the method for model building and is confused with results section. M1: How a dog can be part of a free-roaming population? What is the response variable here? Dog restriction? M2, is regarding if a dog bring wild prey at home?? Model 3 not explained at all. Discussion There is data first given in the discussion and not informed in result section, such as distance of sighting dogs in wild area, among others. Conclusion How authors can be sure dogs and cats interact with wildlife?, only by interview?, there is any confirmatory method such as faecal analysis? Tables and Figures Figure 2. How sighting of dogs was defined??

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

Title: DOMESTIC CARNIVORE INTERACTIONS WITH WILDLIFE IN THE CAPE HORN BIOSPHERE RESERVE, CHILE: HUSBANDRY AND PERCEPTIONS OF IMPACT FROM A COMMUNITY PERSPECTIVE Review round: 2 Reviewer: 1

Basic reporting: commented on the previous version Experimental design: commented on the previous version Validity of the findings: commented on the previous version 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: DOMESTIC CARNIVORE INTERACTIONS WITH WILDLIFE IN THE CAPE HORN BIOSPHERE RESERVE, CHILE: HUSBANDRY AND PERCEPTIONS OF IMPACT FROM A COMMUNITY PERSPECTIVE Review round: 2 Reviewer: 2

Basic reporting: The background and rationale for this research are described clearly, and the importance of exploring possible interactions between dogs, cats, people and wildlife in the Cape Horn Biosphere Reserve (CHBR) is made exlicit. In general, the standard of English expression is good (I have noted a small number of places for improvement below), and figures and tabular materials are clear. I have just a couple of suggestions to make on the basic reporting (in giving line numbers below I refer to the track-changed Word version, not the pdf), including a couple of references that may be relevant: Line 54: The Cape Horn Biosphere Reserve is certainly a long way south, but it isn't strictly the southernmost protected area of the globe. That area would be Antarctica, a great deal of which is protected under international treaties. Lines 74 onward: A recent reference that quantifies the global impacts of domestic dogs on wildlife is: Doherty et al. (2017 The global impacts of domestic dogs on threatened vertebrates. Biol Conservation 210, 56-59). Conclusions: This section reads well and makes several well justified arguments about how the impacts of domestic dogs and cats might be reduced in the CHBR, based on the questionnaire survey results. A recent study by Hall et al. (2016, PLOS ONE 11(4) e151962) made similar recommendations, and more, based on questionnaire surveys carried out in six different countries, and should have some further recommendations of relevance here. Experimental design: The questionnaire surveys and analyses of results appear to have been done well, and the results are presented clearly and fully. Importantly, the sampling and analytical designs allow the study's four main questions to be addressed and, with provision of the questionnaire, could be replicated by later researchers. The third aim could be clarified slightly. This says: "... examine experiences and perceptions of the impact of free-roaming dogs and cats on wildlife and corroborate those with an analysis of prey remains of dog feces". Why could clarification not be attempted with cat feces too? I presume these were too hard to find, but a short explanation would be helpful. Validity of the findings: In general, I think the findings and conclusions are well made, with appropriate tempering of the initial results and conclusions that are drawn from them. Limitations are clearly identified and ways forward presented. This work will hopefully lead to actual quantification of the impacts of domestic dogs and cats on wildlife and other values in the CHBR, and to better mitigation of these impacts in future. Additional comments: I was not a reviewer of the original manuscript, but my reading of the current version (with the track-changes) and of the authors' rebuttal letter suggests that it has been very extensively reworked and revised. It reads well, and most of the concerns that appear to have been present in the original version have been effectively addressed here. I noted just a small number of corrections that could be made to improve clarity or to improve the English expression: Lines 101-102: "... a population of free-roaming dogs and cats ..." should be "... populations of free-roaming dogs and cats ...". Lines 210-211: "(ii) report experienced problems associated with dogs ...". This is not clear; perhaps something like "(ii) report problems reported by respondents that were associated with dogs ..." better describes what is meant. Or, even more simply, just: "(ii) report problems associated with dogs ...". Line 271: Were the 'street dogs' that were being fed on a regular basis owned or feral dogs? Line 309: "Predominant experienced dog problems in town were conflicts ..." would read better as: "Predominant dog problems experienced in town were conflicts ...". Line 470: Is 'provenience' meant to be 'provenance' or something similar? Table 1: Please define what was meant by NAs. Fig. 1: Yendegaia National Park is mentioned in the figure caption, but is not shown on the map. Can its location be added to the map?

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

Title: CHARACTERIZATION AND TOXICITY OF CITRAL INCORPORATED WITH NANOSTRUCTURED LIPID CARRIER Review round: 1 Reviewer: 1

Basic reporting: The article is well written, clear, structured and it is within the field of the journal. The article describes the production of lipid nanoparticles of citral with a good particle size, PI, ZP, EE and biocompatibility. The article is well illustrated. This article can be accepted for publication after a major revision. Experimental design: The design of the production and caracterization of the NLC prepared is well done. The methods are well described. The article is well illustrated. Validity of the findings: The article is well written and it is within the field of the journal. The results and conclusions are acceptable. Additional comments: The article is well written and it is within the field of the journal. The article describes the production of lipid nanoparticles of citral with a particle size, PI, ZP and good EE. The article is well illustrated. This article can be accepted for publication after a major revision. Minor Changes: • Add a list of abbreviations in the end of the article, after the conclusions subchapter (in the abstract have the abbreviation NO?! I know what is but some authors no…. a list of abbreviations solve this problem); • In the introduction, the authors should highlight the advantages of lipid nanoparticles compared to other colloidal carriers. Explain why you chose the NLC and not SLN! Describe the advantages of this kind of second generation of lipid nanoparticles. Authors can see these in the article: (cite the article) Almeida H, Amaral MH, Lobão P, Silva AC, Sousa Lobo JM. Applications of lipid and polymeric nanoparticles in ophthalmic pharmaceutical formulations: present and futures considerations. J. Pharm Pharm Sci. 2014; 17(3)278-293. • Why you chose these solid and liquid lipids to prepare the NLC? Are you sure that are the best choice? Justify! • In the preparation of NLC detail the rpm of the stirring and time. • In the determination of EE% describe the wave-length (nm) used in the UV-Vis spectrophotometer. The same in the in vitro dug release study. • In the in vitro splenocyte viability (MTT) why you use these rage of concentrations of the formulation? Support with bibliography. • ZP values prove that the optimized formulation is not stable over time. Why you did n´t optimized the formulation or add something to improve this parameter? How you justified this? Improve this because your explanation is not enough… Major Changes • In order to improve the quality of the article, the subchapter results and discussion must be united in only one subchapter. It is better for the readers to understand your research.

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

Title: CHARACTERIZATION AND TOXICITY OF CITRAL INCORPORATED WITH NANOSTRUCTURED LIPID CARRIER Review round: 1 Reviewer: 2

Basic reporting: Manuscript title: Characterization and Toxicity of Citral Incorporated With Nanostructured Lipid Carrier Authors: Nordin et al The manuscript describes the physicochemical characterization of the Citral loaded NLC. Works appear to be systematically carried out and trustworthy. The manuscript is worthy to be published. However, some corrections are to be made before a final decision could be made. The level of English in its present form is not acceptable. Besides there are some typos and merged words. Authors should have made a general correlation between the different experimental data set. Some specific comments are appended below: 1. In the introduction section the authors should make some statement as how the present manuscript is going to add further information in the field of NLC research. What is the novelty of the present work? 2. Abstract: Polydispersity index does not have any unit. 3. Authors should justify such a higher extent of entrapment efficiency (99%). Is it trustworthy? 4. The interpretation on the TEM images (Figure 1) is completely wrong. NLCs are elsewhere, which the authors have failed to mark. 5. Authors have not shown the control experiment. This is absolutely essential to prove that the citral release from the NLC is sustained. Also authors should propose as which model is best fitted to the release kinetics. 6. I failed to follow the meaning of 105% cell viability (Figure 3), especially for the blank NLC. What does it signify? 7. Line 399: statement is wrong. Authors should rather justify as how the systems with lower magnitude of zeta potential is stable. Author should specifically mention that the used Tween provided steric stabilization to the NLC formulations. 8. Figure 3: Authors should comment on the optimum concentration of the formulation. 9. The conclusion section should be rewritten. Some future perspectives are worth mentioning. Experimental design: Looks fine Validity of the findings: Alright 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: CHARACTERIZATION AND TOXICITY OF CITRAL INCORPORATED WITH NANOSTRUCTURED LIPID CARRIER Review round: 2 Reviewer: 1

Basic reporting: I accept the changes done by the authors! The manuscript must be published! Thanks Experimental design: I accept the changes done by the authors! The manuscript must be published! Thanks Validity of the findings: I accept the changes done by the authors! The manuscript must be published! Thanks Additional comments: I accept the changes done by the authors! The manuscript must be published! Thanks

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

Title: TRANSLATING GENOMICS INTO PRACTICE FOR REAL-TIME SURVEILLANCE AND RESPONSE TO CARBAPENEMASE-PRODUCING ENTEROBACTERIACEAE: EVIDENCE FROM A COMPLEX MULTI-INSTITUTIONAL KPC OUTBREAK Review round: 1 Reviewer: 1

Basic reporting: No comment Experimental design: No comment. Validity of the findings: No comment. Additional comments: This article describes the enhanced effect of combining WGS information with traditional field epidemiology to inform management of an established KPC producing Enterobacteriaceae outbreak.The text is well written, clear and unambiguous with sufficient relevant references and background provided. General comments The value of the WGS in the context of patient benefit and healthcare economics is difficult to quantify and the manuscript would benefit from estimates of these in this study, i.e. the authors state that further transmission has slowed because of increased intervention but is there an estimate of the healthcare saving and patient benefit because of this? This is a subjective area but if screening had been introduced at an earlier stage would there be an estimated saving in terms of hospital bed days for example? The authors discuss the revision of guidelines for outbreak management since the study was performed, a discussion of further improvements/recommendations and how they would be implemented would be of value. Throughout the manuscript the term real-time is used to describe how WGS informed transmission dynamics and subsequent infection control management. Near real-time is more accurate as it still takes at least a week to take appropriate samples, culture, sequence and analyse the information. The ability to determine whom infected whom in these investigations is an interesting point for discussion and also has potentially difficult ethical/legal considerations that could also be discussed.

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

Title: TRANSLATING GENOMICS INTO PRACTICE FOR REAL-TIME SURVEILLANCE AND RESPONSE TO CARBAPENEMASE-PRODUCING ENTEROBACTERIACEAE: EVIDENCE FROM A COMPLEX MULTI-INSTITUTIONAL KPC OUTBREAK Review round: 1 Reviewer: 2

Basic reporting: The article is clearly written and easy to follow, with an extensive bibliography and appropriate context provided via the Introduction. DNA Data Checks: The raw data and assemblies, along with a useful assortment of sample-level metadata, have been deposited at NCBI and I was able to successfully access them. The accession number is included in the manuscript. Scripts used in the analyses have also been made available via two GitHub repos. Human Participant Checks: The investigation was undertaken by a public health agency as part of an outbreak response. No identifying information is included in the article or in the publicly available data. Experimental design: The article is well-suited to publication in PeerJ, offering an extremely comprehensive investigation of a pathogen of international concern. The objective of the study is clearly described, and the work presents an exemplary model for uniting genomic and epidemiological investigations of hospital-associated infections. Each of the many analyses was done to high technical standards, and described in enough detail for others to be able to replicate the work. Validity of the findings: The conclusions around the origins and the transmission of the outbreak isolates are well-supported by the data, and clearly linked to downstream infection control activities. Limitations of the study are clearly identified. Additional comments: This is wonderfully detailed and fascinating investigation of CPEs circulating in Victoria, Australia over 2012-2015, which links genomic and epidemiological data in a way that very few papers in this domain have. It was a very enjoyable read, and my suggestions for improvement are all very minor and addressing them is left to the author's discretion. Introduction 1. The abstract mentions that the genomic epidemiology investigation proved more fruitful than genomics or traditional epidemiology alone. Perhaps towards the end of the Introduction, the authors could describe what the state of the epidemiological understanding was at the time the genomic investigation was launched - were there obvious gaps that the genomic analysis was aimed at closing? Methods 1. The authors are to be commended for the incredible breadth and depth of their analyses! That being said, there is definitely a lot for the reader to keep track of here, so perhaps a figure outlining the study workflow could be provided to orient the reader. 2. Along those lines, the Bioinformatics subsection contains many analyses - these could be further subdivided into smaller sections to facilitate reading. Results 1. Line 310: Would be helpful to briefly differentiate KPC-2 and KPC-3 earlier in the manuscript for the reader unfamiliar with these designations - perhaps a brief comment in the Introduction, including their incidence in other settings like the US. 2. Line 311: "from one patient" instead of "from the one patient" 3. Figure 1: The annual peaks in Sept/Oct/Nov are interesting and might merit some comment in the Conclusions section. 4. Line 313: delete the "of" 5. Line 315: For the readers who skip the Intro and Methods and head straight to result, it would be helpful to explain what the "Initial Analysis" was - from my reading of the methods, it was a retrospective 2014 look at the isolates from the 29 patients identified to date, with further work happening prospectively. 6. Figure 2: Depending on the actual geography, it might be interesting to colour the facility nodes according to their distance from each other, maybe with the most central Melbourne facility as a saturated colour with the other facilities less saturated as one moves out. This would, of course, only be really interesting if there was some spatial structure to the phylogeny, but it is suggested in the Combined Analysis section. It would be interesting to note Facility F, though, which is mentioned later on in the text. 7. Figure 4: No suggestions here, just wanted to compliment you on this super figure! 8. Line 392: A lot of the TransPhylo transmission events arise from unsampled individuals (white nodes), whereas I imagine the screening in place knowing that KPC was circulating meant that most of the population should have been fully sampled. I think (as a TransPhylo author ;) that this is a bit of a shortcoming of this current implementation – I think it sometimes overestimates the proportion of unsampled cases. This might be worth commenting on, as would noting whether any of the chains it did identify amongst the sequenced isolates (I spot an interesting one involving five B1-harbouring individuals) were reflected in the epidemiology. 9. Line 430: I wonder if a figure summarizing the plasmid content of each isolate might be helpful here, to give a quick visual of which isolates across which species harboured identical or near-identical plasmids. 10. Line 475: What sort of accessory genome changes? 11. Figure 9: I see environmental sampling isolates in this tree that appear to be closely linked to one of the patients for whom multiple isolates were sequenced (bright pink patient) and another singly-sequenced patient. This is interesting, but I don’t see a mention of it in the main text. Supplement: 1. Figure S6: The black (overall) regression line seems to be missing, though the R^2 value is there.

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

Title: DEVELOPMENTAL EFFECTS OF ENVIRONMENTAL LIGHT ON MALE NUPTIAL COLORATION IN LAKE VICTORIA CICHLID FISH Review round: 1 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: The cichlid species pair Pundamilia pundamilia and P. nyererei represents a well-studied model system to identify mechanisms of ecological speciation. In the present manuscript, Wright et al. investigate environmental light-induced plasticity in nuptial colouration in this cichlid sister species pair from Lake Victoria. To this end, both species (and their hybrids) were reared under light conditions, which mimic their natural habitats, and male nuptial colouration was quantified based on photographs of fish. The study finds very limited plastic responses in male colouration, and importantly not in the species-specific nuptial colours (blue and red), which have been shown to be subject to divergent selection by female choice. The paper is nicely written, the methods and statistical analyses are sound and the results are generally interpreted adequately. However, a weak point of the study involves the relatively low sample sizes (9 to 10 individuals per species/hybrid and treatment in Experiment 1, and 3 to 4 individuals per species/hybrid and treatment in Experiment 2). This should be addressed in the discussion when interpreting the results. Please find more specific comments below: - Presentation of results, lines 288-307. Are the results presented here based on individuals from both shallow and deep ambient light conditions? This is not made clear, and given the aim of the study (lines 130-139), it would make more sense to present the results for each treatment separately (also in Fig. 1 & 2). - Methods, lines 148-151. Please explain why you used both F1 and F2 crosses. - Lines 158-160. Differences in body size and species identity between neighbours (and also the number of neighbours) might influence the social rank/dominance among males and thus the expression of nuptial colouration. This was (partly) accounted for in the linear mixed models (line 252), however, whether any of these random effects were significant or not is not mentioned in the results. - Lines 202-203. Quantifying nuptial colouration in cichlids is notoriously difficult due to the fact that fish change colouration quickly when stressed - which might be the case when transferring the fish into a glass cuvette. How did you standardize photographs, i.e. make sure that you didn’t take pictures of stressed fish with faded colours? - Please provide legends for supplementary figures and tables.

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

Title: FORMONONETIN INHIBITS LIPOPOLYSACCHARIDE-INDUCED RELEASE OF HIGH MOBILITY GROUP BOX 1 BY UPREGULATING SIRT1 IN A PPARΔ-DEPENDENT MANNER Review round: 1 Reviewer: 1

Basic reporting: The article is clear and english is correct. Results of figure 2 should be inserted in figure 1 as panels C and D. Similarly, data of figure 4 should be inserted in figure 3 as panels C to F. Data presented in the supplemental figures should be inserted in the main figures. Some references should be added in particular on the effect of resveratrol on HMGB1 release (e.g. Xu et al. Shock 2014; Dong et al. Free Radic Biol. Med 2015). Experimental design: The objective of the study is to assess if formononetin modulates HMGB1 secretion in response to LPS. Overall, the methods are well described, the experiments are well designed with appropriate controls and technical replicates. The authors should provide the quantification of the western-blots in figure 2A-B. In figure 2B, it seems that the total amount of HMGB1 is increased with LPS or formononetin treatment. The authors should analyze HMGB1 mRNA level upon formononetin treatment. RAW264.7 cell line is not the most suitable model to study HMGB1 release and the effect of formononetin on HMGB1 secretion should be confirmed in human primary macrophages. Validity of the findings: The novelty of the study is rather limited as a large number of studies already demonstrated that many herbal compounds inhibit HMGB1 release by preventing its acetylation. It might be interesting to focus on the advantage of using formononetin compared to other similar compounds. Hence, it might be useful to compare the effect of formononetin on HMGB1 release with other similar compounds. In figure 5A, treatment with formononetin alone significantly increased the level of SIRT1 and in figure 1B it seems that formononetin alone decreases the level of HMGB1 in the cytoplasm. It might be interesting to perform a pre-treatment with formononetin and then wash the cells and activate them with LPS in order to evaluate if the effect of formononetin treatment persists in time. From authors conclusions, formononetin and resveratrol appear to modulate HMGB1 release via SIRT1 expression. Hence, why did they observe a synergic effect in figure 5D? If these two compounds modulate HMGB1 release via the same mechanism, no synergic effect should be observed. The authors should also analyze the level of SIRT1 in this experiment. 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: FORMONONETIN INHIBITS LIPOPOLYSACCHARIDE-INDUCED RELEASE OF HIGH MOBILITY GROUP BOX 1 BY UPREGULATING SIRT1 IN A PPARΔ-DEPENDENT MANNER Review round: 1 Reviewer: 2

Basic reporting: The paper deals with the role of formononetin as anti-inflammatory molecule. Basically, formononetin acts by inhibiting HMGB1 release via Sirt1 up-regulation. English form is correct and appropriate. References are accurate. Line 165: Please fix “. At which time” Experimental design: The study is well designed. The results are well presented and understandable. Methods are well described with the exception of statistical analysis, which should be explained in a wider way. Validity of the findings: Authors provide a functional explanation for HMGB1 release inhibition by formononetin. All experiments are well showed, results are consistent and statistically sounding. Conclusions are well written and results support the conclusions. 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: FORMONONETIN INHIBITS LIPOPOLYSACCHARIDE-INDUCED RELEASE OF HIGH MOBILITY GROUP BOX 1 BY UPREGULATING SIRT1 IN A PPARΔ-DEPENDENT MANNER Review round: 2 Reviewer: 1

Basic reporting: The results of the two reference figures should be inserted in the manuscript (as figure 1C for reference figure 1 and as figure 2D for reference figure 2). In addition, it would be useful to highlight in the text that the maximal inhibitory effect of formononetin is observed with a pre-treatment of 24 hours which corresponds to the time of maximal induction of SIRT1 expression upon formononetin treatment (Fig.2B and C). Experimental design: no comment Validity of the findings: no comment Additional comments: The authors replied to all my comments. The results of the two reference figures are convincing and should be inserted in the manuscript (as figure 1C for reference figure 1 and as figure 2D for reference figure 2). In addition, it would be useful to highlight in the text that the maximal inhibitory effect of formononetin is observed with a pre-treatment of 24 hours which corresponds to the time of maximal induction of SIRT1 expression upon formononetin treatment (Fig.2B and 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: FORMONONETIN INHIBITS LIPOPOLYSACCHARIDE-INDUCED RELEASE OF HIGH MOBILITY GROUP BOX 1 BY UPREGULATING SIRT1 IN A PPARΔ-DEPENDENT MANNER Review round: 2 Reviewer: 2

Basic reporting: The authors have reviewed the paper and fixed where requested. Indeed, English form is now correct and references accurate. Experimental design: Statistical analysis section has been enriched and it is now well understandable. Validity of the findings: The paper is clear and results are well written. Experimental section is accurate and well designed. Conclusions are consistent. Additional comments: No additional comments

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

Title: A HISTORICAL LEGACY OF ANTIBIOTIC UTILIZATION ON BACTERIAL SEED BANKS IN SEDIMENTS Review round: 1 Reviewer: 1

Basic reporting: • The paper is overall well written in a clear language. There are a few minor errors (see general comments below). • The introduction relies heavily on a relatively small number of references (particularly Taylor et al 2011), and widening the perspective slightly would not hurt. For example, all the work of Fernando Baquero and José Martinez on environmental antibiotic resistance is entirely left without reference. • The figures are relevant, but Table 2 seems inappropriate and contains much information that does not concern this manuscript. In Figure 2B, it is however unclear how the years were imposed on the PCoA. • I could not find the raw data for the chemical analysis of iron and manganese associated with the manuscript. Experimental design: • The study is original and within the scope of PeerJ. • The manuscript aims to investigate if dormant microbial seedbanks in lake sediments can be used to track the abundance of antibiotic resistance genes in the environment over time. This is an important question for retrospective monitoring and such analysis would fill an important knowledge gap regarding baseline for antibiotic resistance prevalence in the environment. • Although the scope is small (onty two genes – sul1 and tetW – are investigated) this is an important proof-of-concept study, and it conforms to methodological standards. • However, the qPCR for the non-seedbank community would need a better description. • Furthermore, it must be described how the chemical analysis for Mn and Fe in the sediments was carried out. Now, this analysis is just mentioned in passing on lines 241-242 in the manuscript. Validity of the findings: • The data underlying the results seem to be robust and statistically sound. • However, I have a major problem with one of the conclusions by the authors. The idea that tet(W) abundance would be a result of antibiotic pollution (or pollution by human bacteria carrying tet(W) genes) seem quite farfetched to me, given that the lake at the same time (according the authors) underwent eutrophication. Since eutrophication would lead to more anaerobic conditions, and the bacteria co-enriched with tet(W) are obligate anaerobes it seems that the explanation to the tet(W) increase would be increased abundances of these bacteria – not a selection for tet(W) by antibiotic residues. Indeed, some of these bacteria are known to harbour the tet(W) gene. If the authors want to make claims along the lines of “tetracycline exposure caused selection for tet(W) in the sediment communities”, they need to support this hypothesis with chemical measurements of tetracycline in the sediments. • The paragraph on lines 278-301 is entirely speculative, but is presented as if it was well supported by the results. This needs to be modified. Additional comments: • Line 19-20. It is unclear from the abstract why legacy of antimicrobial agent disposal is important. • Line 41: “mitigates” seems to be me to be a weird choice of word in this context. • Line 45-46: As far as I know the idea that ARGs are a fundamental component of microbial ecosystems is a hypothesis (albeit plausible), and should be presented as such rather than as a fact. • Line 50-51: I don’t understand what the authors mean with “further hone naturally occurring antibiotic resistance systems”. Maybe it is the use of the word “hone” that confuses me. • The entire first paragraph of the Introduction mixes the clinical/human society and environmental components of antibiotic resistance development in a quite confusing way, and particularly the lines 51-55 about risks really suffer from this problem. I would recommend the authors to read a recent review in FEMS microbiology (https://doi.org/10.1093/femsre/fux053), which may help in structuring the Introduction better. • Line 56-58: I think that studies by Gerry Wrights lab make an even more convincing case for that ARGs were present before the antibiotic era, particularly these two papers: https://doi.org/10.1038/nature10388 & https://doi.org/10.1371/journal.pone.0034953 • Line 59: “in” -> “over” • Line 71: “made” -> “make” • Line 71: I think it is overstating the importance of sediments by attributing it “a highly likely source of resistance determinants” just because it is diverse. I would recommend that the language of this sentence is toned down. • Line 79: “or” -> “and” • Line 153: Which version of the SILVA database? This matters for reproducibility. • Line 163: “OTUs relative abundance and ARGs frequency” -> “OTU relative abundances and ARG frequencies” • Line 188: Where in the methods section is the total community DNA extraction protocol described? • Line 198: “ARG” -> “ARGs” • Line 239-240: I disagree with the authors here. To me, it looks like the observed changes in tet(W) could be entirely due to the effect of eutrophication (i.e. the increase of anaerobic bacteria). I would rather say that the results of the total community composition imply that eutrophication would be the cause of the tet(W) changes (although it is of course not the only possible explanation) • Line 241-242: Where did the Fe and Mn data come from? How was those levels determined? • Line 255: Insert “make it” after “Lake Geneva”. • Line 257: “ARG” -> “ARGs” • Line 278-280: This sentence is pure speculation, and it should be stressed as such. Actually, I would refrain from making this connection at all, given the vast number of possible confounding factors. • Line 285-301: This entire paragraph is just pure speculation. If the authors want to make a claim that tetracycline has selected for tet(W), they should also quantify antibiotic residues in the sediment. The correlation between medical use and ARGs in sediments in a single lake is not a very convincing argument. • Line 312-313: I disagree with that HGT would be a major pathway for dissemination of ARGs in aquatic environments. There are many environments that are more likely to be hotbeds for resistance transfer. However, waterways are great for dissemination of resistant bacteria, but that has nothing to do with HGT. I recommend the authors to read the review by Baquero et al (https://doi.org/10.1016/j.copbio.2008.05.006) or the aforementioned FEMS Microbiology review (https://doi.org/10.1093/femsre/fux053) to straighten out these concepts, as they again seem to be intermixed here by the authors. • Line 316-319: The relation to penicillin and cotrimoxazole here is an interesting speculation, but how come that sul1 has not been enriched earlier when sulfonamides where widely used in healthcare? This would need an explanation as well. • Line 321-323: How is this last sentence related to sulfonamide resistance? • Line 329: “can” -> “may” • Line 336-337: I would rather spell it out as that the taxonomy of the seed bank community influences the ARG content. • Line 338: Contaminated in what sense? Are these genes really contaminations if they allow better resilience to the bacterial community? • Table 2 contains much information that does not concern this manuscript and could probably be left out. • Figure 2B: How were the years imposed on the PCoA?

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

Title: A HISTORICAL LEGACY OF ANTIBIOTIC UTILIZATION ON BACTERIAL SEED BANKS IN SEDIMENTS Review round: 1 Reviewer: 2

Basic reporting: The manuscript is nicely written and clear. It was very enjoyable to read through the study. Experimental design: The research presented in the manuscript is original and interesting. Also, consistent with the Aims and Scope of PeerJ journal. The research results are interesting and have a good contribution to study of antibiotic resistance in aquatic environment throughout the history of anthropogenic used of antibiotics. Although the methods were described thoroughly, there are some information that would be better to be added in the material and methods, as follow: 1. Correlation of the depth of sediment core with the years of study (Line 98-102), also in Figure 1 and Supplementary Figure 1 (This information was only described in supplementary metadata) 2. Efficiency of DNA extraction from each sample (L 122) 3. Limit of quantification from the qPCR measurement of each gene (L 139) 4. The explanation to use the amount of DNA concentration as normalisation instead of using the amount of 16S rRNA gene Validity of the findings: The finding is well reported and giving new contribution on providing insightful information to prevent the spread of ARGs in the environment. If there is one question, it is in the decision to study tetW gene as the representative of more than 20 types of tetracycline resistance genes that are known. Several studies shown that tetM gene is the most abundant of tetracycline resistance gene found in sediment environments. 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: EFFECT OF DARK SWEET CHERRY POWDER CONSUMPTION ON THE GUT MICROBIOTA, SHORT-CHAIN FATTY ACIDS, AND BIOMARKERS OF GUT HEALTH IN OBESE DB/DB MICE Review round: 1 Reviewer: 1

Basic reporting: Nothing to report here Experimental design: Were the lean mice db/db as well, or another strain? It may be good to fully describe the lines used (db/db is not the complete descriptor)... L186 colon mucosal cells normally implies host cells, which would not be Bacteria! Maybe you mean mucosal samples? Validity of the findings: Figure 1: I am not convinced that showing results at the order level is acceptable. It is way more common to present phylum and genus (family) level. Figure 2 has no place has a main figure, this is not "valuable" data Figure 3: The ANOSIM should give you a p-value which is needed to conclude on the clustering! Figure 4: It would be so much easier if you had the taxon name on each plot! H, L, J, O appear to show very similar values, which raises questions about the statistics... Table 2: I personally have doubts about the validity of Picrust, and seems like the differences you report are all extremely slight. Without a way to test if such differences are not due to stochastic variations in the sampling, I would not report them. Table 3: These results seem completely flawed. There is no way butyrate and propionate are not produced by gut microbes! And the 100X more values in the obese supplemented mice don't make any sense 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: EFFECT OF DARK SWEET CHERRY POWDER CONSUMPTION ON THE GUT MICROBIOTA, SHORT-CHAIN FATTY ACIDS, AND BIOMARKERS OF GUT HEALTH IN OBESE DB/DB MICE Review round: 1 Reviewer: 2

Basic reporting: Discussion could be implemented (see general comments to the author). Experimental design: Seems quite adapted to the question raised. Studies seem competently performed. Validity of the findings: The findings are interesting and seem valid and =adequately interpreted. Additional comments: This is an interesting study, which questions the "power" of the gut microbiota to change metabolism of the host. Basically, from a healthy dieting (cherry powder, fiber-enriched), inducing a healthy microbiota composition (enriched in "beneficial" bacteria), and producing "beneficial" metabolites deriving from fermentation of soluble fibers (short-chain fatty acids), no substantial change in several important gut health parameters of the host was observed in a mouse model of diabetes (db/db mice). This is an important set of results, suggesting that the power of microbiota to significantly alter the host metabolism is limited by the genetic bakground of the host. This study resonates in me with another study in relation with the role of fiber-derived SCFAs to improve host glucose control by activating intestinal gluconeogenesis, the benefits being absent in intestinal gluconeogenesis-deficient mice despite the presence of SCFAs and the modification of microbiota (De Vadder et al, Cell, 2014). This suggests that host genetics may dominate the microbiota composition as for regards host metabolism. The paper would be greatly improved by inclusion of a paragraph od discussion addressing this point

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

Title: SEX DIFFERENCES IN THE USE OF SOCIAL INFORMATION EMERGE UNDER CONDITIONS OF RISK Review round: 1 Reviewer: 1

Basic reporting: Generally the basic reporting is good. A few issues below: - You use sex and gender interchangeable. Sex is biological, gender is cultural. See for example line 77 compared to line 121 and line 345. If you let people self-identify, I suspect you should use gender throughout rather than sex. -Along this same line, you note that you had four options for participants to select for gender including other and prefer not to say. Did anyone select these options? If so, were they excluded from the analyses? Please specify. - Line 116 Typo: of not f -Line 143: You haven't used the term league table before. Perhaps reword to standings list or something like that. -Line 204: I believe you mean Figure 3 in this line. -Line 306 Typo, a period, at the beginning of the line. Experimental design: The methods themselves were fine. However, I have questions about how they relate to the research question. See comments in the validity section. Validity of the findings: I am unclear why you decided to make this a competitive task. It seems like risk preferences may well vary when in a competitive situation versus other situations. I would like to see some information about how being a competitive task or not influences risk preference. In other words, do competitive situations change risk tolerance? My biggest issue is that you are confounding risk preference and score maximization. This is an issue that many studies examining risk preferences have. For example, in the asocial risky condition, since the asocial option is more risky, the social option will lead to higher rewards, since it has a higher expected value. Thus, women may be maximizing their score rather than interacting with risk, per se. In the other conditions, their performance is also consistent with reward maximization. Therefore, an alternate interpretation of your findings is that women are more sensitive to rewards (not punishments as you say in line 349) or that they are more willing to switch strategies when it is optimal to do so. Thus, I find your explanations for this behavior (punishment and lack of confidence) quite unsatisfactory. What I think is perhaps the most interesting is that men continue to avoid social information even when doing so leads to higher scores. So, men are not maximizing their rewards in the asocial risky condition. This suggests to me that men my be more averse to social information than women. Because of the conflation or risk and score maximization, I think what you actually tested is willingness to use social information. You show in your control condition that in labs people avoid social information (this is also supported by the literature). Then, in your social risky condition you show that people continue to avoid social information even when there is a potential, but not guaranteed, benefit. Finally, you show in the asocial risk condition that women, but less so men, are willing to use social information when it is beneficial. I am not sure how you can disentangle risk preferences and score maximization with your existing data. Perhaps there is another control condition you could add, but I am having trouble imagining what that would be. Alternatively, you could restructure the paper to focus on preferences for asocial/social information across various payout schedules. 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: SEX DIFFERENCES IN THE USE OF SOCIAL INFORMATION EMERGE UNDER CONDITIONS OF RISK Review round: 1 Reviewer: 2

Basic reporting: The article is well-written and nicely presented. I found it very clear and easy to follow. The literature review places the work suitably in context. The raw data file was easy to access and understand. I found that the column headers did not line up with the data when I opened the file in Excel (the column headers were misaligned by one cell, relative to where they should have been), but this may have been a quirk of using Excel to open a txt file which might otherwise have displayed correctly in some alternative package. However, perhaps the authors can take a quick look at this and check that everything displays correctly in the most commonly used software packages that they would expect readers to be using. The figures were reasonably helpful. Having looked at the response to reviewers' comments for the previous submission, I think the decision to include the "real" means in one of the figures was prudent, and actually these do potentially cast a slightly different light on the results, as I will elaborate in Section 3. Experimental design: The experimental design is essentially very simple. The key manipulation was that participants were told that one option available to them was risky (operationalised as potentially reducing score, but also potentially significantly increasing it), and the other safe (operationalised as guaranteed not to reduce to score, but with only marginal increases possible). These possibilities were counterbalanced across the option types ("social" operationalised as the ostensive attempt of another participant, and "asocial" operationalised as an alternative source of solution elements not presented as having originated from another's attempt). In a control condition both sources were presented as being safe. The measure of interest is simply the choice made by the participant (which could be variously: social safe over asocial safe; social risky over asocial safe; social safe over asocial risky). In spite its simplicity (and I regard simplicity as compliment rather than criticism!), it is clear from the manuscript how the manipulation makes a novel contribution, in looking at how gender differences in risk sensitivity might interact with increased reliance on social sources of information under conditions of risk. The design is appropriate to tackle this question. From the information provided, the study appears to have been run with due attention to methodological rigour, and the methods are described in sufficient detail. Validity of the findings: The authors' conclusion (that female participants are particularly likely to rely on social sources of information when asocial sources are known to be more risky) appears to be upheld by the data. My only concern here is that I suspect that this might present a somewhat incomplete picture of the results. There are a couple of things that I think the authors might want to consider in order to present a more fleshed out interpretation. I do want to emphasise however that this would entail in one case a speculative interpretation, and in another case, a post-hoc analysis of results. So if the authors take either of these suggestions on board then of course it is important that they make these caveats clear. In addition, given the need for the caveats, I think it is fine if the authors prefer not to stray into this territory at all. However, for me, it would make for a slightly more thought-provoking paper, and one that might be more likely to generate further literature investigating these possibilities in a more a priori fashion. The first suggestion concerns my point about the "real" means in the Figure. I'm really glad the authors included these. Perhaps it makes me very old-school but I much prefer to see real data over the outputs of models. And in this instance I think that it is particularly helpful as these seem to suggest that something quite interesting might be going on which is not captured by the models. It looks like in the control condition (safe, safe), female participants are actually LESS likely than males to opt for the social source of information. This seems quite interesting, and not what one would expect based on general assumptions and theories about gender and social learning? And then in the social risky condition, women actually appear to INCREASE their tendency to select the social source. This is really interesting too, and certainly it seems consistent with the authors' interpretation that female participants are not simply more risk-avoidant. The pattern of results suggests that actually female participants may respond to ANY cues to potential risk by increasing reliance on information from social sources (even when it is that very social source that has been identified as risky). Of course, I may be completely wrong, and this might not be upheld in the data at all. But it looks intriguing, and therefore potentially worth a bit of post-hoc analysis (explicitly flagged as such, of course) to see whether there might be anything in this interpretation. And regarding the interpretation, the authors state quite clearly (and correctly, based on their data), that the female participants are not just more risk-averse. And indeed, in line with this, the effect appears not to be explained by risk aversion anyway, based on the lack of relationship with the personality measures. However, the authors do little to propose an alternative interpretation, and I think this would be merited. What exactly IS driving the effect, if it is not simply risk sensitivity? Personally I would like to see the authors propose an alternative interpretation which might be testable in future research. My hunch would be that female participants are particularly sensitive to something that might be labelled as social shame, or similar. If one turns out to have made a choice that sets one apart from the crowd in a "good" way, then there's no shame. But if one underperforms relative to the crowd then perhaps the sense of having foolishly deviated from established behaviour is possibly more aversive to women than to men. This might even explain why women appear to be slightly more inclined to use the social information even when it has been identified as risky, relative to the control condition where it is identified as safe. It might be the case that the mere possibility of one's score going down at all leads women to rely more heavily on social information (since it is not so much the possibility of having made an adverse decision that is disturbing, as the possibility of being the only one to have done so - and copying another's design guarantees that this will not be the case). Anyway, as previously indicated, these are merely suggestions. I leave it up to the authors whether they take them on board (here or elsewhere). I have no problem with the very neutral interpretation as currently presented. 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: SEX DIFFERENCES IN THE USE OF SOCIAL INFORMATION EMERGE UNDER CONDITIONS OF RISK Review round: 1 Reviewer: 3

Basic reporting: 1. The study presents an experiment exploring the relationships between risk preference and preferences over social or asocial information sources. As such present tense is appropriate. The use of past tense is unnecessary. For example line 108 could be rewritten as: Eighty eight participants are recruited, … give consent… are reimbursed…” etc. I checked several experimental papers on my desk and all follow this convention. Although this is a relatively minor point. 2. Given that the payments are small and deceptive I suspect that wording is very important. I would have found a box listing the choices in full useful. Something like the following but with the actual wording given to subjects. Conditions Asocial information source “consisted of viewing up to 10 previously unseen items in the scrapheap, of which up to three items could be kept for use in the next building Phase.” Social information source: “viewing 3 completed space ships ostensibly built by ‘other participants’ along with the associated scores. These spaceship designs had actually been generated by the experimenter prior to the study using randomly selected tiles, and three out of 12 completed spaceships were presented at random as social sources. The scores for these space ships were also randomly assigned. Participants could choose up to three items from one of the three ships, and these items were automatically added to the participant’s spaceship template in the next phase and could not be removed.” AR Asocial Risky Asocial option “their score could markedly increase or decrease if they visited the scrapheap “some of these items may be broken and useless, but some may greatly increase your ship’s score… your score could go up or down”. Social option: participants were informed heir score could slightly increase or would remain the same “the ships will have the same score as your ship or slightly higher… you will be guaranteed at least the same score as your current ship”. Social risky (SR) Asocial option ““all of these items will help your ship to fly and some of them can slightly increase your ships score … you will be guaranteed at least the same score as your current ship”. Social option ”the ships may have a much worse or much better score than your current ship’s score… your score could go up or down.” Control: (C) Asocial “the ships will have the same score as your ship or slightly higher… you will be guaranteed at least the same score as your current ship”. Social “the ships will have the same score as your ship or slightly higher… you will be guaranteed at least the same score as your current ship”. 3. Results in economics papers typically reproduce the various models in a table. It took some time to adapt to this method but the verbal description is sufficiently careful that a close reading is rewarded and the use of a weighted average fit does reduce the sometimes tedious descriptions of various models. Therefore the article as written could benefit from yet another rewrite but meets the basic requirements of clarity, references and organizational structure. Experimental design: The research question clearly fits within the aims and scope of PeerJ. Human and animal studies on risk, social behavior and gender may ultimately provide clear guidance as to which features of humanity are cultural and which stem from more basic evolutionary pressures. Therefore, the goals are clear and address an identified knowledge gap. The procedures are mostly clear although I would need the precise wording given subjects before attempting a replication. The use of deception is within the norms of psychology but not economics. The researchers are psychologists though and therefore conform to the ethics of their field. Validity of the findings: Unfortunately, I do not trust the findings. Given subjects in an experiment for 20 minutes with little at stake, it may well be that decisions are based on small differences. For example, the description of the social and asocial conditions have one obvious difference - length and complexity. Perhaps the reason both genders prefer the asocial option in the control is because it is simpler. For the social option they have to decide if they believe the scores given spaceships are relevant and which features account for score differences. In the asocial option they have to decide which of 10 items to include. Notice that the social option is social only in the description of information and not any actual social interaction and does not in fact embody any wisdom based on group decision making. On the other hand, there is a significant interaction effect. Women, but not men, prefer the social information source when the asocial information source is described as being risky. I do not see how this can be attributed to complexity. I suspect there is some other small effect I have missed but I do not know that. I do trust the result that those told their spaceship is substandard are more willing to take risk. I would at least mention this is consistent with Kahneman and Tversky's prospect theory. Additional comments: As an economist I find the use of deception difficult to defend. I have tried hard to keep this out of the review but believe you should be aware of the economists' position. Debriefing means subjects eventually learn of the deception and may well participate in a later experiment where they will know not to trust the experimenter. I am not sure why deception is needed here. It seems to be used to save money and time, not inconsiderable motives but not particularly convincing either. If you want non-psychologists to pay attention, you may want to put in a little effort defending why deception is sufficiently useful in this study to outweigh the objection above.

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

Title: SEX DIFFERENCES IN THE USE OF SOCIAL INFORMATION EMERGE UNDER CONDITIONS OF RISK Review round: 2 Reviewer: 1

Basic reporting: Errors from the previous version were fixed. Experimental design: Design is good. Validity of the findings: Now that the authors, in their rebuttal letter, have clarified the bounded risk model, the findings make more sense. However, I would prefer to see this model be introduced earlier in the manuscript, perhaps in the introduction. This model initially confused both myself and a prior reviewer, suggesting this information needs to be highlighted in a better way in the manuscript. Additional comments: No additional comments

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

Title: SEX DIFFERENCES IN THE USE OF SOCIAL INFORMATION EMERGE UNDER CONDITIONS OF RISK Review round: 2 Reviewer: 2

Basic reporting: As before, I am happy with the reporting. Sorry for being annoying about the raw data file, but it still doesn't display correctly for me, regardless of whether on home or office computer, in Excel or R. I still get values such as 18-25 appearing under "Edu", and Undergraduate Degree appearing under "Country". It would definitely be helpful if the Editor could also check whether it displays OK. Sincere apologies if I am just doing something weird with the file. Experimental design: As before, no problems to report. Validity of the findings: I think the handful of new additions to the text help to make the authors' interpretation clearer and more explicit. 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: SEX DIFFERENCES IN THE USE OF SOCIAL INFORMATION EMERGE UNDER CONDITIONS OF RISK Review round: 2 Reviewer: 3

Basic reporting: See previous review. Experimental design: See previous review. Validity of the findings: See previous review. Additional comments: I have checked the responses to my earlier remarks. Adding the screen shots subjects actually see describing the four fold decision greatly facilitates reading and removes many of my objections. I was hoping the authors would defend the use of deception in the article, not just the reply to me. It is as I suspected, it saves time and money. For support you could have used Kahnenman - I forget which article right now - but he defends use of unpaid questionnaires because they are faster. He was able to do a new set each week. Made me jealous. Another possible defense is that the number of experiments in a given location is so small that contaminating the subject pool is unlikely. I recommended minor modifications before and did not demand to see the article again. Glad I saw the screen shots. Seeing more of the program might remove the comments about complexity as well. I believe paper should be accepted.

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

Title: HETEROASSOCIATIVE STORAGE OF HIPPOCAMPAL PATTERN SEQUENCES IN THE CA3 SUBREGION Review round: 1 Reviewer: 1

Basic reporting: In this study de Camargo et al. investigate whether a simple learning rule is sufficient to explain the features that make the hippocampus a suitable candidate for memory formation, storage and retrieval. The authors state that they have used a biologically plausible network model of the CA3 hippocampal subregion consists of 10,000 pyramidal cells accompanied with fast and slow inhibition in a feed-back manner. The authors have found that a network with synaptic weights determined by a heteroassociative learning rule can store and retrieve patterns and also renew the set of already stored memories with new ones. In addition, combining the heteroassociative learning rule with a homeostatic mechanisms, i.e., synaptic scaling, have permitted the storage of new memories while forgetting older ones. The motivation and the findings of this work are interesting. The network they used consists of 10,000 neurons which means that is biologically plausible in terms of neuronal numbers. This work is an accurate exploration mainly three parameters: the sparsity, the connectivity extent and the use or not of the modified STDP rule. Overall, this work seems to me that it is very interesting and well explained. In addition, it is well written and its structure meets the rquirements of this journal. However, I have some concerns about the modelling approach and the findings presentation. Experimental design: The Methodological part is in general very informative, however it is hard to replicate this network as some critical parameters are missing (see below for details). Validity of the findings: The findings are clear presented and the figures are in the majority of the cases accurate and correct. However, if there is a weakness, it is in the lack of any statistical analysis of the results. I suggest that you provide all the statistical tests you have used and also you should report their results (p-values, statistical values etc.). Finally, the conclusions are very informative and they give insights of the significance of this work. Additional comments: Generally, I suggest that you define in the Introduction more clear the difference between hetero- and auto-association memories. If anyone is not familiar with this topic it would find it difficult to follow. In addition, in my point of view it is of high importance to provide the code that is used on ModelDB or any relevant website. Also, Figures 4a and 4b never mentioned in the text either explicitly or as Figure 4 in general. Finally, it is better to use a larger line-spacing while you are in a review process as it would be easier to read. Following are specific comments on methodology part (not in order of significance or appearance in the text): 1. It is widely known that CA3 receives two major inputs from DG via mossy fibers and from EC LII and their role have been studied (Treves, Rolls, 1992). The authors should justify why the do not simulate explicitly the inputs. 2. Related to my previous concern, the authors state that their model is "a biologically plausible model". However, they do use a very simple form of inhibition in the network. I am wondering if by using fedd-forward inhibition as well would eventually change their findings. 3. Using integrate and fire neurons, it is necessary to report the voltage threshold as well as the reset voltage after a spike. 4. NMDA receptors are very important in pyramidal cells and they play a crucial role in memory procedures. There is no justification of the reason they are not simulated in the network. 5. I would like to know how many OLM cells are in the netowork and how many the Basket cells. In addition, in Figure 1 the name Basket appears but there is not in the main text. 6. Justification on the parameters choice is missing. In addition, the reason that the authors have chosen such a large refractory period is not explained. 7. Line 147-148: It is not explained how the difference is subtracted from the rermaining synapses. Randomly? Uniformly? 8. Critical simulation parameters and information should be provided, such as the total simulation time, the programming language and/or the simulator that use. Following are specific comments on the results validity (not in order of significance or appearance in the text): 1. Although Figure 6 is one of the key finding of this work, I believe that the way the findings are presented in this graph is somehow confusing. I highly suggest this figure to be re-made. 2. In Line 251, they say that "retrievable patterns increased significantly", however there is no reference to any statistical test in the whole paper. I highly encourage the authors to report the statistics af any comparsion they have done, as well as to report which test is used in every case. 3. Typo in sparsity legend in Figure 2 (right side, on the top)

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

Title: HETEROASSOCIATIVE STORAGE OF HIPPOCAMPAL PATTERN SEQUENCES IN THE CA3 SUBREGION Review round: 1 Reviewer: 2

Basic reporting: de Camargo et al. studied the performance of a heteroassociative coding scheme, combined with synaptic scaling, in pattern sequences storage in a biologically plausible model of the hippocampal CA3. They looked into pattern learning of the network, the ability to retrieve stored pattern sequences upon presentation of a partial cue, as well as the renewal of stored sequences as new memories are learned. The objectives of the research article are sound, and the report of the findings is also clear. While the authors discuss a lot about the pros and cons of heteroassociative and auto-associative networks, it would be good if more background on their definition and their network architectures could be provided. It is unclear to me how “to combine the 30 properties of auto- and heteroassociative networks” (line 29-30), and why “feedback connections from CA3 to Dentate Gyrus or between different CA3 areas would store sequences of memories in heteroassociative connections, and recurrent connections within single CA3 areas would work as auto associative connections” (line 32-34). On the other hand, the authors want to show that a heteroassociative coding scheme would suffice to account for the features that make the hippocampus a reliable network for memory. In line 36, it is not clear what the authors want to say with "representing sequence of neuronal ensembles which are sequentially activated bounded by theta cycles”. In line 71, it is better to use the term "oriens lacunosum-moleculare” once and then O-LM later as abbreviation. In line 190, STDP-induced LTD is abbreviated as SS-LTD, but in the context SS-LTD seems to refer to synaptic scaling combined with STDP-induced LTD. Please clarify. Experimental design: The design of the model fits well to the aim of the study. Most part of the network model is clearly described. However, the authors shall verify the model scheme and the use of parameters. Why are NMDA conductances not considered in the model? Earlier work on CA3 suggested that CA3 exhibits attractor dynamics due to the abundance of recurrent excitation, and NMDA conductances are needed to support attractor dynamics. In line 67, "The network has two kinds of feedback inhibition, mediated by fast and slow GABA channels (Pearce, 1993), modeled as direct connections between every pair of pyramidal neurons.” There is basket cell in Figure 1 but the inhibition is implemented as direct interaction between pyramidal cells. It would be helpful to the readers if you add a model scheme of how exactly the model is implemented, and include all kinds of inputs the CA3 circuit receives. In line 75, does “neuron” refer to pyramidal neuron? In line 77, a membrane time constant of 2ms is a bit small. Can you justify it? In the equation below line 79, is the negative sign missing in the exponent? In line 83, the step size is set to 0.1ms, which is a bit big compared with a membrane time constant of 2ms. Have you verified that the simulation results converge? In line 106, is the external noise between neurons independent? In line 109, a rate of 0.8 Hz during pattern retrieval in the simulations seems to be slow, compared with the firing activity in Figure 4. In line 115, as the computational demand is not that high for point neurons, it will be interesting to check the memory capacity and performance as the network size is varied. In line 144, could you add one or two sentences to describe what heterosynaptic long-term depression is? In line 145, is the additive synaptic scaling rule the same as the usual normalization? Why the rule is applied after 100 sequences, not after every sequence? In the formula below line 172, what if all neurons o_i(t) are active? Shall it be normalized by sqrt(n_p*n_o) instead? Validity of the findings: The report of the findings is clear in general. There are a few minor issues that the authors should address. In line 185, "storing a pattern sequence increases the total synaptic weight of each neuron, which is compensated by the synaptic scaling rule”. Is it true only if you have no or weak LTD? In line 258, the connectivity extent of 0.4 to 1 looks a bit high. What is the biological range? In Figure 4 right column, there are 10 peaks in 4(e) but 7 patterns are used. Could you explain? What is the function and advantage of theta rhythm in the model, does it work without theta rhythm? In line 211, "the benefits of using less neurons per pattern” are stated. How about the disadvantages? The benefits come with expenses. Additional comments: I think the article by de Camargo et al. could be accepted after addressing the comments above.

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

Title: HETEROASSOCIATIVE STORAGE OF HIPPOCAMPAL PATTERN SEQUENCES IN THE CA3 SUBREGION Review round: 1 Reviewer: 3

Basic reporting: The language of the article as written is mostly fine and understandable, but there were some sticking points for me as a reader that confused me until I was able to infer the intended meaning much later in the paper. (My annotated copy details my struggles as I read through from the beginning.) A major ambiguity for me was “connectivity extent”, as I thought at first “extent” was used as “level” or “value”, in which case I would drop “extent” altogether and just refer to “connectivity”. However, it became apparent that it meant the initial connectivity of the network, specifically the initial fixed fan-in connectivity fraction per neuron. Since “final connectivity” is referred to later in the paper to indicate connectivity after learning, I would strongly suggest replacing “connectivity extent” with “initial connectivity” and expand the methods definition to make the distinction more explicit. Another major ambiguity was “sequence completion” versus “pattern completion”: this paper deals only with sequence completion yet uses the term interchangeably with pattern completion. I take “pattern completion” to indicate autoassociative dynamics that retrieve a full pattern based on a degraded cue, so for much of the paper I was waiting for the authors to demonstrate autoassociative processing in their model. I marked instances in my annotated copy, but Lines 116, 294, 296, 383, 388 are examples. You’ll note I figured out this was a purely heteroassociative model at Figure 4 where the full cue pattern is not itself retrieved (the overlap curve is flat for the cue patterns in Figure 4e). The abstract and introduction refer to the “coding scheme” being studied (Lines 11 and 51), but to me this is a very ambiguous phrase and I didn’t understand what was being referred to. It seems that the authors meant to refer to heteroassociative processing as a whole as a “coding scheme”. Under that interpretation, the main hypothesis of the paper translates to whether heteroassociative learning (with homeostatic synaptic scaling) suffices to account for the role of the hippocampus in memory, with the implication that the alternative “coding scheme” of autoassociative dynamics may be unnecessary. If that is the case, the results of the simulation study fall far short of supporting the stated hypothesis. This is not an issue with the scope of the simulation study, more that the hypothesis interpreted this way is, I believe, overly and unnecessarily broad. I would like to see “coding scheme” replaced with a more specific and clear phrase and the hypothesis narrowed down to the more specific questions addressed by the study. Another ambiguous term was “renewal” used in the context of “renewing the set of stored sequences”. I did not understand what was being “renewed” in this usage, but came to sense the authors meant the abstract set of currently available (or “stored”) sequences was “refreshed” or “updated” as new sequences replaced old sequences. I originally thought “renewal” meant that old sequences were somehow reinforced, which I believe is the opposite of the intended meaning. Given its key place in the abstract and introduction (Lines 49 and 52), I would suggest replacing “renewal”/“renew” with clearer terminology (whether “updating”/“update” or similar would suffice I’m not sure). A number of sentences do not follow the scientific logic of the results and seem to editorialize unnecessarily. For instance, in arguing for “heteroassociative” processing, the authors seem to go to lengths to argue against autoassociative dynamics, for which there is substantial evidence and which do not need to be repudiated to discuss heteroassociative effects in sequence learning. This is further reinforced by the broad and dichotomous hypothesis in the abstract as I mentioned above. There are autoassociative effects of hippocampal memory that cannot be explained by the sort of heteroassociative network studied here (cf. the flat cue pattern retrieval in Figure 4e). “Biologically plausible” (in abstract and Line 59). I really do not like this phrase, it is overused by modelers (including me in the past!) to the point it doesn’t mean anything; I much prefer to see specific language supporting why the details of the model constitute an appropriate approach to the scientific question under study rather than this blanket assertion of “plausibility”. I object to the consistent description of LTD throughout the paper as “STDP-induced” or “STDP-based” LTD. There is no STDP in the network training. The authors describe a time-windowed learning rule perhaps inspired by STDP as indicated by the citations to the STDP literature, but it is not an actual implementation of STDP, and it is wrong to describe the LTD rule that way. The optional LTD in the learning rule is simply a time-windowed heterosynaptic LTD. The breadth of literature references is generally fine, if overly dependent on John Lisman’s work. I would suggest that W. B. Levy’s studies of hippocampal sequence learning would provide additional valuable and instructive background, particularly his 1996 paper (Hippocampus 6, 579-90). The structure of the paper is fine. However, the abstract does not do enough work. In addition to my comments above on the abstract, it seems to end at the point where the results of this study should be summarized and put into context for the readers. As it is, the abstract provides (too much) background, an overly broad hypothesis, and a simplistic description of the “biologically plausible” model. The entirety of the results are captured by the phrase “these hypotheses hold”, but there is no elaboration of actual results, how they support the hypotheses, any problems, or what the take-away conclusion of the study should be. This abrupt end to the abstract is a disservice to potential readers of the paper. Given the above caveats with the scope of the hypothesis, the study is self-contained, relevant to the questions posed, and constitutes an appropriate unit of publication. Minor points of language: - Line 17, the definition of “autoassociative memory” is wrong, referring to “association between different memories” when it should be “association between the features of a given memory” (or similar) - Lines 42 and 425, I believe “temporally” was supposed to be “temporarily” - Line 36, the phrase “Different from” should be “In contrast to” in idiomatic English - Line 28, “representing” should be “represented by” unless my understanding is wrong - Lines 230 and 238, “bifurcation” is the wrong word, maybe “transition” or “maximum” - throughout, “less” is used when “fewer” is correct (with discrete noun plurals), I’ve marked most instances in the annotated copy. Ibid. with “smaller” and “lower” for numerical values - Line 42, “third feature”. It is unclear how the authors are counting the features of the hippocampus being introduced; I didn’t realize there was a “first” or “second” - Line 48, “acceptable”. Saying an effect is “acceptable” for CA3 is presuming fundamental knowledge of the actual purpose and role of CA3; this is tautological and teleological. One could say it is “consistent with” a “hypothesized role” of CA3. - Lines 205 and 207, the authors never define “crosstalk”. Is it a form of memory interference? Does it mean anything beyond “memory pattern interference”? - Line 253, “neurons per pattern” doesn’t make sense to me, did the authors mean “connections per neuron”? I don’t think the pattern size was changing there. - Lines 413-415 and 426-428, I’ve highlighted these sentences in the discussion as being vague and unclear. They should be elaborated more clearly or dropped. - Line 345, missing noun after “autoassociative” - Line 422, missing noun after “heteroassociative” - Line 435, missing “are” Experimental design: This a primary research manuscript pertaining to Biological Sciences, which is appropriate to the Aims & Scope of the journal. I do have concerns with the statement of the research question, as I discussed in the Basic Reporting section. It is overly broad and dichotomous, forcing an unnecessary counterpoint to autoassociative learning when the history of research in this area suggests that these dynamics are not mutually exclusive. I would like to see a narrowing of the scope of the research question in a way that makes it more specific and attuned to the subtleties of homeostasis, synaptic modification, and temporal interactions in sequence learning that are actually probed in the simulation studies. After reviewing the methods descriptions and a shallow review of the source code, the technical standards for the simulation study are in-line with the typical research output in this field of study (spike-based networks of sequence learning with various synaptic modification rules). For a simulation study like this, the only route to reproducibility is source code release and it appears that the source code is available (or will become available) on figshare with a DOI. However, the authors’ methods descriptions have a number of minor issues. My annotated copy is complementary to the points below. Network model: - The Figure 1 caption refers to the inhibitory “neuronal populations” but the description and figure graphic suggest that a single global interneuron (basket cell) is used to control activity. Line 71 mentions “O-LM” cells for inducing theta oscillations, but not how many are in the model, or why more than one are needed since they would presumably have the same activity. All of this should be clarified. - Line 68 describes the feedback inhibition as “direct connections between every pair of pyramidal neurons”, but that would constitute feedback excitation. Something is wrong here, unless it was implied that the “direct connection” was through the global interneuron, which is something that should be made explicit. - Figure 1 shows the O-LM interneuron driven by the pyramidal neuron output, but it is described in the text as a spike generator. The figure should be fixed to remove those pyramidal/O-LM connections if they don’t actually exist. - Line 72. It is unclear what “intrinsic” firing rate means or why the theta frequency of 5 Hz was chosen. That would be considered atypically slow theta in rodent hippocampus - Line 72, “It” (referring to one or multiple O-LM cells) is said to “modulat[e] the generation of the theta rhythm”, but it IS the theta generator, so it is unclear what modulation is being described here - Line 79, “neural adaptation” is too broad and should be specified as, I believe, “spike frequency adaptation” - Irep equation should have exp(tspk – t) instead of exp(t – tspk), otherwise the hyperpolarizing current would be exponentially increasing with time Synaptic model: - Ik equation should have a halfwave-rectified \Delta{t_s} otherwise it would be negative after the spike but before the delay has finished Pattern sequences: - Line 111, the authors should specify whether pattern sampling is with or without replacement. The patterns should be orthogonal, which would be sampling without replacement. If not orthogonal, justification will be needed - Line 113, the definition of “sparsity” is actually a definition of “density”. Sparsity should be defined as the fraction of inactive cells in the pattern (the authors define it as the fraction of active cells). Instead of changing the numbers and figures, it would be easiest to change “sparsity” to “input density” (or similar) throughout the paper to convey the meaning correctly. - Line 124, typo “s1-s2-s2-…” - Lines 115-118, the authors justify a higher input density than de Almeida’s 2007 estimates for their 10,000-neuron simulations, but it would be nice to see an analysis of larger simulations (e.g. 30K neurons) that may allow sequence learning with more “realistic” input densities to see if network size was the limiting factor Learning rule: - Line 132, 15-ms presentations for off-line training seems to presume that the sequence has already been learned, in order to compress events into gamma cycles. I suppose the authors are considering an ordered set of place cells that fire in sequence within a theta cycle due to phase precession. However, this should be clarified in text and made explicit to readers with and without background knowledge of theta sequences - Weight update equation is unclear. How is the initial ± supposed to be interpreted? Is there a forward pass for LTP followed by a backward pass for LTD? - Also, weight update is clearly not based on spike-timing differences. As I mentioned above: it is not an STDP rule, it is only a sequence-adjacency rule. This approach is valid for studying the parameters of sequence learning, but it is not valid to refer to this as “STDP-induced” LTP/D throughout the paper. STDP can have complex consequences and effects that are not accounted for with this rule. - Line 142, I think this is the beginning of my “connectivity extent” problem described above. I did not understand why the authors related the initial connectivity to that of a “highly connected developing nervous system”. New episodic memory sequences are not learned from an initial state corresponding to a developing nervous system. The authors should clarify that they train these networks from a highly connected state with many sequences until stabilizing at a lower connectivity, effectively simulating a “lifetime” of episodic sequences until maturity. More clarity about this training regime would help. - Lines 153-154, “by anatomical or physiological reasons” is confusing. I’m not sure what it means since the model only contains synaptic connections Validity of the findings: The findings of this simulation study have high face validity: I believe the results are relevant to the research question(s) that the authors aimed to study. Further, the paper as presented demonstrates substantial content validity: Many of the parameters most relevant to the narrow scope of “pure” heteroassociative learning in a network model are studied in an interesting and inter-related way that helps to bridge a knowledge gap in the combination of homeostatic and experience-dependent forms of synaptic modification in a cognitive neural system. The authors’ lack of consideration of autoassociative processing in this system reduced the potential content validity, since there may be critical interactions (e.g. with other hippocampal inputs or subregions) that contribute to sequence learning as such and were not addressed by this work. However, it is good for the field to have studies like this that explore particular dynamics of interest. The data, consisting mostly of static network analysis and network simulation outputs, appear to be robust and statistically sound within the standards of the field. As with the abstract, which ended abruptly, the paper does not conclude in a way that summarizes the results or relates those results back to the specific research questions. The authors should consider trimming some of the vague discussion and adding a strong conclusion that ties specific results back to specific knowledge gaps revealed by the research question. Given my concerns about language and structure above, to make this possible the research question will need to be reframed and better specified throughout at least the abstract and introduction in any subsequent revisions. 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: HETEROASSOCIATIVE STORAGE OF HIPPOCAMPAL PATTERN SEQUENCES IN THE CA3 SUBREGION Review round: 2 Reviewer: 1

Basic reporting: The language is clear and not ambiguous. After revisions the authors have taken into account my comments and more specific, they give a clear definition of auto- and heteroassociative networks. In addition, the authors have given a link of a webpage where anyone can download the source code. The only disagreement with the authors is that even if the learning process is performed online, in my opinion, NMDA current should be included in the model as it is widely know that plays an important role in dendritic integration in pyramidal cells in CA3 and CA1. However, for the specific questions that the authors have tried to answer it is valid to ignore this parameter in order to reduce the parameter space. It would be interesting though to see if any change would arise by incorporating the NMDA component to this model. Taken all into account, I think that this project is almost ready for publication. However, I have some minor comments about the construction of the figures. In my opinion it is better to produce graphs without top and left axes (Figure 6 axes are nice) for consistency and clarity. Moreover, in Line 197, where authors refer to MATLAB I think that you should include also the version and also the computer specifications (CPUs, RAM) where the code has run. Typically, MATLAB (R2016a, The Mathworks, Natick, MA) form is used. Additionally, some minor suggestions are given below: Line 33: missing noun in "autoassociative" Line 78: "send connections ..." with "is connected with every pyramidal neuron via GABA synapses..." Line 84: "is dependent" with "depends" Line 125: "to mean that" with "to refer to" Line 219: "would be performing" with "would be" Experimental design: no comment Validity of the findings: no comment Additional comments: no comment

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

Title: HETEROASSOCIATIVE STORAGE OF HIPPOCAMPAL PATTERN SEQUENCES IN THE CA3 SUBREGION Review round: 2 Reviewer: 2

Basic reporting: The authors have updated the manuscript according to reviewers' feedback on language use. The manuscript is in general well-written. However, there are still some issues with the use of words. In line 26, replace “the cell preferred place” by “the cell’s preferred location”. In line 51-52, for “since an increase in the number of stored memories leads to a catastrophic interference effect”, do you mean an increase beyond the capacity limit? In line 155, “We decrease all synapses from each neuron by a fixed amount”. I guess the authors mean to reduce all synaptic weights. In line 279-280, “For the initial random weights, we used the values of 2.5, 280 3.0, 6.0 and 4.0 for the initial connectivities 1.0, 0.8, 0.6 and 0.4, respectively” is confusing. In line 301, “larger then” should be “larger than”. In line 329, “latter” should be “later”. Experimental design: In the METHODS, the authors reported the learning rule first and then connectivity. It seems more natural to describe the network connectivity before the learning rule. The readers want to know how the network is like before what changes to make. In line 92, shall it be for all t > ts + tdelay? In line 194, what are the combinations of parameter values? Validity of the findings: In line 266-268, "If we consider the estimated connectivity of 20%, which full connectivity we would have to use an initial weight of 2.0, resulting in about 1400 retrievable patterns.” Did the authors mean “with full connectivity”? Also in line 268-271, the description is not totally clear. Are the readers supposed to look for the initial weight corresponding to 20% connectivity from Figure 3A given the initial connectivity, and then figure out the number of retrievable patterns from Figure 3B with the corresponding weight and the given initial connectivity? If so, it would be helpful to indicate the 20% connectivity and mark the values used for comparison in the figures. In line 411 and 413, what are errors refer to? Additional comments: There are some very long sentences in the article. For example, in line 15-19, “Based on these models, Rolls et al. (1997) proposed that the CA3 subregion could work as an autoassociative memory, where neurons from the same pattern have recurrent excitatory connections between them, which enable associations between features of a memory, retrieval of stored memories from presentation of partial cues, and noise tolerance during memory retrieval.” Could the authors consider breaking them down to improve readability? It seems that the authors dropped “extents” following the feedback from Reviewer 3, but there are still a few occurrences (e.g. in line 347).

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

Title: HETEROASSOCIATIVE STORAGE OF HIPPOCAMPAL PATTERN SEQUENCES IN THE CA3 SUBREGION Review round: 2 Reviewer: 3

Basic reporting: The authors substantially improved the clarity of the text and the results. The revision and letter demonstrated a comprehensive treatment of points from the initial review. The arguments about auto- vs. hetero-association have been smoothed out and provide better context, including additional references, and the central question of the study was more prominent and more clearly defined. The abstract and discussion now have proper conclusions that will certainly help readers. I believe the manuscript now meets the journal’s criteria for publication. There were a few places where “extent” was still used instead of the new “initial connectivity”. There were also some minor typographical issues that I have marked in my review copy that should be fixed prior to publication. Experimental design: The model design, especially regarding the implementation of interneurons and inhibition, has been given appropriate detail and clarified in the network figure. The expanded details about the number of simulations, means, and standard deviations in the figure legends has increased the transparency about the study design and findings. Validity of the findings: As in my initial review, the modeling study has both face and content validity for the central question of the paper. The additional language about the relationship with auto-associative processing established a narrower focus that helps to allay my prior concerns about overly broad claims. 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: BIOTEA: SEMANTICS FOR PUBMED CENTRAL Review round: 1 Reviewer: 1

Basic reporting: I enjoyed reading the manuscript. The presented work is sound and highly practical. The manuscript is well written. I commend the authors for semantically processing articles from >7K journals and making all the data and software code available. In my opinion the submitted manuscript should be accepted for a publication after suggested minor revisions: - ‘the NER service provided by  the NCBO Annotator’ The accuracy of many other NER tools is higher than of this one, see for example tools provided by NaCTeM. Those other tools also are using ontologies. Please explain your choice. - Overall, there is no discussion of text mining (TM) as a closely related area of research. Of course, there are principle differences with what the authors are producing, but many TM steps can be re-used by Biotea. - I would prefer to see more examples and discussion o how Biotea can be used. - Line 64: the first mentioning of Biotea – you need to provide more explanation what it is. The same about hypothes.is - Proofread the manuscript, examples: Lines 48-49: add missing gaps; Line 65: “representation, idem. that “, etc. etc. Experimental design: no comment Validity of the findings: no comment Additional comments: no comment

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

Title: BIOTEA: SEMANTICS FOR PUBMED CENTRAL Review round: 1 Reviewer: 2

Basic reporting: The authors do an excellent job of explaining what they mean by their term "RDFize". However, I do not see the need to coin a new verb, especially considering the authors later turn the this new verb into a noun: RDFization. I think it best editorial practice if they refrain from coining a new term and instead refer to RDF generation or RDF creation as is appropriate. Suggested grammatical changes: Lines 280-281. "We are using hierarchical ... using the cosine distance as the metric" Line 409. "The resulting dataset is over 150 Gigabytes in size" Line 421. Parameterize (actually suggest rewriting this sentence - it's not completely clear) Experimental design: No comment Validity of the findings: No comment Additional comments: Just a suggestion. You note several software dependencies for using Biotea - Maven, Java and Eclipse. These particular programs tend to have very specific versions for various OS platforms. I think it would be helpful to your audience and potentially increase the usage of Biotea if you were to provide a preconfigured Virtual Machine image using Ubuntu. Virtualbox provides such customized VM's for various purposes (http://www.oracle.com/technetwork/community/developer-vm/index.html), as well as Bitcurator (https://www.bitcurator.net/)

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

Title: BIOTEA: SEMANTICS FOR PUBMED CENTRAL Review round: 1 Reviewer: 3

Basic reporting: The subject of the manuscript is one that is important to the future of science, an one that is close to my heart: the improved reporting of scientific knowledge through the use of semantic technology. There is a clear and urgent need to improve the reporting of scientific knowledge, and it is scandalous that so much public and charitable money is spent on science that cannot properly be used because it is inaccessible to computers. The Introduction and background are clear, and the literature is well referenced and relevant. Clear, unambiguous, professional English language is used throughout. The figures are relevant, high quality, well labelled and described. Experimental design: The models, services, software and datasets are available. Validity of the findings: The authors demonstrate the utility of Biotea in two examples. This is the weakest part of the manuscript: • Example 1 concerns the retrieval and clustering of papers annotated with the SNOMED CT term ‘renal 
cell carcinoma’. Unfortunately, the three papers investigated have little to do with ‘renal 
cell carcinoma’, although it is true that this phrase occurs in all of them. However, the authors do a good job of describing why papers 3862691 and 3862582 are more similar with each other than with 3899087. • Example 2 involves the creation of a very long SPARQL query, but the results of the query are not described. The SPARQL query would be very hard for a domain scientist to generate without the use of some tool. I recommend that two other examples are used to demonstrate the utility of Biotea. Around line 255 the manuscript describes the mapping between Biotea and SIO concepts: ‘encapsulating the original data type property value; thus, a bibo:pmid with the value “28300141” is 
mapped to the object property sio:has_identifier, this is linked to the class sio:identifier 
that is related by means of sio:has_value to the actual PMID “28300141”.’ I don’t see how this mapping captures the key information that the identifier comes from PubMed . 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: SATELLITE TAGGING HIGHLIGHTS THE IMPORTANCE OF PRODUCTIVE MOZAMBICAN COASTAL WATERS TO THE ECOLOGY AND CONSERVATION OF WHALE SHARKS Review round: 1 Reviewer: 1

Basic reporting: The paper is clearly written and concise. Background information provided are relevant, the paper is relatively well structured, except in the abstract. Specific comments are provided below. I did not identify issues with the figures, tables and raw data. However, I made some comments (general comments) for Fig. 1 that remains difficult to read. Results and hypotheses are relevant. Experimental design: I have no comments here, except for the description of the methods that are lacking, particularly for aerial surveys. There is a temporal mismatch between aerial survey and satellite tagging data. Since artisanal fisheries are dynamic in time and space, and that prey availability (influenced by the complex oceanography of the Mozambique Channel) also greatly varies between years, you need to clearly show how comparable both datasets are. I know the authors cite Cliff et al. (2007) for details on the sampling for aerial survey data. However, more (even brief) information would be needed to enlighten the reader. You also need to refer to a map with the sampling design. Validity of the findings: I have reservations on the use of gillnet data, and about the compatibility of aerial survey and satellite tag data. More details are provided in the "general comments" section. Movement data are robust and statistically sound, and conclusions are well stated. However, conclusions on the overlap between gillnets and whale sharks are speculative and should be identified as such. Additional comments: Abstract The abstract lacks of structure. The first sentence should be removed, and I would rather include one more sentence on aerial survey methods. A justification of why you combined aerial survey and satellite (even very brief) tagging methods would be good to add too. A better presentation of the question(s)/hypothesis is critically needed here too. Introduction L. 59-61: this sentence (last of 1st §) seems to be out of place. Should be removed. L. 66-67: “ecological drivers of their movements is poorly understood” might be better. L. 67-71: very general statement here, which also seems to be incomplete (how about predation risk?). General statements like this one should be in 1st §. 2nd § should keep focusing on whale sharks. L. 81: “in review” paper should be removed (replaced by unpublished data), same for Normal et al. (l. 84, 89) L. 96-106: anthropogenic threats might explain the observed decline. However, oceanographic conditions and changes in productivity (and prey availability) at feeding aggregations might have affected whale shark abundance. It should not be ignored here, and I think anthropogenic threats are obvious in this region (particularly bycatch in drift gillnets), but other drivers in abundance should be mentioned, even briefly. L. 123: and “underlying environmental drivers” would be (a bit) better. Materials and methods There is a temporal mismatch between aerial survey and satellite tagging data. Since artisanal fisheries are dynamic in time and space, and that prey availability (influenced by the complex oceanography of the Mozambique Channel) also greatly varies between years, you need to clearly show how comparable both datasets are. L. 140-141: I know you cite Cliff et al. (2007) for details on the sampling. However, more (even brief) information would be needed to enlighten the reader. You also need to refer to a map with the sampling design. L. 227-228: what are net dimensions and where are they set? What is the mesh size? What kind of gillnets are used? It looks like these are bottom set since their location is very coastal. Unless you prove otherwise, I doubt whale sharks can get entangled in these, whereas large meshed drift gillnets can. L. 232: “boat-based” surveys? Looks like these net locations were recorded opportunistically. More information on sampling is needed here. How representative this sampling is? Not sure if the sampling really reflects the distribution of gillnets in the study area. Results Fig. 1: very few gillnets were recorded, so I wonder how relevant it is to link their distribution and whale shark distribution and movements. You also mention in the methods that gillnets were recorded during “boat-based” surveys, but it is not clear whether gillnets on the map were recorded during flights or at-sea surveys. L. 286: How did you find the animal? L. 295: I think “range” would be better than “band”. L. 366-367: sounds speculative. Any reference supporting this? What about thermoregulation? L. 396-397: speculative. This statement needs to be. Discussion L. 325-332: it doesn’t look like you are really discussion the spatial overlap between gillnets and whale sharks. It really convinces me that this dataset might not be relevant for the scope of the paper. L. 347: “in revision” papers should not be cited, same for submitted (l. 422). L. 433: analyses that were performed do not clearly show the overlap between gillnet fisheries and whale sharks.

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

Title: SATELLITE TAGGING HIGHLIGHTS THE IMPORTANCE OF PRODUCTIVE MOZAMBICAN COASTAL WATERS TO THE ECOLOGY AND CONSERVATION OF WHALE SHARKS Review round: 1 Reviewer: 2

Basic reporting: the manuscript is well written and easy to read. I'm uncertain if PeerJ allows "in revision" articles to be cited. Experimental design: The limited aerial survey effort combined with diving behavior limits the utility of these surveys. The short track duration and resulting data set on movement of tagged whale sharks limits the ability of this research to determine anything other than short term movement and habitat use. While the data are presented well and used to interpret selection of inshore habitats there is limited discussion on long term habitat use combining satellite and visual observations. By addressing the limitations of the short term data and acknowledging some of the issues around gaps in movement data for ~ half the year I think the manuscript would be greatly improved. Methods of random models was not adequately addressed and I could not view figure 2 in the supplementary material. More detail on how the models were generated including the code used to generate these models would be useful. Validity of the findings: See previous comments on limitations of aerial survey data as well as limitations of whale shark movement data due to short track duration and no data on movements for ~ 5 months of the year. More could be done to determine level of overlap with gillnets sets along the coast. The analysis would benefit from a seasonal component to movement analysis - did sharks occupy different areas at different times. The horizontal movements are very descriptive and while data are limited, more should be done to try and determine any patterns related to season or environmental conditions. Additional comments: Introduction Line 70 – there are more relevant papers on reproductive movements than the one cited. Please provide additional examples. Line 84 – does the journal allow manuscripts in revision to be cited (Norman et al. in revision)? Line 137 – 139: the amount of aerial survey effort is very limited and amounts to ~2 flights per year over 5 years. Furthermore the survey area is incredibly narrow. It is difficult to see how such a survey could provide data relevant on abundance given the variability in whale shark ‘availability’ to aerial surveys. It is well known that Whale Sharks spend considerable time in deep water (even when close to shore) making them impossible to see from the air. The variation in the number of sharks visible during any one flight makes it difficult to reconcile abundance estimates. Line 243 – the large number of sharks seen on one day highlights the variability in whale shark abundance/sightability which do not always reflect true population size. Line 291 – 297: that temperature data obtained from SPOT tags is limited to days when temperature histogram data were recorded and while there is a nice range of data presented, the authors need to acknowledge that these data are not reflective of the total range of temperature experienced by the sharks. The number of time at temperature messages received per day should be presented in a table to illustrate over what time period these data were recorded. Figure 2: it is unclear from this figure whether the duration of the tracks influenced offshore movement. It would be good to colour code days since tagging to illustrate whether sharks that moved off the shelf were tracked for longer or whether this just represents individual variation. Most offshore movement seem to have occurred in summer suggesting some seasonal shift. KUD estimates from satellite telemetry of whale sharks is problematic given low detection rates combined with periods of limited or no detections over multiple days. It is therefore important to present detection data in terms of number of fixes per day to enable the reader to interpret KUD estimates which will be biased by periods when the sharks were detected more frequently either due to favourable environmental conditions or because the sharks were spending more time at the surface swimming at slower speed resulting in more fixes. Figure 2. the end of the track of shark MZ471 appears to be from the tag floating on the surface. Figure 4 is misleading as the total number of transmissions will be a function of how many tags were deployed as well as weather conditions. The number of transmissions should therefore be an average per tag not the sum of all tags. Figure 5: it would be good to see a figure of the randomised tracks to determine how realistic/unrealistic they are compared to recorded track. Line 309-321: the fact that tags didn’t record depth limits the use of these data and this section appears to be overly long. I suggest shortening this section to focus on the main point - It is clear from the temperature data that sharks that moved off the shelf experienced a wider range of temperatures most likely due to vertical movements into cooler waters. It would be nice to try and match these temperatures with known depths if there are any oceanographic data available for this region. Line 334-347: the short duration of the tracks and the fact that combined tracks do not span more than 7 months is a major limitation of the research. This limits the ability to make overarching comparisons and management implications need to be taken into context. While it is clear that sharks spend a disproportionate amount of time close to shore for some part of the year and return to the region over multiple years there is still uncertainty over where the sharks go and more needs to be done in the discussion to piece together this research combined with previous work. Line 363-364: the limited time animals were tracked for needs to be acknowledged here. This statement should include the period animals were tracked. Line 387-390: the lack of temperature stratification in relatively shallow coastal waters limits the ability to determine diving behaviour from temperature alone. The statement that little diving took place is therefore not supported by the data as there isn’t any data on diving behaviour in shallow coastal waters. It should also be acknowledged that data on temperature is not collected frequently by SPOT tags (how often temperature data were obtained is not presented so I’m assuming that the transmission of these data was limited as is often the case when prioritising location transmission over temperature data). Line 401 – 404: given that there is very limited data on vertical movement that is inferred from temperature data it seems a long bow to claim that these data support a hypothesis that vertical movements in whale sharks relate to foraging behaviour. I suggest removing this statement. While there is limited evidence to suggest that whale sharks are occasionally captured in gillnets the numbers captured per year or the frequency of these events are not presented or discussed (although it appears that efforts are underway to quantify this). Gillnets are usually highly size and shape selective and designed to mesh certain sized sharks/fish. The shape of a whale sharks head would make it virtually impossible to be meshed in a gill net and while there is the potential for entanglement the likelihood of this occurring is small. It is also not clear how much overlap there is between where the nets are set (what is the average net length) and the habitat whale sharks use. Data from satellite tracks could be used to determine the average distance offshore and therefore to determine overall overlap with gill nets. It would be good to see this attempted given that data are available. Furthermore this coastline is not protected and setting gillnets up to 500 m offshore in swell and over reef would be very difficult. I acknowledge that even low levels of fishing mortality could be unsustainable, however given that no data on the probability of capture are presented the link between population declines and gillnets appears over-stated. A more detailed explanation of how the gillnets are set (e.g. are they set from boats, are they set offshore in mid water, are they anchored or set as tangle nets, what mesh size is used and what water depth of water they are set in) is required to enable the reader to contextualise this information. Overall, this manuscript is well written and the analysis are sound. The short track duration is problematic and at times the conclusions in relation to habitat use, diving and temperature profiles are over-stated given the limited track length as well issues with how much data are obtained from satellite tags.

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

Title: SATELLITE TAGGING HIGHLIGHTS THE IMPORTANCE OF PRODUCTIVE MOZAMBICAN COASTAL WATERS TO THE ECOLOGY AND CONSERVATION OF WHALE SHARKS Review round: 2 Reviewer: 1

Basic reporting: The paper is really clear and unambiguous. Background information provided is sound and sufficient, and the paper (including the abstract) is well structured. Experimental design: The research is suitable for the scope of the journal. The research question is well defined and relevant, and the results fill an identified knowledge gap. The investigation is rigorous, methods have been greatly improved. Validity of the findings: Data is robust and sound, and conclusions are well stated. Additional comments: The paper has been clearly improved and the authors have addressed 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: SATELLITE TAGGING HIGHLIGHTS THE IMPORTANCE OF PRODUCTIVE MOZAMBICAN COASTAL WATERS TO THE ECOLOGY AND CONSERVATION OF WHALE SHARKS Review round: 2 Reviewer: 2

Basic reporting: The authors have done a good job responding to the reviewer comments. Experimental design: no comment Validity of the findings: see comment below regarding gillnet mesh size and implications for whale shark entanglement Additional comments: Overall the authors have done a good job addressing reviewer comments. There is still a lack of understanding about gillnets and this needs to be addressed. 8-10 cm (3-4 inch) mesh is not large. This size gillnet is used to target small - medium sized fish (~30 – 50 cm TL) depending on the body shape. Nets this size are not even used to target small sharks and these nets do not have the capacity to regularly entangle sharks > 1 m TL. They certainly do not have the capacity to regularly entangle whale sharks unlike large mesh tangle nets (mesh size 20 – 40 cm) which have been implicated in whale shark capture in other parts of the world. I acknowledge that these nets may entangle the occasional whale shark and that this could have long term consequences but these nets simply will not results in frequent entanglement. It is unclear whether observations but S. Pierce relate to mortality/entanglement along the Mozambique coastline or other parts of the world. It is also not clear if sharks were entangled in similar/same sized mesh nets. This needs to be made clear as entanglement and injuries reported by Speed et al. are not specific to Mozambique. I suggest changing "large mesh" to small or medium size mesh to better reflect mesh size and the selectivity of different sized mesh.

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

Title: EFFECTS OF PERCEPTUAL SIMILARITY BUT NOT SEMANTIC ASSOCIATION ON FALSE RECOGNITION IN AGING Review round: 1 Reviewer: 1

Basic reporting: The crucial summary of the design in Experiment 1 appears to have a grammatical error (line 100); also what is a list lure? If it was in the list is it a lure or a target? We only find out at line 109. The method may benefit from a design section that is self-contained. I found the method very hard to follow and I feel that it would benefit from figures depicting each of the three conditions. I am unfamiliar with the way that interactions are reported intertwined with main effects and cannot discriminate between original analyses, follow-up analyses and analyses based on different dependant variables. Nested parentheses caused further confusion. The y-axis is not labelled on the Figure. All four panels on the Figure have different maximum y-values, which hinders comparison. Experimental design: I could not fully follow how words were placed into each condition and into study and test list in the 2nd paragraph of the material section in Experiment 1. Is the word ‘novel‘ being used to label non-studied words on line 113 or does this refer to the fonts? There seems to be sufficient detail to replicate if one was prepared to devote a significant amount of time to unpack the information. I did not see any control for the degree of congruency between fonts and words, which could have differed between study and test items and between conditions. Validity of the findings: My lack of comprehension of what was actually being measured restricts my comments on the validity of the statistics although individual F-values appear to have been reported correctly. The Bayes factor analyses were a good way to account for multiple tests. The general discussion is fairly long and is therefore not always closely linked to the results. Additional comments: In some respects I feel that I may have missed parts of the article due to my lack of understanding, particularly of the results sections. However, I likely made more effort with the paper as a reviewer than would a normal reader and the lack of clarity in the article prohibits my recommendation for publication. It seems as though the main point of interest is age differences in perceptual (font) versus semantic (associative strength) lures. However, Experiment 1 had the same perceptual presentation across all 3 manipulations of semantic relatedness. Experiment 2 controlled for this by also manipulating font uniqueness and the take home message is that perceptual lures can lead to greater age deficits than semantic lures. As far as I understand it, this could be conveyed in a much briefer write-up that excludes a full statistical exploration of the data and that has a shorter 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: EFFECTS OF PERCEPTUAL SIMILARITY BUT NOT SEMANTIC ASSOCIATION ON FALSE RECOGNITION IN AGING Review round: 1 Reviewer: 2

Basic reporting: The paper is generally very well-written and structured. Minor issues: - The authors could expand on the relevance of remember/know judgments in the introduction (l.64). - Figure 1, together with its caption, should be more self-explanatory; please add Y-axis labels. - l. 173-174 missing p-values - l. 183 incorrect p-value - l. 315 should be table 2 - Table 2: How was false recognition to font only lures in older adults computed/corrected? Shouldn't this value be closer to .38? Experimental design: The study seems carefully designed. Yet, for the author to fully understand the design the clarity of reporting the methods of Experiments 1 and 2 could be improved (e.g., how many lists and items of each type were used and how exactly were they combined to create the materials for each condition; perhaps a table could more easily illustrate the construction of materials; an appendix could be addedd that provides the full list of words and fonts to increase replicability). Validity of the findings: My major concern is that, instead of false memory, the results might reflect strategic differences between age groups in how they (mis)understood the recognition instructions. There was no instructions regarding the role of fonts at test. Did age groups differ in their understanding of the relevance of fonts to recognition judgments? I.e., older adults may have understood the similarity between studied and test fonts as relevant (instead of to-be-ignored) for the recognition test. If so, their recognition of lures would no longer be interpretable as "false" memory. A perhaps related puzzling finding is that, in Experiment 2, older adults had increased perceptual false memory (font only condition), as well as increased semantic false memory (unique font condition) - but when both factors were combined (correlated font condition) the age-related false memory increase was eliminated. This needs to be discussed, perhaps with the differences in true recognition in mind. A third issue concerns statistical analyses. To interpret null results, the authors report Bayes Factors in some cases, which is helpful for interpretation. In other cases, however, they still interpret null findings without reporting BF, especially for ANOVA analyses. I would recommend to add the missing BFs that are easily computed from JASP (or to avoid interpreting the respective null findings). In interpreting BF values, the authors unfortunately decide that a BF > 3 represents strong evidence, but that a BF < 3 only yields inconclusive evidence. I believe there are two problems with this interpretation: first, there is little agreement that BF > 3 is strong (e.g., http://www.nicebread.de/grades-of-evidence-a-cheat-sheet/). Second, the BF value is a continuous measure of evidence (or relative belief updating), and lends even less support for introducing arbitrary cutoffs or decision criteria than the p value; we all know that the difference btw significant and non-significant is itself not significant, and analogously (but for different reasons) a small difference in BF (e.g., between 3.3 and 2.1) is itself not (strong) evidence for a difference in support for two hypotheses - but such a difference might be directly tested by targeted model comparisons in some cases. Finally, the authors report that they used robustness checks to test whether their result patterns depend on the choice of priors. They do not, however, report detail on these analyses or their results. 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: EFFECTS OF PERCEPTUAL SIMILARITY BUT NOT SEMANTIC ASSOCIATION ON FALSE RECOGNITION IN AGING Review round: 2 Reviewer: 1

Basic reporting: Improved (see below) Experimental design: Same as original submission. Validity of the findings: Same as original submission. Additional comments: I feel that the clarity of the article has improved and that there is a clear take-home message that is sufficiently evidenced and of interest to ageing researchers. The new figure aided clarity. I still feel that this simple message could be presented with fewer statistics and measures but I was better able to follow this version.

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

Title: EFFECTS OF PERCEPTUAL SIMILARITY BUT NOT SEMANTIC ASSOCIATION ON FALSE RECOGNITION IN AGING Review round: 2 Reviewer: 2

Basic reporting: The authors have added Bayes factors for some non-significant findings that are interpreted but not for all of them. Please supply additional analysis for... - Both experiments: - non-significant difference in forward digit span between age groups - False recollection of novel items - Experiment 1: - adjusted false recognition report BF for non-significant post hoc tests - Experiment 2: - True recognition: report BF for absence of interaction effect - True recollection: Report statistics for group and interaction effects including BF for non-significant findings. - Also, double check that the reported BF = 0.96 is correct (seems surprising given the strong effect in the frequentist analysis, partial eta² = .25, p < .001) - Experiment 2, adjusted false recognition: Double check BF for effect of interaction given that the frequentist analysis was non-significant. Experimental design: no further comments Validity of the findings: - The authors have clarified in the method section that participants received no instructions regarding the relevance of font. They rightly state that it is unlikely that older participants responded solely based on fonts. However, as previously pointed out the possibility remains that older adults may have considered the font relevant in addition to the words and more so than younger participants. In fact, the possibly impaired ability to ignore fonts that the authors discuss in lines 528-539 makes a similar point. Strategic or involuntary processing of fonts both have consequences for the interpretation of the findings. In case of strategic differences it would be hard to attribute the findings to "false" memory. This caveat to the interpretation should be discussed. - The authors interpret the age-related increase in perceptual false recognition as being caused specifically by false recollection---being accompanied by a recollective experience rather than feelings of familiarity due to semantic knowledge---(e.g. l. 219-221 or 457-459) because the analysed "remember" responses follow the false recognition pattern. To validly make claim that recollection is selectively driving the effects, the authors would need to show that the same pattern is absent in know responses. Moreover, in the analysis of false recollection in Experiment 1 the ANOVA is followed up by post hoc tests of age effects for each condition. However, it appears that, contrary to all other analyses, the authors do not adjust the alpha level for multiple comparisons (alpha = .017). Had they done so, the age-effect in the font only condition would not reach statistical significance. This would further weaken the claim that the effect is driven by recollection. - After correct adjustment of false recognition rates in Experiment 2, the group x condition interaction is no longer significant and the evidence for the interaction effect is anecdotal. Hence, the interpretation of differences between subsequently significant and non-significant post hoc tests of age effects per condition is questionable. Similarly, in the subsequent across-experiment analysis the authors report extreme evidence for the group x condition interaction but do not report whether they found evidence against a three-way interaction with experiment. Given the ambiguous results of Experiment 2 such an interaction would call into question whether the group x condition interaction was indeed replicated. Additional comments: - We appreciate the authors' sharing of raw data and stimulus materials. - The authors may consider moving lines 144-167 to a data-analysis subsection of 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: THE EARLY MIOCENE BALAENID MORENOCETUS PARVUS FROM PATAGONIA (ARGENTINA) AND THE EVOLUTION OF RIGHT WHALES Review round: 1 Reviewer: 1

Basic reporting: Authors' English is not adequate. The text is rich in syntax errors and misspellings and needs a profound revision by a native speaker. Use of commas and abbreviations is not consistent, names of authors cited in the literature are often misspelled. Editorial and linguistic revisions are highly needed. No comment. No comment. The article is self-contained but some of the results cannot be accepted unless significant revisions of the methods. Suggested revisions are provided in the comments to the authors. Experimental design: No comment. No comment. Problematic use of published methods to infer total body length in Balaenidae. Characters used for orientation of the skull insufficiently described. Suggested improvements are providedin the comments to the authors. Method used for orientation of skull insufficiently described and impossible to replicate (acute angle not defined; posterior wall of nasal fossa not defined). Suggested improvements are provided in the comments to the authors. Validity of the findings: Good validity as far as the anatomical description of Morenocetus parvus is concerned. Phylogenetic analysis must be supplemented with information about the authors' hypotheses of character evolution used to order a high number of character states. Body size reconstruction inadequate based on problematic use of the cited methodology. Suggested improvements are provided in the comments to the authors. No comment. Conclusion are well stated based on the methods used but such methods must be discussed and revised as far as body size reconstruction and phylogenetic analysis are concerned. Improvements are related to changes in the methods as suggested in the comments to the authors. No comment. Additional comments: This manuscript includes the re-description of an important fossil balaenid, that is Morenocetus parvus. The anatomical description is generally good, as only minor changes are needed to better explain the concepts presented by the authors. The organization of the manuscript is substantially good and the text does not need to be shortened, I only suggest moving the Comparative Skull Analysis before the Phylogenetic Analysis as it provides evidence then used to support the nodes found in the cladograms. The fossil described is important as it represents the oldest record of Balaenidae whose original description is from 1926 and is inadequate for being used in modern phylogenetic works. Unfortunately, the manuscript shows several problems and, in my opinion, cannot be accepted for publication in its present form. I list such problems below and then discuss all of them: 1) Insufficient description of the characters used for the orientation of the skull; 2) Many character states used in the phylogenetic analysis are ordered but no explanation is provided suggesting that the authors had preconceived hypotheses of character evolution; 3) The equation used for body size reconstruction of Balaenidae was not developed for such a group thus results are expected to be wrong. 4) The English is not adequate and needs a thorough revision as it shows too many syntax errors and misspellings that, sometimes, make understanding difficult. The English needs a thorough revision by a native speaker. Please, check verbs, their accordance with subjects; check commas in references; be consistent in your way to cite literature; check authors’ names (e.g., Kellogg includes two g). Please, do check also in the supplementary information files. In its current form, the English is highly incorrect. For example, on p. 8 lines 46 and following: Balaenids has been consider a key group in understanding of baleen whales evolution, because is the oldest surviving lineage of crown Mysticeti, as suggested by their early… This sentence must be changed into the following (or an equivalent): Balaenids have been considered a key group in understanding baleen whale evolution because they represent the oldest surviving lineage of crown Mysticeti as suggested by their early Materials It is not clear why the referred specimen is not assigned to Morenocetus: is your decision based only on age assessment? Your decision must be based on morphology as you cannot be sure about species longevity for Morenocetus and you do not provide a quantitative assessment of the different age of the referred specimen. If it is not from Miocene, what is its age? How can you be sure about the fact that the observed differences in Nitrogen and collagen content exclude a Miocene age? Miocene spans from 23 to 5.3 million years ago: are the differences you found indicative of a difference in age of more than 15 million years? Methods Physical maturity: the study by Walsh & Berta (2011) was based on balaenopterids and it is not clear whether the chronology of suture closure that they detected on balaenopterids may be directly applied to Balaenidae. They applied to Balaenidae but, without a study of balaenid embryos an fetuses, their application to Balaenidae could be wrong. I strongly recommend authors to cite this problem in the direct application of Walsh & Berta (2011) method to Balaenidae. Orientation of skull: Character 1 is not clear to me. What does it means that the posterior portion of the nasal fossa is perpendicular to the transverse axis of the skull? In lateral view? In dorsal view? Do you mean the anterior end of the nasal bones in dorsal view? Or do you mean the fundus of the nasal fossa (formed by ethmoturbinates) according to Mead & Fordyce (2009)? Character 2 depends on the whole orientation of the skull and may be a character typical of Eubalaena australis. How can use such a character for determining the orientation of different balaenid species? Character 3 must be defined more clearly. You must provide a value in degrees of the acute angle otherwise your character does not work. Acute angle is an angle included between 1° and 89°: please specify. Is Buono’s personal observation based on dissection? In this case, it is fundamental to understand your methodology otherwise character 3 cannot be accepted. Phylogenetic analysis: The use of ordered characters suggests that the authors have an idea of how such characters evolved. In many papers, authors do not explain why they decided to treat some characters as ordered. I think that this should be avoided because it seems to me a way to get optimized phylogenetic searches based on preconceived hypotheses of character evolution. In my opinion, authors must explain why they treated characters 1, 4, 5, 25, 35, 37, 41, 58, 79, 86, 99, 104, 107, 114, 121, 172, 176, 182, 186, 204, 212, 226, 229, 231, 252 as ordered. This is a high number of ordered characters. Moreover, the authors use terms not described by their reference (Mead & Fordyce 2009): as an example, medial lobe of the tympanic bulla is not described there. Please, refer to the literature you cited or cite the relevant literature where the terms were defined. Body size reconstruction: Pyenson & Sponberg (2011) developed the equation cited by the authors for stem Mysticeti that, in their work, included baleen whale species not belonging to crown Mysticeti. This means that the formula cannot be used to predict body size of Morenocetus, Balenella brachyrhynus, Balaena montalionis, Balaenula astensis and Eubalaena shinshuensis. The authors must remove all the parts of the manuscript related to this research and use different methods as the results are not based on the appropriate methodology. Geological setting It would be better to define authors’ concept of early Miocene. I guess that they mean Aquitanian-to-Burdigalian but more detail would greatly help in understanding. The authors cite U-Pb and Sr-Sr absolute datings: please cite the dates in million years to clarify the results of these analyses. A clear specification of the age of morenocetus parvus would be greatly helpful. I strongly recommend authors to change the generic ‘early Miocene’ date into a more detailed age horizon (e.g., Aquitanian, Langhian). Emended diagnosis If the total length was estimated based on the equation cited in the Methods section, it must be removed as the equation is not appropriate for the reconstruction (see above). Is ‘the narrow exposition of the squamosal’ observed in dorsal or lateral view? I suggest changing exposition into exposure. Bisconti (2000) published a redescription of Balaenula astensis showing what he called ‘lateral squamosal crest’ that corresponds to the supramastoid crest of Mead & Fordyce (2009). The authors stated that the supramastoid crest is absent in Balaenula astensis but this statement may be easily questioned. It would be better to state that in Balaenula astensis the supramastoid crest is reduced. The same character should be reviewed and changed accordingly in the character list in the Supplementary information file. Authors must check the English because their text is rich in syntax errors. Description Preservation: The authors use the term “corroded” which refers to chemical attack to the bone. Is that the correct term to be used here? If so, which kind of chemical attack occurred? Physical maturity: check the English. Rephrase the very long last sentence to help understanding. Supraoccipital: the references to the stages of suture fusion described by Welsh & Berta (2011) are for balaenopteroids. The authors must state that. As an example, on p. 23 line 388 they should write what follows: (corresponding to stage SR4 of balaenopteroids). Basisphenoid-presphenoid: on p. 25 line 448, the authors used abbreviations to indicate length and width. I recommend writing length and width as abbreviations are not defined in the previous lines or in the Methods section. Pterygoid: authors used ~ or ≈ inconsistently throughout the manuscript. I recommend being consistent. Frontal: p. 31 line 588: how can the authors explain the presence of the rugose surface on the orbit (a character of calves) in an adult or subadult individual? Periotic: p. 34 line 648: what about the tractus spiralis foraminosus? Phylogenetic analysis Check the English carefully. P. 35 line 663: please, define the posterior border of the zygomatic process (of the squamosal, I suppose). P. 37 line 708: note that in Figs 2 and 3 of Bisconti (2000), the narial process of the frontal of Balaenula astensis is absent thus it is impossible to accept it as a synapomorphy of the clade including Balaenula astensis, Balaenella, balaena and Eubalaena. Moreover, it the presence of such a process is questionable in Eubalaena australis based on several illustrations. The analysis results also in the paraphyly of Balaenopteridae as Eschrichtiidae is included within it. I suggest moving the Comparative skull anatomy before the Phylogenetic analysis as it provides evidence used to code character states and, in the end, for supporting nodes in the cladograms. Discussion The authors make comparisons to an unpublished skull (MPL 5-21) and use characters of such a skull as evidence against the monophyly of Balaenoidea. However, they must illustrate what they are referring to otherwise their evidence cannot be accepted. I recommend illustrating the characters they are describing in the text or remove the text about MPL 5-21 to avoid ambiguity. I must note that the character under discussion in this paragraph is related to the development of the coronoid process of the dentary. It must be remarked that the use and codings of ontogenetic characters should be carefully discussed as in many cases may be problematic. Panchen (1994 in Hall, Homoplasy, Academic Press) strongly recommend to use adult morphologies to establish homology and cites authoritative references. I agree that ontogenetic data may be used but only in cases in which complete developmental paths are known but this is not the case of Balaenidae and Neobalaenidae. Orientation of the skull: I recommend to remove this text unless the characters discussed in the Methods are better defined. On p. 43 the authors use the italic for a title. Is this correct? P. 43 line 866: anterior process of the periotic? Please, specify to which bone belong the characters mentioned in this paragraph. Moreover, this whole paragraph is to be carefully revised, as the English is inadequate and rich in misspellings and wrong syntax. P. 46 line 918 and followings: Pyenson & Sponberg (2011) applied their methodology to the reconstruction of two extinct cetaceans including one balaenopterid (Balaenoptera siberi). In their paper, they are honest and clear in stating that the bizygomatic width overestimated the total length of B. siberi and that corrections are to be made in order to obtain the true length of the specimen under discussion. In this sense, the methods of Pyenson & Sponberg (2011) must be used carefully and with full complement of statistical corrections, they suggested. Finally, they developed specific regression equations to predict the total body length of specific clades of mysticetes: stem mysticetes and Balaenopteroidea. They did not develop specific equations for Balaenidae and, judging from their Fig. 3, stem mysticetes do not include Balaenidae. For these reasons, the predicted body sizes of the present manuscript cannot be accepted. Figures Figure 4. The supraoccipital fossa should be named external occipital fossa according to the text. Figure 5. The lateral border of the exoccipital seems wrongly placed. In fact, in Fig. 5A (right part), such a border corresponds to the lateral edge of the sternomastoid fossa which is not the case, in my opinion. A thinner line located more dorsally seems to better correspond to the lateral border of the exoccipital. Moreover, it is possible to indicate the posterior process exposure in the right part of Fig. 5A. In Fig. 5B the authors should indicate the parietal-frontal suture and the squamosal-exoccipital suture which are clearly visible. Figure 6B. The authors should indicate the parietal-frontal suture. The lateral border of the exoccipital indicated in this figure does not agree with that in Fig. 5. Please, decide and correct. Figure 7. Postglenoid must be changed into postglenoid process. Figure 11. Proximal opening of the facial canal must be changed into endocranial opening of the facial canal according to Mead & Fordyce (2009) which is the reference cited by the authors. Figure 13. In the reconstruction of the skull of Balaenella brachyrhynus the shape of the premaxilla is different from the photographic representation provided in the original description. In the latter, there is a sharp edge between the anterior border of the narial fossa in lateral view and the portion of the rostrum that is anterior to such an edge has a different orientation. In the authors’ reconstruction, the horizontal portion of the premaxilla is too long if compared to the shape of the premaxilla of the original description and the change in orientation of the premaxilla seems not as marked as in the original description. I recommend the authors to provide a better reconstruction of Balaenella brachyrhynus showing the right details of the rostrum. From a different viewpoint, I would recommend the authors to remove this illustration unless they provide better-grounded characters for getting correct skull orientations for Balaenidae (see above).

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

Title: THE EARLY MIOCENE BALAENID MORENOCETUS PARVUS FROM PATAGONIA (ARGENTINA) AND THE EVOLUTION OF RIGHT WHALES Review round: 1 Reviewer: 2

Basic reporting: See below in general comments. Experimental design: See below in general comments. Validity of the findings: See below in general comments. Additional comments: I am very pleased to see this manuscript. A more proper description and thorough analysis of Morenocetus parvus was long overdue. This manuscript does both things, which makes this a much needed contribution to our understanding of marine mammal diversity from South America, and the evolutionary history of balaenids. This work will no doubt be of interest to paleontologist, neontologist as well as anyone interested in the evolutionary history of baleen whales. The manuscript is generally well written and I commend the authors on the high quality images, illustrations and analysis. However, I do have some minor concerns that are outlined in detail below and that I hope the authors take into consideration. Specific comments: Lines 87-89: I suggest changing the beginning of this sentence from “In latest Bisconti’s (2005) analysis…” to “In the latest analysis (Bisconti, 2005)…” Lines 182-184: I suggest the authors to be cautious when using the implied weight method as it may not necessarily result in the best topology. See the papers by Congreve & Lamsdell (2016) and Puttick et al. (2017) where they discuss the inconsistencies and lack of accuracy of using implied weights. For example see the different position of Mauicetus parki in Fig. 12B, as the sister taxon of Plicogulae and not as a balaenopteroid as in previous analyses (e.g. Marx & Fordyce, 2015). See also Tsai & Fordyce (2016) which show similar discrepancies with this taxon in the equal vs implied weight results. Congreve, C.R. &. J.C. Lamsdell. 2016. Implied weighthing and its utility in paleontological datasets: a study using modeled phylogenetic matrices. Paleontology 59:447-462. Puttick, M.N., J.E. O’Reilly, A.R. Tanner, J.F. Fleming, J. Clark, L. Holloway, J. Lozano-Fernandez, L.A. Parry, J.E. Tarver, D. Pisani & P.C.J. Donoghue. 2017. Uncertain-tree: discriminating among competing approaches to the phylogenetic analysis of phenotype data. Proceedings of the Royal Society B 284:20162290. Line 275: Is there a full name for Cremonesi? Line 290: I suggest changing “… and lateral edges of supraoccipital straight.” to “… straight lateral edges of the supraoccipital.” Line 312: correct typo here “… having ths supraorbital…” Line 315: “thicked orbital rim…” did the authors meant “thicker orbital rim…” Lines 379-382: In figure 4B the “external occipital fossa” is labeled as “Supraoccipital fossa” please update the figure (or text) so that they are consistent. Lines 567-569: There seems to be a discrepancy with the first half of this sentence “The supraorbital process is short transversely and long anteroposteriorly:” however, it looks more like the supraorbital process is long transversely, and short anteroposteriorly, not the other way around. Line 770: period missing after Eubalaena spp. Line 782: add period after Balaenula sp. and only italize genus Lines 792-793: separate “Discussion…” section from preceding paragraph. Line 876: remove space between Chaeomysticeti and comma Line 887: remove space between “stasis/” and “morphological” Line 893: I suggest changing “Mysticetes” to “mysticetes” Line 912: montalionis is not completely italized Line 914: typo at end of line “extinc” Line 915: typo, change “diferent” to “different” Lines 927-930: see also Slater et al. 2017

You are one of the reviewers, your task is to write a review for the article. You will be 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 EARLY MIOCENE BALAENID MORENOCETUS PARVUS FROM PATAGONIA (ARGENTINA) AND THE EVOLUTION OF RIGHT WHALES Review round: 1 Reviewer: 3

Basic reporting: Some minor grammatical and spelling issues are present and the text would benefit from some language editing in places. Experimental design: Se below. Validity of the findings: See below. Additional comments: This manuscript by Buono et al. provides a long-awaited redescription of the early right whale Morenocetus parvus from the lower Miocene of Argentina. Right whales have often been interpreted as being the most conservative extant baleen whales owing to this species and its early occurrence, predating most other families by nearly 10 My. This paper includes a well-written, detailed, and long overdue morphological description of Morenocetus as well as a reconsideration of the history of certain hallmark anatomical features of balaenids and a phylogenetic analysis. I have no major concerns with the manuscript and think that it deserves publication. I have a list of minor comments below, and suggest minor revision. Some English language editing is necessary for some sections - but note that with a few exceptions, the language does not seem to impact clarity. Congratulations to Monica and colleagues on getting this manuscript completed! Kind regards, R.W. Boessenecker, Ph.D. 46: change to "Balaenids have been considered" 51: replace cf with "in comparison to" 53: Extremely large head: perhaps give some brief statements on cranial length as a percentage of body length 54: length of baleen plates in meters? 69: confirmed by whom? In the spirit of thoroughness, a few citations for previous phylogenetic analyses to have included Morenocetus would help. 120: is MLP 5-21 Pleistocene or Holocene instead? Be specific. 135-136: "to avoid its destruction" do the authors mean that removing the periotic would cause irreparable damage, either to the earbone, or the skull? 141: delete the comma after ’specimens' 142: change "based" to "description focused" 171: "list of characters was modified..." how? how many characters were removed? how many added? why? A separate section of text outlining and justifying these changes is necessary. 199: could BZW be estimated and scaled based on ratios of BZW to exoccipital width in other balaenids? Geologic setting: I thank the authors for including an informative stratigraphic column and not shying away from geological context. 280: narrow exposure; report as a % of exoccipital width? 281: change deeply to deep 288: quantify this as a % of postorbital or exoccipital width. 292: change to "crest-like parieto-squamosal suture" 297: narial process of frontal 299: lateral tuberosity of the periotic? please clarify 304: lateral hypertrophy of the body of the periotic? 307: supramastoid crest extending all the way to tip of zygomatic? please be more specific about the condition in Eubalaena and Balaena. 312: Differs; "ths" to "the" 313: frontal gradually sloping away ventrolaterally 314: half of distance: unclear, please clarify. 373: does not reach the level of the preorbital process? 386: approximately 388: delete the extra comma after 774. 442: Which neck flexor muscles? Be specific - Schulte 1916 might be a good enough reference. 466: hamuli 473: in comparison to what other taxon? 481: I think there's an extra space between 774 and the comma 498: alisphenoid 517: I notice that the authors use both "periotic fossa" and the problematic term cranial hiatus. According to Mead and Fordyce 2009 the latter term is poorly defined and they introduced the term periotic fossa to replace it; are the authors using it in the correct sense, or as the pit that the periotic sits within? 527: Should an unpublished thesis be cited here? I personally don't think so. 562: I strongly think that the "frontal ridge" proposed here is the orbitotemporal crest, which has shifted anteriorly within Crown Mysticeti. Many stem balaenopteroids like Parietobalaena and Pelocetus have an orbitotemporal crest shifted anteriorly on the frontal. It's been a while since I have read relevant literature on muscle insertions on mysticete crania, but I recall that a dissection of Caperea prior to the 2013 Marine Mammal conference in Dunedin revealed that the temporalis inserted upon the entire dorsal surface of the supraorbital process of the frontal, terminating at a low ridge at the same position as what I have identified as the orbitotemporal crest. 577: change to "in extant balaenids" 586: dorsally concave? 615: change in to "to be" 626: obtuse angle - please quantify 684-685: suprameatal part of periotic not hypertrophied - Morenocetus appears to still have quite a large body of the periotic. This should be quantified, perhaps as a maximum transverse width expressed as a percentage of pars cochlearis width. Relative to an eomysticetid, the body of the periotic is enormous. 687: change specialized to derived? 718: unclear 812: A relevant citation that should probably be cited here for correct skull orientation as far as interpreting skull morphology is Yamada et al. 2006: Memoirs of the National Science Museum, Tokyo 44:1-10. 865: change to "exhibits" 876: derivate to derived 878: change to "hypertrophied body of periotic" 890: change to "major mysticete clades" 893-894: "this..." please rephrase. 894: Aquitanian or Burdigalian? 897-898: Unclear. Figure 13 is a picture of skulls; does this refer to Fig. 14? What is the taxonomic inclusion of Balaenopteroidea? Which balaenopteroids are present in the Chattian and Aquitanian? Some citations of specific examples should be provided in the text since these questions arise from lack of clarity in the figure and caption. 900: occur 942: The authors should include a table of Aquitanian and Burdigalian mysticetes, summarizing the lower Miocene record. 950: citations needed. 952: Paleobiology database is not a reasonable source to cite. Please cite published articles. 946-952: A recent paper by RE Fordyce and myself reports an eomysticetid from the lowermost Miocene of NZ and makes some of the same statements provided here in the "Pending Issues" section, and may be a worthwhile citation. RW Boessenecker and RE Fordyce, 2017. Cosmopolitanism and Miocene survival of Eomysticetidae (Cetacea: Mysticeti) revealed by new fossils from New Zealand. New Zealand Journal of Geology and Geophysics DOI: 10.1080/00288306.2017.1300176

You are one of the reviewers, your task is to write a review for the article. You will be 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 EARLY MIOCENE BALAENID MORENOCETUS PARVUS FROM PATAGONIA (ARGENTINA) AND THE EVOLUTION OF RIGHT WHALES Review round: 2 Reviewer: 1

Basic reporting: Professional English used throughout. Methods and discussion not completely unambiguous. Important changes to be made as in the General comments for the authors. Literature references are mainly correct. Some additional citations needed as suggested . Field background and context are provided correctly in most of the cases but with some necessary changes to be made. The article structure is OK. Some problems with a few figs as suggested in the General comments for the authors. The paper is self-contained. Relationships between hypotheses and results to be revised by the authors. Experimental design: Original primary research within aims and scope of the journal. Research question well defined, relevant and meaningful. The paper fills an important knowledge gap as a redescription of Morenocetus is very welcome task in the field. The investigation is mostly rigorous but fails in a number of points as discussed in the General comments to the authors. Methods are described with sufficient detail about some aspects. Other aspects need clarifications. Validity of the findings: The description of Morenocetus is surely an important and valid finding. Anatomical data are robust and controlled. Other aspects need clarification. See General comments to the authors. Conclusion are well stated as far as anatomy and phylogeny are concerned. Other aspects (i.e., reconstruction of body size) to be clarified and discussed in better way. Additional comments: I think that the a new description of Morenocetus parvus would be very welcome. All the students of mysticete phylogenetics would be happy to have such a description made in modern terms and with current criteria. Unfortunately, there is a number of issues that have to be made clear or that need additional scientific research. I will be more explicit below. Moreover, the newly added texts required a new review round that resulted in the need for further clarification in many points. I am sorry if this will delay the publication of this manuscript but I honestly feel the changes I request are really necessary. I will try to be highly specific in my review and will provide also illustrations to better express my thoughts. Review of “The early Miocene balaenid Morenocetus parvus from Patagonia (Argentina) and the evolution of right whales” by Mónica R. Buono, Marta S. Fernández, Mario A. Cozzuol, Jose I. Cuitiño, and Erich M. G. Fitzgerald The manuscript is now improved as far as the English is concerned and the new details added provide better quality and clarity. There are still some parts of the manuscript that, in my opinion, need to be changed in order to better present the scientific content and to ameliorate readers’ understanding. I will discuss these below. The line numbers are based on the Word file with tracked changes. 1. A new paper was recently published dealing with a revision of “Balaena” belgica that established the new species Eubalaena ianitrix (Bisconti et al. 2017, PeerJ). The authors cited such a paper several times in the text but they still use “Balaena” belgica in their text. Unless they can provide a criticism to the taxonomy of Bisconti et al. (2017), they must change “Balaena” belgica into Eubalaena ianitrix throughout the text and in Fig. 12. 2. In the differential diagnosis, the authors must make comparisons to Eubalaena ianitrix. 3. In Fig. 12, the authors must change Eschrichtius gastaldii into Eschrichtioides gastaldii unless they can justify their assignment to Eschrichtius. 4. In Fig. 13, the premaxilla of Balaenella is still drawn imprecisely. I attach an image for giving the authors an idea of what I am saying (Fig. R1). Moreover, it is not clear how they reconstructed the length of the rostrum of this species given that its anteriormost portion is not preserved. Finally, the dorsoventral diameter of the maxilla in lateral view approaching the lateral process is undersized with respect to the photographs published in the original description. Based on that, the reconstruction of this skull is not done thoroughly, in my opinion, and should be done more precisely or removed. 5. Line 90. It is not clear to me why the authors did not cite also Bisconti (2000, Palaeontographia Italica, 87: 37-66), Churchill et al. (2012, Marine Mammal Science, 28: 497-521), and Bisconti et al. (2017, PeerJ) in the comprehensive analyses of mysticete phylogeny focusing on balaenids. 6. Lines 171-175. To be honest, I did not find any reference to the specimens you dissected or analysed to understand the pattern of sutural closure in balaenids in the Supplementary Information. You should provide (at least in the form of a table in the Supplementary Information) the specimens you analysed (species, repository and number), their ages and the pattern of sutural closure observed in each of them. I note that Welsh and Berta dedicated a whole paper to this subject while you are discussing it very quickly and without providing the necessary information. Otherwise, we simply have to trust you. We could do that but it is not a scientific procedure in a strict sense. 7. Line 206. In the revised text, the authors write about “dissection of extant balaenids”. What balaenids? From the manuscript, it seems that only specimens of Eubalaena australis were dissected. Is this correct? What about the other species of Eubalaena? What about Balaena mysticetus? It seems that only character 1 was tested with Balaena mysticetus. If the authors do not have data about these species, they should carefully apply their method to the fossil specimens as a significant taxonomic sample of living balaenids was not tested. I strongly recommend to use a more dubitative textual communication approach undermining that your results (regarding the orientation of the skull) are acceptable if the landmarks can be applied to all the living balaenids at least. Unfortunately, currently that is not known and the resulting orientations may be not as precise as desirable. I guess that this point will be the focus of criticisms by other authors if published as is, especially because the authors are very bold about their own results. 8. Line 211. accurate reconstruction of the branching order of the sister groups may influence the reconstruction of character state evolution. The authors have to be aware that their work results in a hypothesis of phylogenetic relationships of Mysticeti with focus on Balaenidae. They decided to order some characters thus suggesting that a number of evolutionary trajectories are known (which should be demonstrated). Thus they accept that Balaenopteridae and Eschrichtiidae are merged together. They have to remark this in the text. 9. Line 226. It is a pity that the present authors pruned the original dataset as Wagner (2000, Evolution, 54: 365-386) and Heath et al. (2008, Journal of Systematics and Evolution, 46: 239-257) convincingly argued that more taxa are highly useful in the reconstruction of phylogenies in groups whose diversity is mainly formed by extinct taxa. Including more taxa should increase the probability to retrieve the correct phylogeny. I strongly suggest to include the whole dataset by McGowen et al. (2008) and Marx and Fordyce (2015). 10. Line 352. Actually, there are three different specimens of Balaenula that have been assigned to two different species and to an unnamed taxon. These are Balaenula balaenopsis, Balaenula astensis and the Balaenula sp. from Japan. As the taxonomy of these three specimens is debated, in the diagnosis, the authors should state clearly which is the Balaenula they are comparing to Morenocetus or they should state which definion of Balaenula they are referring to. 11. Line 665. Change disecction in dissection. 12. In the comparative skull morphology, comparisons to Caperea and Miocaperea would be useful because Morenocetus resembles these species closely in the high position of the orbit, in the lateral exposure of the posterior process of the periotic and in some aspects of the supraoccipital morphology (especially the anterior border). 13. Line 888. The authors shows that the narial process of the frontal is present also in Balaenula astensis and use this character as a synapomorphy of a larger clade. I attach here a figure illustrating the different morphologies of the narial process of the frontal in Balaenula astensis, Balaenella brachyrhynus and Balaena mysticetus supporting the view that these three taxa exhibit at least two different morphologies of this character (Fig. R2). Based on that, I suggest that the authors have to subdivide the character about the narial process into two different characters: 1) presence/absence of the narial process; 2) relative development of the narial process (short/elongate). In this way, they can correctly represent the morphological diversity of this feature in balaenids. 14. Line 902. To my knowledge, based on direct examination of several specimens, the crest at the parietal-squamosal suture is not present in Eubalaena glacialis and Balaenula astensis. Is this proposed synapomorphy a result of character optimization made by the computer program or the authors actually found such a crest in specimens of these species? Being positive the second option, please, cite repository and numbers of the specimens because that would be very useful to other authors. 15. Line 1127. Actually, Balaenella is not a historical form as it was described for the first time in 2005. Balaenula astensis received two different revisions in 2000 and 2003 based on computer-assisted phylogenetic analyses and morphological re-descriptions in modern terms. It is evident that the authors do not agree with these descriptions and revisions, and this is OK, of course. However, in my opinion, they should re-phrase this last sentence in order to avoid to be in error by considering Balaenella a historical form. I suggest them to mention Balaenula balaenopsis and Balaena primigenia, as examples of historical forms. 16. In the Supplementary Information, List of the specimens studied for the anatomical and phylogenetic analysis, please, note that Balaenula astensis preserves both periotics and both tympanic bullae (Bisconti 2003, Cranium 20: 9-50). 17. The same list is presented in an inconsistent way: some specimens are associated to a list of materials while others are merely cited. A consistently compiled list would be more useful to understand what exactly you examined. 18. Literature cited in Supplementary Information not in alphabetical order. The authors have to write it in alphabetical order. 19. The last point I want to discuss is about the reconstruction of the body size of Morenocetus and, more generally, the way the authors discuss their work and the work of other authors. In fact, in my opinion, this is a case in which the discussion is made in a misleading way and gives the readers ambiguous information. The authors insist that the method developed by Pyenson and Sponberg (2011) is the best available method at the moment. This is certainly true about the reconstruction of body size in basal mysticetes and balaenopteroids; this is certainly not true about balaenids. In fact, such a method consists in an regression equation developed from a large dataset of mysticete measurements that, unfortunately, does not include a single balaenid specimen. As a wealth of published literature recognizes that the body proportions of balaenids are different from the other mysticete families, it is obvious that one should not expect that such an equation can retrieve accurate results for balaenids. In the introduction of the present manuscript (line 54 in the newly added text), the authors acknowledge that one of the characterizing features of living balaenids is their body proportions in which the head is about one-third of the total body length. This character is not observed in any other mysticete. The head length:body length ratio is based on a large number of measurements available in early literature and summarized in more recent papers (Cummings 1985, Handbooks of marine mammals, vol. 3, pp. 275-304, Academic Press; Reeves and Leatherwood 1985, Handbooks of marine mammals, vol. 3, pp. 305-344, Academic Press) and is usually accepted as a matter of fact. I appreciate that the authors of the present manuscript recognized that the Pyenson and Sponberg (2011) equation was not specifically developed for balaenids (line 247). However, I strongly disagree with the statement in the rebuttal letter in which the authors state that “there is no basis for assuming that ‘the results are expected to be wrong’”. In fact, the results are expected to be wrong because: (1) living balaenids (and we can measure only complete skeletons from the living species) have different head:body length ratio from other mysticetes and (2) the Pyenson and Sponberg equation is not based on balaenids. Thus, the Pyenson and Sponberg equation provides results compatible with an expected head:body length ratio different from that of balaenids. In the rebuttal letter, the authors write also that “this equation was recently applied with success to Balaenidae in a major synthesis of mysticete body size evolution (Slater, Goldbogen and Pyenson 2017)”. However, Slater et al. (2017) did not provide the original dataset of the measurements they used to base the equation used in their paper; they provided only a table with the estimated body lengths of a number of mysticetes. Therefore, it is not possible to know if their equation is based on a dataset including balaenids or not. In this sense, their reconstructions of the body sizes of balaenids is hard to accept as presented in such a paper. In the end, the use of that equation in other papers is not per se a source of support for the validity of the equation itself. In my opinion, the authors may publish their results in the way they want bearing in mind that this means that their paper will be prone to strong criticism in future publications by other authors. Another aspect of the text about the reconstruction of Morenocetus body size is around the discussion of the paper by Bisconti et al. (2017). From the present manuscript, it seems that Bisconti et al. (2017) used alternative methods to the Pyenson and Sponberg equation and that they accepted the results of these methods without discussion. This does not correspond to truth. If the authors read carefully the paper, they can find that Bisconti et al. (2017) used several methods for inferring the body length of Eubalaena ianitrix including the equation of Pyenson and Sponberg (2011). Moreover, Bisconti et al. (2017) discussed pros and cons of each method and concluded that none of the available methods is exempt from problems. Bisconti et al. (2017) acknowledged that their reconstruction of the body mass was probably wrong and discussed their results in a broad sense. The harsh criticism to the paper of Bisconti et al. (2017) made by the present authors does not add anything to the discussion of the methodologies about body size reconstruction in fossil balaenids and misleads the readers by ignoring the real content of the work published by Bisconti et al. (2017). I strongly recommend to rephrase the discussion of the Bisconti et al. (2017) work from line 1067 onwards in order to correctly represent the real content of that paper. It is also possible that the application of the Pyenson and Sponberg (2011) equation by Bisconti et al. (2017) was done better than in the present manuscript as Bisconti et al. (2017) made the corrections suggested by Pyenson and Sponberg (2011), i.e. they reduced the results by 47% and 37% in order to get a range of body size variation, while the present authors present only one result. Is the value obtained by the present authors corrected as suggested by Pyenson and Sponberg or not? To be honest, I was unable to replicate the results of the present authors when I checked the results presented in the present manuscript. In fact, the application of the equation of Pyenson and Sponberg to a bizygomatic width of 570 mm allowed me to find a total body length of 7.46 m. After the changes suggested by Pyenson and Sponberg (i.e., reduction of 47% and 37% of the initial result), I found two body lengths for Morenocetus: 3.96 m and 4.7 m that are different from the results of the present manuscript (5.2 m). The application of the same equation to a bizygomatic width of 620 mm (specimen MLP 5-15) resulted in a total body length of 8.06 m and, after the corrections, in a body length range included between 4.26 and 5.16 m that is different from the result of the present manuscript (5.6 m). In light of these new results, the discrepancies with the Bisconti et al. reconstruction of the body length of Eubalaena ianitrix seems more reasonable: 3.96-4.7 m for Morenocetus and 5-7 m for E. ianitrix which is almost twice of Morenocetus. By the way, I noticed that the reconstructions of the body lengths of other fossil balaenids made by the present authors by using the equation of Pyenson and Sponberg differ from the published reconstructions made by Slater et al. (2017, see their supplementary information). In fact, in the present manuscript, the total body length of Balaenella brachyrhynus is 7.9 m while Slater et al. found that this whale was 7.2 m in length; Balaena montalionis is 7.2 m in this manuscript but the reconstruction made by Slater et al. found this whale being 4.87 m. Moreover, judging from published measurements, Balaenella brachyrhynus seems smaller than Balaenula astensis. In fact, the supraoccipital length of B. brachyrhynus is 310 mm (Bisconti 2005) and that of Balaenula astensis is 350 mm; the maximum diameter of the foramen magnum of Balaenella brachyrhynus is 64 mm and that of Balaenula astensis is 75 mm. How can Balaenella brachyrhynus be longer than Balaenula astensis in total body length? In fact, in the reconstruction of the present manuscript, the total body length of Balaenula astensis is 6.6 m and that of Balaenella brachyrhynus is 7.9 m. I cannot understand these discrepancies and strongly recommend the present authors to calculate again the body length of Morenocetus and other balaenid species and, eventually, to discuss these discrepancies in the Discussion section. In my opinion, due to the above observations, the manuscript is still in need of a serious revision. Figures attached to this review are provided in a separate file.

You are one of the reviewers, your task is to write a review for the article. You will be 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 EARLY MIOCENE BALAENID MORENOCETUS PARVUS FROM PATAGONIA (ARGENTINA) AND THE EVOLUTION OF RIGHT WHALES Review round: 2 Reviewer: 2

Basic reporting: See below in general comments. Experimental design: See below in general comments. Validity of the findings: See below in general comments. Additional comments: I am very pleased to see the revised version of this manuscript. The authors have properly addressed all the issues brought up by myself and the other reviewers. As stated in the first review of this work, a more proper description and thorough analysis of Morenocetus parvus was long overdue. This manuscript does both things, which makes this a much-needed contribution to our understanding of marine mammal diversity from South America, and the evolutionary history of Balaenidae. The whole discussion of the orientation of the skull of balaenids is very helpful and will hopefully be followed by anyone else working on this group. The authors did an outstanding job in the description of the specimens and related figures. Furthermore, the detailed discussion on the morphology and relationships of Morenocetus parvus and Balaenidae is helpful and thorough, especially making the tie-in between the text and the character/state used in the phylogenetic analysis. The manuscript is well written and I commend the authors on the high quality images and illustrations (which are extremely helpful and detailed). This work is no doubt of interest to paleontologist, neontologist as well as anyone interested in the evolutionary history of baleen whales.

You are one of the reviewers, your task is to write a review for the article. You will be 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 EARLY MIOCENE BALAENID MORENOCETUS PARVUS FROM PATAGONIA (ARGENTINA) AND THE EVOLUTION OF RIGHT WHALES Review round: 2 Reviewer: 3

Basic reporting: OK Experimental design: OK Validity of the findings: OK Additional comments: The authors have done an excellent job in addressing all of my comments and I recommend acceptance of the paper - and look forward to seeing it in print. Kind regards, R.W. Boessenecker

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

Title: TRANSCRIPTOMIC ANALYSIS IDENTIFIES GENES AND PATHWAYS RELATED TO MYRMECOPHAGY IN THE MALAYAN PANGOLIN (MANIS JAVANICA) Review round: 1 Reviewer: 1

Basic reporting: This manuscript provides a very thorough accounting of the data, methods, and results. The narrative could be improved by more clearly linking the transcriptomics approach with both evolutionary (e.g. comparisons with carnivores) and species management/conservation questions and implications. The manuscript would benefit greatly from the assistance of a native english speaker to ensure correct grammatical usage and to make several parts clearer. There are a few spots where as written, it would appear that the correct meaning is not conveyed. Experimental design: No comment. Validity of the findings: By the nature of the study design, the comparisons of what is observed in ONE pangolin and only a certain number of tissues is compared against databases from multiple species and individuals and settings. Correlations that are highlighted need to done emphasizing that these point to areas where further research might be very useful for theoretical and practical purposes. 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: TRANSCRIPTOMIC ANALYSIS IDENTIFIES GENES AND PATHWAYS RELATED TO MYRMECOPHAGY IN THE MALAYAN PANGOLIN (MANIS JAVANICA) Review round: 1 Reviewer: 2

Basic reporting: This is a very interesting and informative manuscript dealing with the transcriptomics of an unusual and little-studied mammal. In general, the manuscript is fairly well-written, although the English grammar does require some attention and I have tried to point these instances out in the accompanying annotated document. In general, the manuscript is well-composed, although there were numerous instances where statements were made but not supported by a suitable reference. Again, I have tried to highlight these instances in the accompanying document. Experimental design: With specific reference to the study animal, I would like to see more information included on the specimen used. Ideally, the authors should include the mass and/or length of the study animal to give some indication of its age. Also, I would like to see more information on where this individual was sourced, specifically whether it was a captive-bred individual, a wild individual that was maintained in captivity (and if so, how long was it in captivity for?), or a completely wild individual. I believe that the origin of the individual could have a direct effect on the conclusions drawn from this study, as each scenario has different levels and origins of stress, and the diet of the individual (especially if it was sourced from captivity) could also affect the transcriptome classes recorded. These factors also need to be considered when comparing your results to those of Yusoff et al. (2016) – was their study animal also sourced from captivity? Validity of the findings: I would like to see the results of this study compared and contrasted to the study by Yusoff et al. (2016), especially as this is the only other study (to the best of my knowledge) that has investigated transcriptomics in a pangolin species. Despite these studies looking at predominantly different organs (with the exception of the liver, which is common to both studies) I do feel that comparisons can be made, especially with regards to the proportion of transcripts that were assignable to each ontological class, as well as the proportion of transcripts assigned to each molecular function and cellular component class. Additional comments: Please see the accompanying document for more specific comments. This is a very interesting study that will definitely be a valuable addition to the growing body of literature on this threatened species, and I look forward to reviewing a revised version of this manuscript.

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

Title: TRANSCRIPTOMIC ANALYSIS IDENTIFIES GENES AND PATHWAYS RELATED TO MYRMECOPHAGY IN THE MALAYAN PANGOLIN (MANIS JAVANICA) Review round: 1 Reviewer: 3

Basic reporting: Minor grammatical errors occur in the manuscript. Some are listed in the 'General comments,' below. References to other studies that have successfully linked dietary requirements to transcript profiles would be useful, if they exist -- see 'General comments'. Experimental design: The question was well-defined, and meaningful. The experimental design was constrained by the availability of samples -- biological replicates would presumably have been useful, but impossible to obtain. Validity of the findings: In the General comments, I suggest some places where the links between the results and the conclusions ought to be bolstered. This might be achieved with the addition of references to studies in other species, or detailed explanations. Alternatively, greater qualifications might be added to the conclusions addressing uncertainties. Additional comments: The manuscript by Ma et al. describes an analysis of transcriptomes from several tissues of the Malayan pangolin. Previously, no transcriptome has been completed for the salivary glands or intestine of this species. Here they were sequenced with the aim of providing information about the genes involved in myrmecophagy, and learning more about dietary requirements of this species, whose conservation might benefit from ex situ breeding. This motivation is outlined quite well in the introduction. The methods used to perform the transcriptome sequencing and assembly seem to me to be largely sound. I have a number of suggestions for the manuscript, but otherwise think it would make a worthwhile contribution to PeerJ. Overall, my most serious comment is that I was sometimes uncertain that the results justified inferences that were drawn in the discussion. It is an important goal to look for hints about pangolin dietary requirements, but in some cases I think greater clarification, qualification, or references to relevant literature would be helpful. As an example, on Line 373, I found the section on the amino acids to be highly interesting. It suggests that the absence of transcripts for arginine synthesis might mean they are incapable of making this amino acid, so that arginine might be added to pangolin diets in captivity. (An aside: Line 376 seems to contradict this -- "These results suggested that M. javanica may synthesize arginine de novo." Wonder if this shoud say "not synthesise"?) However, I kept wondering, in the case of Arginine, what does the absence of transcripts mean, especially when the experiment was not replicated? How likely is it that transcripts involved in Arginine biosynthesis were present, but not observed due to low abundance, and random chance? Have studies in other species drawn correct (verified) inferences about dietary requirements based on transcriptomic profiling of different tissues? Could the available reference genome be interrogated more carefully to try to confirm the absence of genes, to corroborate this inference? Note, I do not suggest removing comments on this result from the discussion, which seems potentially informative, but it would be good to see it considered a more thoroughly, or qualified more carefully. Moderately important points: Line 145-146: Are there parameter values used for tophat that could be supplied to make the analysis more reproducible? Line 154-157: Little information is provided about the databases, to explain why the transcripts are annotated using all of them. Do they offer complementary insights, or just a greater chance of locating a match for a given transcript that is observed? Line 181-182: It is not clear what the "ratio of the transcripts" refers to... Line 225-228: Terms such as 'cell' (Line 225) and 'cell part' are used without much explanation. For example, does 'cell' mean expressed in the cytosol? Line 334: The analyses of differences in expression between tissues are interesting, but the results probably ought to be interpreted somewhat cautiously, since there is no (technical or biological) replication. Perhaps a qualification could be added to the methods or discussion. Minor corrections / suggestions: Throughout: Mohamad Yusoff et al. 2016 -> Yusoff et al. 2016? From Yusoff et al. 2016: "How to cite this article: Yusoff, A. M. et al. De novo sequencing, assembly and analysis of eight different transcriptomes from the Malayan pangolin. Sci. Rep. 6, 28199; doi: 10.1038/srep28199 (2016)." Line 43 myrmecophage -> myrmecophagy Line 44 protection seems awkward here. Line 48 eutherians and placentals seems redundant.. perhaps "are unique among plancental mammals" Line 92 suggestion: "the last two years" Line 97 "popping up like mushrooms" perhaps too informal?

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

Title: TRANSCRIPTOMIC ANALYSIS IDENTIFIES GENES AND PATHWAYS RELATED TO MYRMECOPHAGY IN THE MALAYAN PANGOLIN (MANIS JAVANICA) Review round: 2 Reviewer: 1

Basic reporting: This manuscript has been greatly improved since its original submission, and the authors are to be commended for this. I found the manuscript to be well-written, unambiguous and for the most part easy reading. Experimental design: The methods and experimental design appear to be appropriate. Validity of the findings: No comment. Additional comments: I have a few minor suggested edits to improve the manuscript, which are detailed below: Abstract Line 43: Suggest deleting “of”. Sentence would read “Our study is the first to investigate the molecular mechanisms underlying myrmecophagy in M. javanica.” Introduction Lines 50 & 51. Gaudin et al. (2009) revised the taxonomy of pangolins, restricted the genus Manis to the four Asian species while assigning the two African tree pangolin species to the genus Phataginus and the two terrestrial African pangolin species to the genus Smutsia. These results have also been subsequently confirmed with molecular techniques (see for example du Toit et al. 2014), and this nomenclature has been adopted by most researchers and taxonomic groups, including the IUCN Pangolin Specialist Group. I therefore suggest that this taxonomy be adopted here as well. [Gaudin, T.J., Emry, R.J. and Wible, J.R., 2009. The phylogeny of living and extinct pangolins (Mammalia, Pholidota) and associated taxa: A morphology based analysis. Journal of Mammalian Evolution 16(4): 235–305.] [Du Toit, Z., Grobler, J.P., Kotzé, A., Jansen, R., Brettschneider, H. and Dalton, D.L., 2014. The complete mitochondrial genome of Temminck's ground pangolin (Smutsia temminckii; Smuts, 1832) and phylogenetic position of the Pholidota (Weber, 1904). Gene 551(1): 49–54] Lines 55–58. Pangolins have been reported feeding on far more than four ant and one termite species, as is reflected in the articles that are cited. The four ant and one termite species listed all originate from the Pietersen et al. 2015 reference. I suggest that this sentence either be revised to reflect the full list of ant and termite species that pangolins are reported to feed on, or more preferably just state that pangolins feed exclusively on ants and termites (and keep the current references). Line 73. Replace “the’ with “a”. The sentence will read “…habitat as a result of deforestation” Results Line 203. Remove “compared with the exons” Line 361. You need to state where this “referred liver” originates from. I suggest including the "Yusoff et al. 2016" reference before “Figure S8”. Line 367. What previous study does this refer to? Include the Yusoff et al. 2016 reference here as well. Discussion & Conclusion Line 378. Insert “the” between “between” and “small”. The sentence will read “between the small intestine and liver.” Line 401. “Our” should not be capitalized. Line 440. Remove the space in “amino -acid” Lines 441 & 442. I suggest combining these two sentences to improve the flow. This can be done by combining the sentences to read “Malayan pangolins are endangered mammals for which captive breeding provides an opportunity to study the molecular mechanisms of myrmecophagy.” Figures Figure 3. Please write the full name (not just the abbreviation) of each tissue type in each of the axis legends. Tables Table 1. Replace “ID” with “Organ” Table 2. Replace “ID” with “Organ”

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

Title: FUNCTION OF JARID2 IN BOVINES DURING EARLY EMBRYONIC DEVELOPMENT Review round: 1 Reviewer: 1

Basic reporting: Please double-check the grammatical mistakes in this paper. Experimental design: Design good Validity of the findings: The paper is concise, well written and contains some interesting findings. Additional comments: In this paper, authors investigated the function of JARID2 during bovine embryonic development. They studied the expression pattern during early embryonic process by using qRT-PCR and immunofluorescence and found that the cleavage rate and blastocyst rate were decreased after small interference mediated knocking down of JARID2, which is maily caused by dysregulation of pluripotent related genes expression in 2-cell stage. Finally, they detected the H3K27me3 level and found its level was significantly decreased in all the early embryonic stage. 1. The authors used double-stranded RNA to knockdown JARID1, did the authors detect the change of mRNA level of JARID1 after knockdown？ 2. Manuscript should be written clearly in uniform line spacing. 3. Please move “how to analyse the fluorescence intensity” in line 257-258 into the M&M section. 4. Please double-check the grammatical mistakes in this 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: FUNCTION OF JARID2 IN BOVINES DURING EARLY EMBRYONIC DEVELOPMENT Review round: 1 Reviewer: 2

Basic reporting: This study identified the expression pattern of JARID2 in bovine oocytes and early embryonic stages and suggested that JARID2 played a key role in early embryonic development by regulating the expression of OCT4, SOX2 and c-myc via the modification of H3K27me3 expression. These results provide new data for the improvement in bovine in vitro embryo culture and understanding the regulation mechanisms in early embryonic development. However some contents need more modifications and explanations. 1. Some common mistakes or different words in English writing. These should be modified. I suggest that authors should check the manuscript carefully and try to avoid any mistakes like these: Line 88 “MII stage”, but “M2 stage” in Fig 1 legend. Line 181, “antibody (Abcom)”, is it “Abcam”? Company name and country should add to the manuscript like Line 161 “siRNA (Gene Pharma, China)”. 2. The Introduction Part. The authors do not have to write the Introduction contents with extra title like “1. DNA methylation” and “2. Histone modification”. I suggest the authors rewrite this small part based on the important factors of histone modification. 3. Fig 2B. The description of Y-axis is missing. In Fig 1B, Fig 4A and Fig 4B, the magnification or standard bar should be provided. 4. The Discussion Part. This part should be more improved. The authors use lots of contents to describe the function of JARID2, H3K9 and H3K27. I think these contents should belong to the Introduction. More discussion like what’s its potential meaning of expression in cell nucleus and the synergy/different effects from this study would attract more readers’ attention. I think contents in line 314-329 are useful. Experimental design: Experimental design is well done. 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: FUNCTION OF JARID2 IN BOVINES DURING EARLY EMBRYONIC DEVELOPMENT Review round: 1 Reviewer: 3

Basic reporting: This manuscript investigated the function of JARID2 during bovine early embryonic development. JARID2 has been shown to be an important epigenetic regulator for embryonic development via histone modification. Although there have been many reports on the function of JARID2 during early embryonic development, the current study systemically examined the expression pattern of JARID2 in bovine and analyzed its possible regulatory roles on the expression of genes important to the maintenance of stem cell. 1. English writing In general, the manuscript is clear, intelligible and professional. However, the writing could be greatly improved if the manuscript could be reviewed by a native English speaker. The followings are some examples or suggestions: A. It would be consistent if the authors could use past tense to present or discuss their data and use present tense to discuss published data. However, it seems that the tense is randomly used, not consistent. B. Line 41: after knockdown JARID2…………..after knockdown of JARID2 or after JARID2 knockdown. C. Line 42: each early embryonic stages, suggest that JARID2 plays a key role in early embryonic development…………………early embryonic stages, suggesting that D. Line 55: Early embryonic development is when cell division and differentiation are most active and when there is large-scale transcription of zygotic genes……….. Early embryonic development is characterized by the most active cell division and differentiation with large-scale transcription of zygotic genes. E. Line 261: the jumonji histone family….. the jumonji histone demethylase family F. Line 266: JARID2 is a special histone…………. JARID2 is a special histone demethylase member 2. Introduction The introduction is well referenced. However, the introduction could be better structured by making the general introduction of epigenetic regulation more concise and focusing on JARID2 related studies, especially in terms of what is not known. In addition, the significance of the current study should be better defined. 3. Discussion The discussion could be more concise with focus on the significance of current findings. 4. Legend for figure 2-4. Present tense should be used in figure legend. Experimental design: The experiment is well designed with valid controls. Validity of the findings: The results are sound with valid statistical analysis. The quantity of siRNA in cells of blastocysts should be much lower due to siRNA partition during cell division. It should be cautious in explaining the gene expression data without knowing whether enough siRNA exists to interfere with JARID2 in cells of blastocysts. Additional comments: No comment

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

Title: EVALUATION AND IDENTIFICATION OF STEM RUST RESISTANCE GENES SR2, SR24, SR25, SR26, SR31 AND SR38 IN WHEAT LINES FROM GANSU PROVINCE IN CHINA Review round: 1 Reviewer: 1

Basic reporting: * There are some sentences that are ambiguous and must be corrected and written in clear English and must conform to professional standards of expression. Examples: lines 114-116, 117-120, 131, 141-145, 173-174, 186-187 * Citation in the text has o follow the standards Examples: line 182, 186-187 * Headers of some of the tables did not explained well the contents of the respective tables. Examples: Tables 4 and 5 need footnote what is "+" and "-". There is a correction for the headers of Tables 1, 2 and 3 that is highlighted in the attached annotated PDF. * Needs specific conclusion based on on the obtained result rather than just putting a general conclusion at the end of the abstract and at the last part of the conclusion section (highlighted in the annotated PDF). Experimental design: * The materials and methods is good except line 69 needs some clarification. Validity of the findings: * Needs a specific conclusion based on the obtained result and the original research question. Additional comments: * Use the right scientific names Example: line 30 * Better to keep the list of cultivars in he table rather than long listing (42 cultivars) in the text Example: lines 104-109, 152-155 * Put marker names in italics Example: lines 174-176, in some of the references * There are some corrections and comments in the attached annotated PDF

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

Title: EVALUATION AND IDENTIFICATION OF STEM RUST RESISTANCE GENES SR2, SR24, SR25, SR26, SR31 AND SR38 IN WHEAT LINES FROM GANSU PROVINCE IN CHINA Review round: 1 Reviewer: 2

Basic reporting: The manuscript will profit if corrected by a native speaker. References in the text and at the end are in agreement, as well as figures and tables Experimental design: The manuscript is withing scope and aims of PeerJ, objectives of work are clear. Material and method: lack of information about symptoms and scores which specified for each level of diseas. Even in figure 1 not all type of scores have been included. More details about used markers is missing. Missing data about used wheat cultivars and their pedigree, how they are related. Validity of the findings: For this reviewer the amount of information provided is not sufficient for publishing in PeerJ, but if authors can provide more data about used cultivars, infection types using more races and more Sr markers, then the manuscript could be reconsidered. Additional comments: The paper "Stem rust seedling resistance genes evaluation and identification of Sr2, Sr24, Sr26, Sr31 and Sr38 in wheat lines from Gansu Province in China" describes analysis of seedling resistance to 3 stem rust isolates at 75 local wheat varieties followed with marker analysis being diagnostic to 5 known stem rust resistance genes. Authors found that 42 or 66.0% of tested wheat varieties are resistant to tested races of Puccinia graminis f. sp. tritici, while molecular marker analysis showed that 13 cultivars likely carrying Sr2; 25 carrying Sr31; and 9 Sr38. No cultivar was found to have Sr26 and Sr24.

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

Title: EVALUATION AND IDENTIFICATION OF STEM RUST RESISTANCE GENES SR2, SR24, SR25, SR26, SR31 AND SR38 IN WHEAT LINES FROM GANSU PROVINCE IN CHINA Review round: 1 Reviewer: 3

Basic reporting: The English is not professional, but the attached revision will help. Raw data of seedling infection types was not shared. Experimental design: Experimental design is sufficient. Validity of the findings: Data largely robust, but seedling infection type data should be provided. Additional comments: The manuscript is largely well done. Please address the specific comments in the attached revision to help improve the manuscript.

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

Title: EVALUATION AND IDENTIFICATION OF STEM RUST RESISTANCE GENES SR2, SR24, SR25, SR26, SR31 AND SR38 IN WHEAT LINES FROM GANSU PROVINCE IN CHINA Review round: 2 Reviewer: 1

Basic reporting: Greatly amended. Experimental design: Good. Validity of the findings: Fair. Additional comments: Line 288 to 291 need revision (words are repeated). The molecular genetic markers linked to associated with Sr2, Sr24, Sr25, Sr26, Sr31, and Sr38 were used to detect the occurrence of these genes in 75 major wheat cultivars (lines) in Gansu Province in this study. Change to: "The molecular markers associated with Sr2, Sr24, Sr25, Sr26, Sr31, and Sr38 were used to detect the occurrence of these genes in 75 major wheat cultivars (lines) in Gansu Province in this study" In general the conclusion seems need paraphrasing; Suggestion: Breeding resistant cultivars is an economic and effective way to protect wheat from disease. The development of molecular technology facilitated the identification and utilization of molecular markers for durable resistance breeding, leading to increased crop production. The molecular markers associated with Sr2, Sr24, Sr25, Sr26, Sr31, and Sr38 were used to detect the occurrence of these genes in 75 major wheat cultivars (lines) in Gansu Province in this study. The results showed that 35 tested cultivars might carry one of these genes. This information can be used in breeding for stem rust resistance in the future.

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

Title: EVALUATION AND IDENTIFICATION OF STEM RUST RESISTANCE GENES SR2, SR24, SR25, SR26, SR31 AND SR38 IN WHEAT LINES FROM GANSU PROVINCE IN CHINA Review round: 2 Reviewer: 2

Basic reporting: The manuscript meets PeerJ standards. Experimental design: The manuscript meets PeerJ standards. Validity of the findings: Data are robust. Conclusions agree with data. Additional comments: The authors addressed all of my concerns. I have some additional edits to improve the English of the newly added sections of the manuscript: Abstract – change “a donor parent” to “donor parents” Line 125 – use “genes” instead of “gene” Line 311 – use “adult plant resistance” Line 312 – use “chromosome arm” Line 350 – use “Wheat plants carrying stem rust resistance gene Sr25 were susceptible to several…” Line 352-353 – change to “to wheat chromosomes 7D and 7A from Thinopyrum ponticum (references).” Line 357-358 – use “, but with white flour (Bariana…” Line 358-359 – “The use of this gene in wheat programs is increasing for… and the Ug99…. potential yield increases….conditions” Line 416 – delete “linked to”

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

Title: THE EFFECTS OF ARBUSCULAR MYCORRHIZAL FUNGI AND ROOT INTERACTION ON THE COMPETITION BETWEEN TRIFOLIUM REPENS AND LOLIUM PERENNE Review round: 1 Reviewer: 1

Basic reporting: a. Statistical results are reported in insufficient detail. Each test corresponding to the results reported in the figures needs to be reported in a table with F-values and exact p-values. Results of multiple comparisons should be reported by putting letters above bars in the figures to identify significant differences among treatment combinations. b. The objectives paragraph (L61) is overly brief. Articulating a clearer set of objectives will assist the authors in restructuring the results and discussion sections. The objectives as written imply practical goals related to mixture development which do not reappear in any detail in the discussion. c. The results section is is very hard to follow, and overly focussed on minor points relative to the main effects in your study. Please revise to more clearly focus on the main issues that you want the reader to see in your figures and tables. A substantial reduction in length may help to focus. d. The dataset provided is not complete, as the individual biomass of Lolium and Trifolium in each pot does not appear to be reported, only total biomass. Also as noted below, the number of lines in the dataset (124) does not match the reported experimental design (100 pots). e. what the error bars on the figures represent (i.e. std. dev, 1 std error) needs to be reported. No error bars are provided on figure 5. If the estimated/actual productivity values were calculated on a pot by pot basis (they should be I think), then error bars and statistical testing can be done. f. excessive numbers of decimal places reported in the tables. g. While the paper is generally well written, extensive editing is needed to ensure correct English grammar and smooth reading flow. h. minor editorial points i. please thoroughly check the ms. for typos (e.g. missing spaces between words on L2, L30, L31) ii. L37 not clear what “higher utilization efficiency in light” means iii. L61 not clear what competence ability is iv. L105 colonized by n-fixing microbes? v. L129. More explanation needed. vi. the reference list needs to be carefully checked for misformatting and typos. Experimental design: a. I don’t understand the pot layout based on the description in the text and figure 1. In particular, nowhere is it stated what the “plant growth pillar” is. To me it appears that in the -roots treatments plants are confined to mesh columns with some volume of soil outside the columns, while in the +root treatment the whole soil volume is available. This design is problematic because the +root plants have more rooting volume available (the equivalent of the soil in the columns plus the volume outside the columns) than the –root plants (only the volume of the columns). This is an issue as there is lots of literature out there showing that available rooting volume can influence the outcome of these types of experiments. b. description of the experiment (100 pots) does not match with the provided dataset (124 rows). c. Statistical analysis is not completely described or justified. Given the design a 3-way ANOVA followed by multiple comparisons is all that should be needed. I don’t understand why the 2-way ANOVA or t-tests are mentioned. d. L93. The reasoning for the mowing treatment is not clearly stated. I understand that forage species would routinely be mowed/grazed, however removal of the aboveground biomass is likely to trigger changes in the plant aboveground interactions that may have belowground implications. Validity of the findings: The most important issue for the authors to deal with is the potential confound between the +root and –root treatments in terms of available rooting volume, as this may substantially affect our interpretation of the root treatment and the root by ratio and root by amf interactions. Otherwise, the underlying data in this study appear sound; the primary issues are in reporting and presentation as noted elsewhere in this review. Additional comments: a. Overall this study has substantial merit. Interactions between Lolium and Trifolium are very well studied, thus re-examination of the many questions that have been investigated in this system under slightly different conditions is very useful. b. I found the discussion to be quite superficial. Given the amount of research on these two species, and the number of pairwise interaction experiments involving roots, I was expecting a much wider ranging and more nuanced discussion. c. I am surprised to see no citation to Angela Hodge’s work which has extensively used these species in a variety of experimental settings including studies involving AMF.

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

Title: THE EFFECTS OF ARBUSCULAR MYCORRHIZAL FUNGI AND ROOT INTERACTION ON THE COMPETITION BETWEEN TRIFOLIUM REPENS AND LOLIUM PERENNE Review round: 1 Reviewer: 2

Basic reporting: I am not a native English speaker but it seems to me that the level of English used in this paper is really not good enough for a scientific publication. There are a lot of conjugation, sentence structure and grammatical mistakes. It also seems that sometimes the wrong vocabulary is used (e.g. competence instead of competitiveness? Or maybe for regeneration competence toward herbivory? But then you will have to explain more about that). The text is thus difficult to read. Therefore the English used here needs to be reviewed and improved. There is not enough background on the two plants used in this experiment (their symbionts? one is a grass, one is a legume what does it mean for nutrient use?) and on the reasons why it is important to study them (sustainability, mix of grass and legume generally leads to a bigger productivity than monoculture and to a higher efficiency of nutrient use…). In addition, some concepts are not well explained or introduced like the plant “competence” or like the “transgressive over yielding” for instance. Some papers cited in the introduction and in the discussion have conclusions that do not match to what is said in this draft. Some of the papers cited are quite old, (more than 20 years old). It could be nice to have newer papers cited. It is not that the older papers are not relevant but it is more because the investigation techniques have changed a lot and also the land management (in term of fertilization for instance). The overall structure of the article conforms to an acceptable format and contains all the standard sections. The figures are relevant to the content of the article. However the quality of the Figure 1 should be improved: there is a missing parenthesis and also there are ‘arrows’ at the end of each word that should not be there I think. The legends of the figures are not detailed enough. Significance codes should be added. All the raw data has been made available. Experimental design: The research presented here fits to the aims and scope of PeerJ. However even if the knowledge gap being investigated is identified (i.e. ‘…many studies have explored the mechanisms of interspecific competition between T.repens and L.perenne, their works more focused on aboveground biomass, light competition, less is done with regard to their root interactions and soil microorganism effects.’), there is no clear hypothesis stated in this paper. The investigation seems to have been conducted rigorously and the experimental design seems nice but it is not well explained. There is missing information and it is not possible to re-create the same experiment just by reading the materials and methods which is described here. No AMF and/or no root physical interactions are non-realistic conditions in the field/nature so one could question the relevance of such a study, but it can be interesting considering fundamental knowledge. Validity of the findings: The results presented here on the effect of AMF inoculation, roots interaction and the ratio of the two plants in mixture on L. perenne and T. repens biomasses and the conclusions made regarding the transgressive over yielding are relevant. The data on which the conclusions are based are provided. Hypotheses should be appropriately stated before to make any conclusion. Some of the conclusions stated in this paper are made a bit quickly and are more speculations. Some of them are also going too far compared from what the results indicate. Additional comments: Overall, I suggest that you have a native English speaking colleague review your manuscript. Some examples where the language could be improved include lines 27, 38, 45, 99,123, –and also especially in the results and in the discussion part. The current phrasing makes comprehension really difficult!!! Introduction: The main issue is that there is no hypothesis stated at the end of this introduction. Then you should add some background on L. perenne and T. repens and on legume/grass mixtures in grasslands. Why mixing both of these plants is important. What is the aim of doing that. L27: You should use 'In comparison with...' or 'compared with... instead of 'In comparing with'' L28: You should check what is said by Nyfeler et al. in the publication you cite, it is different from what you wrote. Nyfeler et al. is about positive interspecific interaction and not about competition. L36: Del Rio et al. 2016 : You should check again what is written in this publication: they are not working on L perenne and T. repens. L39 : Goldberg and Landa 1991: You should check again what is written in this publication: they are considering seed biomass, germination, aboveground biomass, the density of the neighborhood plants etc... and not root trait and soil nutrient concentration. L46 & L51 : I think you should avoid using « ...and so on... » in a scientific publication. L49 : You talk about the effect of moisture on L. perenne, it could be nice to also add the effect of moisture on T. repens. L58: « extraradical hyphae radiating from plant roots for plant-plant interaction” It is difficult to understand what you mean, you should rephrase or explain more in detail. L60: Wagg et al. 2011 You should check again what is written in this publication. Wagg et al. work on two other species than T. repens & L. perenne. L63: “AMF inoculation helps some species for nitrogen absorbing by changing their AMF colonization...” It is difficult to understand what you mean, you should rephrase or explain more in detail. L66: It would be nice to explain the reason why AMF, root and their interactions affect T.repens and L.perenne in their mixtures need to be further tested. L67: In this last paragraph of the introduction you should add the hypotheses you have. Materials and methods: L76: you should explain better the factors and levels of your treatments. L79: you should precise that your are only testing the root physical interactions as roots also interact through their exhudates and you cannot avoid them to go through the mesh you are using in the experiment. L80: “In total, 20 treatments were randomly assigned with 5 replications” you already wrote that on L75. L80,81: I think there is a mistake in the height and diameters of the pots. Later on (L84) they are 10 times bigger. L82: you should precise that you only keep the root from physical competitive interaction. L83: give more details on the seed sterilization. L84-86: It is difficult to understand how is design the experimental set up even with the figure 1: What is the plant growth pillar for? I think you should describe better the experimental set up. What is the size of the meshbags in which are the plant fort the –root interaction? What is the overall tray size? I suggest you add this information. L84: where is the soil from? I suggest you add this information. L88-89: are these the characteristics of the soil itself or from the miture with loess, sand and peat? I suggest you add this information. L92: how long were the day and night duration? I suggest you add this information. L93: could you explain the reason why you mowed all the seedlings after 2months of growth? L99-L102: I suggest that you give more details about the nature of the AMF inoculum.You should add the total number of spore per pot and also tell if there is an equal number of spore of each of the AMF species that you used (did you use an inoculum with all 5 strains already mixed or did you use five inocula and did you mixed them yourself?) L107- 111: In this paragraph you explain how to measure the AMF colonization in the soil. However later in the text, (in the paragraph starting L 131, in the figure) you talk about AMF root colonization. I suggest that you change that to make the text coherent. You should precise that you are using the dry aboveground and belowground biomasses. Results: You should rewrite this section adding for each result the details of the statistical analyses you did (name of the test, P-value...). You should also indicate, each time there is a significant difference if it is increasing, or decreasing and in which proportion. Discussion You should rewrite this section with a better english and a better structuration of the conclusions you make on each result. I also suggest that you avoid doing speculation or if you do, you should say it. L198: Lantinga et al. 1999, Barillot et al. 2014. You should check again what is written in these publications. It is different from what you wrote: they are not saying that the resource competition is only related to lighting absorbing rather than root interaction. L213: you are saying that T. Repens has a better nitrogen assimilation because of root interaction; but you never talk about the symbionts of T. Repens. I suggest that you discuss more about the nitrogen fixing bacteria that are more responsible for the N content of T. Repens than the root interactions. You also need a proper conclusion where you explain after doing this experimentwhich treatment is the best to obtain transgressive overyielding and why it is important to know that for grassland management. Figures: I suggest that you add a figure with the nitrogen content in the plants in the different treatments. You should add more details in you legends: number of replicates, test realized. ..You should also explain the significance code : what does mean A and B in the table 3 , what does mean *, **, *** in the figure 2, 3 and 4. Figure 5: there is no error bar is that normal? Raw data: What is the ratio on the column G? For the root and shoot biomasses: why in total? You should add the unit of measurement. For TN and LN: I suggest that you explain the unit. It is a percentage but a percentage of what?

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

Title: THE EFFECTS OF ARBUSCULAR MYCORRHIZAL FUNGI AND ROOT INTERACTION ON THE COMPETITION BETWEEN TRIFOLIUM REPENS AND LOLIUM PERENNE Review round: 1 Reviewer: 3

Basic reporting: The English language in this manuscript should be improved. Currently, it was difficult for me to discern what was meant in several places. For example, I had an especially difficult time understanding what was meant by sentences starting at lines 29, 34, 44, 75, 84 and 180. Given these issues, it was difficult for me to determine the main purpose of the experiment. If I follow the logic correctly, the researchers are trying to determine how the presence of a mutualist (AM fungi) and belowground interactions influence intra- and inter-specific competition between a forb and a grass. While the citations lead me to believe competition between these species has been tested, the authors state that no study has tested the effects of AMF on competition. In addition to the main text, I though the explanation for the figures could use some clarity. For example, I am not certain I understand how the root interaction treatment was established, and Figure 1 did not help me much. I have some additional comments below, but, focusing on the figure, what is the osculum? Is that a hole in the bottom of the pot? I’m not sure I understand the placement of the plant growth pillars in the two treatments. It makes sense that they are around the plants in the root exclusion treatment, but what is the rationale for the placement in the treatment where root interactions are allowed? Also, I’m not sure that the dimensions for the pillars in the two treatments are accurate because it doesn’t seem possible that they are the same size in both treatments. For Figures 3 and 4, I’m not sure that I understand the x-axis. Is root biomass denoted below the zero line? If so, negative values for biomass do not make much sense. Also, I think I was confused by Figure 4 because “total biomass” typically means the sum of above- and below-ground biomass – not the sum of biomass for both the forb and the grass. Finally, while raw data was supplied, I could not find all of the data and I believe that the column information could be better reported. For example, I’m not sure I understand what information is presented in columns “Ratio” “Shoot in Total” “Root in Total” “TN%” and “LN%”. I think that “Shoot in Total” and “Root in Total” refer to the total above and belowground biomass for the pot, and “TN%” and “LN%” refer to the %N for the forb and the grass, respectively. If that is correct, where is the biomass data for individual plants or individual species in each pot? Also, what about the AM fungal colonization/hyphae data (more on this below)? Experimental design: As mentioned above, it was a little difficult for me to follow the motivation and justification for the experiment. However, I believe that the experimental design is interesting, and the manipulation of AMF and belowground interactions on top of a manipulation of species relative abundance is powerful for determining how these factors influence the relative importance of intra- versus inter-specific competition. That being said, I’m not sure that I understand how belowground (root) interactions were manipulated. How was the mesh installed to prevent root competition? What are the gaps described in line 85? I am also uncertain of some details with the AMF manipulation. How were AM fungi measured? The procedure described in the methods describes quantification of the length of extra-radical hyphae (hyphae outside of the plant root). However, the figures and results describe percent colonization of the roots, which would involve staining the fungus in the roots and measuring what percentage of the root was colonized. Which was the case? If extra-radical hyphae were quantified, what portion of the soil was used? In either case, the efficacy of the treatment could be determined. In other words, did the AMF+ pots have greater colonization/hyphae than AMF- pots? Finally, I’m not sure I followed the methods for the statistical analyses. It states that ANOVAs were performed, but there wasn’t any information presented on whether AMF influenced plant biomass. Also, it seems like the relevant metric to look at in Figure 3 is biomass per individual plant of each species in each pot. For instance, the fact that the forb’s biomass increases with the number of forb individuals in the pot isn’t that interesting – but the response of the individual plants tells you something about how the treatments influence relative performance. Validity of the findings: In the introduction, the addition of an AMF treatment was indicated as one of the major advances of this study. However, the effect of AMF on plant biomass (and competition) was not assessed. I’m not sure that I understand why this was not reported. Additional comments: I believe that the design of this experiment is sound and can yield very interesting results. For instance, I was very interested in knowing how AMF influence the balance of intra- and inter-specific competition for these two species. I also think that the experiment touches on very important and timely topics in ecology: coexistence, biodiversity-ecosystem function, community consequences of mutualisms, above-below ground interactions. A solid rewrite should be able to more clearly place this work in the context of some of these ideas, making the results more accessible to a broader audience.

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

Title: THE EFFECTS OF ARBUSCULAR MYCORRHIZAL FUNGI AND ROOT INTERACTION ON THE COMPETITION BETWEEN TRIFOLIUM REPENS AND LOLIUM PERENNE Review round: 2 Reviewer: 1

Basic reporting: Much improved from the prev. version. Still a few typos here and there. minor point – please provide exact p-values in Table S1 Experimental design: The description of the design is now well described; I no longer have concerns in this area. Validity of the findings: Some remaining concerns with the statistical analyses As described in L167-172, the authors use a 3-way ANOVA with all interactions. This is a correct approach. They then, however, (L173 and on) remove the AMF fixed effects from the model. I do not understand why they do this as it should be possible to test the main effects/interactions involving other treatments in the complete model. Running a separate model, while not strictly incorrect, overly complicates the analysis. Further, on line 176 the authors report running T-tests to compare “variations” between root interaction treatments. Two problems here, first, I assume that the authors are actually comparing means, and second, the main effects in the 3-way ANOVA should provide the answer. The dataset and experimental description have been updated to match total number of pots (124). I am having trouble adding up the df in table S1 to reconcile the total experimental size however. Given that there are 20 pots with no T repens in the dataset and 26 with no L perenne, shouldn’t the error df. be different between the results for the two species? It is critical that ambiguities like this be resolved for me to me able to trust the reported statistical results. Additional comments: This is my second review. The manuscript is much improved, and most importantly the authors have clarified their experimental design to make clear that the plants were grown in equivalent per-plant root volumes. The writing is much clearer now, and more concise.

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

Title: THE EFFECTS OF ARBUSCULAR MYCORRHIZAL FUNGI AND ROOT INTERACTION ON THE COMPETITION BETWEEN TRIFOLIUM REPENS AND LOLIUM PERENNE Review round: 2 Reviewer: 2

Basic reporting: The English level of the draft has really been improved since the first submission. It is really easier to understand the manuscript but there are still a lot of conjugation, sentence structure and grammatical mistakes, so I suggest that you check again thoroughly the English. In addition to the language, the structure and the content of the manuscript have also been really improved. The background and the cited literature have been completed and corrected like it was asked. They are still some missing information in the Statistical paragraph and some paragraphs of the discussion should be rewritten. The overall structure of the article conforms to an acceptable format and contains all the standard sections. The figures are relevant to the content of the article. Relevant hypotheses have been added. All the raw data has been made available exept for AMF root colonization data. All the line numbers I am referring to during the review are from the manuscript with tracked changes. Experimental design: This part has also been really improved. There is still some missing info: L105: you should add the gps coordinate of the station. L111: you should add details on the nature of the AMF strains you purchased. Was it a from a spore solution? Was it from a root-organ culture? L124: you should add the concentration of the rhizobium inoculants: how many bacteria per μl? L153-154: ‘Roots of one plant individual were frozen at –20°C for determining AMF root colonization’: I don’t really understand. Do you mean one plant individual per microcosm? One plant individual per species per microcosm? So, in total, how many root system per treatment did you use for estimating the mean AMF colonization? It would be nice to add more details. In the statistical analyses part: About your data, did you check the normality and homogeneity of variances? If so which tests did you use? For the GLM analysis, What was the most appropriate family wise errors for your data? And then, from the GLM, how were analyzed your data? L196: you should precise that LSD stands for least significance difference. With which test did you analyze the data from the AMF root colonization? Validity of the findings: The data on which the conclusions are based are provided except for the AMF root colonization. Conclusion are well stated and linked to the original research questions & limited to supporting results. Some of the conclusions stated in this paper are still made a bit quickly and are more speculations. Additional comments: L13 :” These results showed T. repens…” there is a missing word: These results showed that T. repens… L15: regulates L16: makes (or you add a ‘s’ to ‘interaction’ lines 14 and 15.) L17: plant-plant L17: ‘involved in’ instead of ‘associated with’ L23: ‘Plant communities with more species’…’: you could replace with: Plurispecific plant communities are usually… L31-33: Mixtures of legumes and non-legumes which have been widely used in mixed sowing pasture and agricultural intercropping system can achieve better performance in productivity and stability…. L39-40: Grass–clover mixtures of Lolium (grass) and Trifolium (legume) are considered as model system in agricultural and natural grassland ecosystems…. L44: ‘had a strong effect…’ L45: ‘L. perenne increased its N absorbing along with increased soil moisture and further enhanced its competitive ability, while T. repens had weaker N response at higher soil moisture…’ You could replace with: L. perenne N absorption increased along with soil moisture and further enhanced its competitive ability, while T. repens displayed a weaker N response at a higher soil moisture…’ L47: ‘studies’ instead of ‘works’. L50: missing s at ‘root interaction…’ or ‘occur’ L51: Which previous study? The one from Dennis and Woledge or the one from Davidson and Robson? I think you should just write: ‘It was found that…’ or ‘Dennis & Woledge (1987) and Davidson& Robson (1990) found that…’ L59, 283, 293, 298: ‘a higher…’ L59: ‘…fast growing root system…’ L60:’… a higher light utilization efficiency…’ L65: I would remove ‘species’ L70: can you explain? How does it enhance the competitive power of T. pretense? L 73:’ratios’ L86: ’ To investigate the amount of nitrogen fixed by T. repens and potentially transferred to other plants…’ L92: ‘The eight…’ L93: Instead of: ‘were close to container wall and evenly located in container ‘ you could write: ‘… were evenly located close to the container wall.’ L97-98:’Thus, the total growth space for the roots of the eight plants was the same in microcosms without root interaction and in microcosms with root interaction.’ L133: ‘… into each…’ L137: ‘… into the opposite…’ L143: ‘…of the microcosms… ‘ L152: ‘all plants’’ L191: remove the apostrophe after ‘its’ L192: ‘alter’ and not ‘altered’ Results: L203 and 207: it should be p> 0.05 L203, 207 and 208: you should add the name of the test used in parenthesis before the p-values. L203: Unless you’ve only used 1 plant per treatment to measure the root mycorrhizal colonization, you should write ‘The average mycorrhizal root colonization…’ and if you only measured the colonization on one plant per treatment then you cannot conclude anything from that. L205: you should indicate the mean percentage of root colonization in the +AMF treatment L214, 215: you should add the name of the test used in parenthesis before the p-values. L228: '...by 21%...' add the statistical test and the p-value. L229:'...by 12%...' idem L233: '...by 64%...' idem L240: you should add the name of the test used in parenthesis before the p-values. L242, 244 &247 & 248: ' add the statistical test and the p-value after the% L251,254, 255: you should add the name of the test used in parenthesis before the p-values L258: ' Shoot N content of L. perenne significantly decreased when its planting ratio changed from monocultures to mixtures in any ratio with T. repens': add the statistical test and the p-value L208: ‘Mycorrhizal root colonization of L. perenne increased significantly along with the increasing proportion of T. repens in mixtures.’ L216-218: you should reformulate like: ‘ When the planting ratios ranged from T2:L6, to T4:L4 and T6:L2, the aboveground biomass of T.repens with root interaction respectively decreased from 47%, 46% and 42% in comparison with no root interaction (T-test, p < 0.05; Fig. 2a)’ L218: remove 'while' L229:'... indicating that...' L231: '...of L. perenne significantly increased...' L235: replace by :'indicating that the intraspecific root interaction...' L239:'yields' L250:'... the RYind of L. perenne was increased with the increase in the proportion of T. repens in mixture...' , replace by : '... the RYind of L. perenne increased along with the proportion of T. repens in mixture...' L263:'... exert contrasting effect...' L264: remove 'of these 2 species' L266: 'legumes' L267 :' grasses' L268: ' which should enhance' L269: 'as a lower' L271:' it is' and not 'it's' L282: ' in regard to' or 'regarding' but not 'in regarding to' Discussion: L293: 'which resulted in ..' this is too affirmative L307: 'It indicates that L. perenne has much stronger root interaction than legume T. repens', you should explain and develop more this affirmation. L294: 'contributes' L296: 'suggesting', 'confers' L296: you yould reformulate the sentence: '... suggesting that this extra N conferred a competitive advantage to T. repens...' L299: You should rephrase: '... resulting from its N fixing rhizobial symbionts...' L301: 'suggests' L289 -303: You should rewrite this paragraph and improve the English. L307: instead of 'was mainly from' : 'was mainly due to ' L311: 'nutrients' L319:'participates in' or ' contributes to' instead of 'helps' L322:'decreases the one from T. repens...' L324-325:' thus they could sustainably cohexist.' L329:'suggest that', 'is a key driver' Figures and data: Fig S1: you should add letters in top of the bars indicating if they are significant differences or not. In the legend of the figure you should add the number of replicates used per treatment and the test used to compare the root colonization in the different treatments. Raw data: you should add the data on the AMF root colonization in the table In the description/title of the figures you should add what the error bar. In the figure 2, even if you indicate the significant differences between +Root and –Root, it is not possible to know if they are significant differences between the planting ratios. I suggest that you put different letters to indicate these differences.

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

Title: EXAMINING THE STRESS-BURNOUT RELATIONSHIP: THE MEDIATING ROLE OF NEGATIVE THOUGHTS Review round: 1 Reviewer: 1

Basic reporting: 1. Although the writing is generally clear, the manuscript could benefit greatly from a thorough proofreading (and modification where necessary) by a native English-speaker. 2. The nature of the interaction and conjunctive moderation noted on lines 124-125 should be described. Similarly, the nature of the mediation and moderation mentioned on l. 147-154 should be described. 3. On line 132, it should be "negative cognitive triad." Experimental design: 1. Evidence in support of the validity of the CSALSS, ATQ, and ABQ should be summarized in the Results section. Validity of the findings: 1. Although the cross-sectional nature of the data are duly noted in the Discussion section on lines 430-434, language that implies that causal relations were investigated and established is used throughout the manuscript (e.g., "influencing" on line 67, "source of burnout" on line 411, "sources of negative thoughts" on line 423). Such language should be removed from the manuscript. 2. Similarly, the process described on lines 89 to 97 cannot be examined with cross-sectional data. In a cross-sectional design, the terms "independent variable" (line 287) and "dependent variable" (line 288) should be replaced with "predictor variable" and "criterion variable," respectively. Even a longitudinal design (as suggested on lines 432 to 434) would not address the issue of causation, although confidence in the model might increase. 3. Given the relatively minimal reductions in beta values in the mediation analyses, the findings should be described as reflecting only partial mediation (as they are in the Abstract). With partial mediation, the statement on lines 359 and 360 is not true. 4. It is plausible that the three sets of variables (i.e.,life stress, negative thoughts, burnout) could be causally related in multiple ways. For example, life stress could precipitate burnout, which could lead to negative thoughts. It would be of interest to see how other potential models pan out. Additional comments: 1. The applied implications, which begin on line 440, are not necessarily wrong, but they seem premature in light of the correlational nature of the results.

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

Title: EXAMINING THE STRESS-BURNOUT RELATIONSHIP: THE MEDIATING ROLE OF NEGATIVE THOUGHTS Review round: 1 Reviewer: 2

Basic reporting: This study tried to identify the stress-burnout relationship and the meditating role of negative thoughts to explain such relationship in the Taiwanese student athletes. Such investigation seems to be meaningful to support cognitive-affective model of athletic burnout which was developed in the western society and verify validation of the model in order to apply it to the different country which has different cultural norm. Moreover, this study is aiming at test meditating effect of negative thoughts between stress and burnout in the athletic context and it is strength of the current study. However, there are many points that should be addressed and corrected throughout the entire manuscript. More details are indicated at below and the authors should revise them. * In the entire manuscript, many grammatic errors, awkward sentences, and illogical paragraphs have been found and indicated in the pdf manuscript by the reviewer. Therefore, it is highly suggested that the manuscript should be edited by English professionals before resubmitted. 1. Introduction -It is too long and very confused to catch up what the authors want to describe. For instance, negative thoughts, just only one research variable in this study was described in about 3 pages from page 14-17. It needs to be concisely shorten. -Moreover, the necessity of the study was not enough to support this study. It also needs to add some more sentences. - The purpose of the study should be clearly indicated. 2. Methods and Results These sections have been relatively logically written. Results section in particular is well described the findings. However, some awkward sentences have been found. 3. Discussion It has been relatively well described. However, it is also too long, almost 10 pages. The authors should concisely rewrite. 4. References It is 15 pages and therefore should be shorten (suggested less than 40 references). Experimental design: 2. Methods and Results These sections have been relatively logically written. Results section in particular is well described the findings. However, some awkward sentences have been found. Validity of the findings: 2. Methods and Results These sections have been relatively logically written. Results section in particular is well described the findings. However, some awkward sentences have been found. Additional comments: This study is timely appropriate to identify the stress-burnout relationship and the mediating role of negative thoughts for the Taiwanese athletes. In addition, the findings will contribute to develop psychological knowledge and athletic performance in Taiwan. However, there are many aspects that should be corrected or addressed by the authors in entire manuscript. Therefore, it is not enough to be considered in publication without major revision. I suggest that this manuscript should be resubmitted after revision.

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

Title: EXAMINING THE STRESS-BURNOUT RELATIONSHIP: THE MEDIATING ROLE OF NEGATIVE THOUGHTS Review round: 2 Reviewer: 1

Basic reporting: I appreciate the efforts of the authors in addressing my concerns and those of the other reviewer. The manuscript is very much improved as a result of the changes that have been made. Experimental design: In my previous review, I should have noted that evidence in support of the validity of the CSALSS, ATQ, and ABQ should be summarized in the Measures section (not the Results section, as I mistakenly wrote). What support for the validity of the scales, independent of the results of the current study, has been obtained? Validity of the findings: Although I appreciate inclusion of the sentences on lines 439-445 about examining other models with the three sets of variables, it would be ideal if the authors could report some of those analyses in the current paper since they have the data to do so. Findings from such analyses could strengthen confidence in the main cross-sectional findings, particularly if other models don't work as well as the one tested in the main analyses. 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: EXAMINING THE STRESS-BURNOUT RELATIONSHIP: THE MEDIATING ROLE OF NEGATIVE THOUGHTS Review round: 2 Reviewer: 2

Basic reporting: It is witnessed that the manuscript throughtout the entire texts has been clearly improved. Experimental design: These sections have been relatively logically written. Results section in particular is well described the findings. Validity of the findings: The findings will contribute to develop psychological knowledge and athletic performance in Taiwan. Additional comments: It has been enoughly improved throughout the entire manuscript and so I believe that the manuscript is OK to be considered in publication without revision.

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

Title: THE FIRST OBSERVATIONS OF ISCHNOCHITON (MOLLUSCA, POLYPLACOPHORA) MOVEMENT BEHAVIOUR, WITH COMPARISON BETWEEN HABITATS DIFFERING IN COMPLEXITY Review round: 1 Reviewer: 1

Basic reporting: Overall a good paper on a neglected subject. I was not able to open the Excel supporting dataset. Experimental design: These are cryptic animals that live under rocks (pebbles, cobble, boulders), usually ones at least partially buried in clean sand, and also on rock covered with a thin layer of sand. While it is commendable that the observations were done without disturbing the habitat (and therefore altering their behaviour), there is no indication of what proportion of the population actually come up on to the top of the substrate and therefore can be seen, compared with animals that stay below the sand surface. Validity of the findings: There is mention of “drifting” dispersal as adults – this is a rare behaviour in chitons and is an escape response to disturbance, and is found commonly in only a few species – I. smaragdinus is not one of them. Species that use this live in mobile boulder fields usually in areas commonly subject to water movement sufficient enough to move the rocks. The vast majority of chitons respond to disturbance by clamping, then crawling into shadow. The very few species that use “drifting” react to disturbance by releasing from the substrate, curling up and falling down into crevices, then uncurling and reattaching – a useful survival behaviour if the substrate is badly disturbed. It is very obvious if this behaviour is exhibited by a species. Rather than just mention this as a possibility, it would have been very easy to see if this species exhibits this behaviour or not, and specify this. They mention that this species has “an atypical carnivorous diet” - while the general opinion of chitons is that they are grazing herbivores, this is definitely not the case in Australian waters where grazing herbivores are the minority – this has been known since Kangas & Shepherd 1984, and is stated in the ABRS Fauna volume amongst other references. The commonest diet is grazing omnivores, grazing carnivores like I. smaragdinus are quite common. Also, this is not the only species the emerge from the sand onto rock to graze at night, there are quite a few species that do this in SA, just not ones studied by Kangas & Shepherd. 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 FIRST OBSERVATIONS OF ISCHNOCHITON (MOLLUSCA, POLYPLACOPHORA) MOVEMENT BEHAVIOUR, WITH COMPARISON BETWEEN HABITATS DIFFERING IN COMPLEXITY Review round: 1 Reviewer: 2

Basic reporting: This is a well-written study showing basic movement patterns of a common intertidal chiton species and linking this to adult distribution. Figure 1b is very blurry – do you have a better example? Figure 2, 3 and 4 or their captions needs to specify sample (total number of boulders at each site for Fig 2, total number of chitons for Fig 3, total chitons measured for each metric in each substratum for Fig 4) Experimental design: The experimental design is sound, with some careful considerations made by the authors (e.g. omitting data from chitons that interact with each other, inclusion of tortuosity of movement), but there are a few clarifications that should be made in the Methods as follows: Clearly define substratum type categories and be consistent with terminology. You mention cobbles in the Results, but the description of the study area doesn’t define them. Clearly state how many substratum categories, their names and definition. If cobbles are not included in these, then remove mention of them from other section. Line 122: specify that this task was undertaken during the day. How did you take photos at night? Presumably with a flash? How do you control for possible effects on chiton? Validity of the findings: The findings are well-linked to general patterns of adult distribution, with appropriate and cited speculation as to reasons for the results. Line 260-261: how does this fit with other intertidal species’ known preferences? Additional comments: Abstract: Please mention where this study took place (intertidal, South Australia) Abstract, line 32: Specify the different habitat types in parentheses Abstract, line 43: Please use ‘adult dispersal’ here to avoid confusion with larval ecology. You clarify this well in the ms, but the abstract stands alone. Throughout ms: commas needed to separate independent clauses (e.g. Line 57, line 62). No commas needed for dependent clauses (line 79) Line 277: specify what these ‘novel methods’ are.

You are one of the reviewers, your task is to write a review for the article. You will be 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 FIRST OBSERVATIONS OF ISCHNOCHITON (MOLLUSCA, POLYPLACOPHORA) MOVEMENT BEHAVIOUR, WITH COMPARISON BETWEEN HABITATS DIFFERING IN COMPLEXITY Review round: 1 Reviewer: 3

Basic reporting: This is a concise and coherent manuscript that tells a reasonably simple story (based on only a few days of field sampling) but tells it well. It contains clearly articulated a priori hypotheses which are then adequately tested. As noted by the authors, there is not much published information on the ecology of underboulder chitons (though I am aware of some unpublished Masters theses on this topic), so there isn't much to directly compare their findings with. However, I did feel that a more in-depth consideration of the recorded movement patters of chitons from other habitats could have been included in the Discussion (see notes on the manuscript itself) . Experimental design: There are 2 components to this study - the dispersion patterns of this chiton determined by overturning boulders/cobbles at 2 sites and counting the attached chitons underneath them; the movement patterns of chitons at one site recorded via time-lapse photography during night-time low tides. Both components have been adequately carried out, with appropriate collection of field data and statistical analyses of those data. The graphs and tables are well presented and clearly illustrate the main findings. There are a couple of minor queries on the ms itself, but no major improvements are required. Validity of the findings: The data are robust, appropriately analysed and the conclusions generally sound. I did feel, however, that some commentary was required about possible movements of this species outside of the only situation that was investigated (ie night-time low tides) - what happens at high tide for example? There is not much speculation in the manuscript and when it occurs it is clearly identified as such. The authors quite rightly suggest that the technique they used has considerable merit for conducting further studies of movement/dispersion of cryptic species. Additional comments: A simple yet illuminating study that provides important new information on a group of molluscs that has traditionally been hard to study. The material is worthy of publication.

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

Title: BROAD SPECTRUM PESTICIDE APPLICATION ALTERS NATURAL ENEMY COMMUNITIES AND MAY FACILITATE SECONDARY PEST OUTBREAKS Review round: 1 Reviewer: 1

Basic reporting: The article is very readable, though too long. Raw data is not shared. Don't know if this is mandatory. Not an expert in terrestrial ecotoxicology, cannot comment on whether literature background is exhaustive. Experimental design: Design largely sound but has shortcomings, see below Validity of the findings: Discussion too long. Additional comments: The paper describes the effects of two pesticide treatments on pest and beneficiary invertebrates over several seasons. The study design is largely sound, but see major comment below. The article is very readable, but pretty long given its contents. Major comment The data analysis seems flawed to me. The authors seem to treat the replicates within a block as independent. However, they are not and the block design does not help here. Generally, there are two replicates per treatment, thus inferential statistics seem misplaced to me. At least in aquatic mesocosm studies this would not work, and I don't see why sampling the same treatment strip multiple times would result in treatment replication. Moreover, the sample size would be even smaller if factors such as the crop type or amount of pesticide applied would be included in the analysis (I don't suggest to do this, but the sample size is too small to draw general treatment conclusions based on statistical inference). Minor comments 19 "community ecology" specify, what you examine 29f point out what is pest and beneficiary 35 add whitespace 36ha 60 and but 92 Begin sentence with capital letter 138 replace comma with . 187 should be "were" 189 how was the kriging done? Should also be kriged data 209 delete "studies" 235 what kind of null model? 257 suggest to move "in total" to beginning of sentence 218 suggest to rephrase "this contrasts to" 308 whitespace missing (pest community) 391 replace methodologies with methods 395 remove "()" 406 species (spelling) Figure 1 you could put the scale directly beside the figure so as to clarify that each width of treatment is 78 m Figure 4 Spelling should be "shows" instead "dhows" Figure 5b control could be included in plot

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

Title: BROAD SPECTRUM PESTICIDE APPLICATION ALTERS NATURAL ENEMY COMMUNITIES AND MAY FACILITATE SECONDARY PEST OUTBREAKS Review round: 1 Reviewer: 2

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: This study represents an impressive amount of sampling and sorting to address a question of great interest and which requires considerable commitment to address with field studies. Represents an impressive attempt on the part of the authors to identify the factors affecting pest and natural enemy abundances in a field study. It would be useful to be provided with information as to relative abundances of taxa considered eg the importance of the observed a significant impact on predatory beetles (carabids and staphylinids) is related to their abundance. See also Ll310 Ff clearly abundance of these taxa is relevant to effect size also L315 with reference to predatory bugs and wasps. This information could be inserted to Table 2. The authors indicate they aim to sample ground dwelling invertebrates but include several beneficials (eg lacewings, ladybirds, syphids) and even pests (eg aphids) for which this is a suboptimal sampling method. Some mention of this should be made. Include consideration of the role of increased rainfall and change of crop to canola in increase in slug abundance observed. Is there potential for increased slug abundance in the final sampling be influenced by the increased rainfall in that season? Natural enemy and beneficial are used interchangeably throughout ms. Greater consistency should be aimed at especially in referring to results where tables and figs use beneficial rather than natural enemy. L84 delete c L85 suggests L135-137 check font Ll138-139 Meaning unclear-“Seed treatments and fungicides were not applied during this long term experiment, only herbicides to limit confounding non target impacts from other pesticides”. Re write to clarify L149 L why was double application of chlorpyrifos expected to increase impact on beneficials? Reference or justification required Ll152-153 repeat of LL143-144. Please delete L173 add information clarifying the outcome of sorting and grouping eg resulting in 13 taxa (variously order, family, species) classified as “beneficials” and 11 taxa of “pests” (see Table 2 for detail). And use consistent term in table “taxa” rather than groups L198 species richness-all taxa? Both “pest” and “beneficial” In results need to list the taxa as included in analyses Table 2 “Beneficial and pest communities and groups defined for this study” These are the individual Taxa referred to in the text (L) I presume. This needs to be made clear and the same terminology used. Change caption to read” taxa” rather than “groups” and make clear these are the discrete units used in analysis Caption Table 2 insert space indicates no available information Results 115 different species or taxa collected but this is reduced to about 30 listed in Table 2. Needs more information n methods eg 6 species included in “carabid beetles” or similar L157 add placed into X taxa for analysis L281 earthmites (figure 3) abundance looks unaffected by treatment except for towards end of treatment? And earwigs higher abundance fr earlier sampling dates? Insert to caption for figure 2 “..species richness insert X taxa of beneficials and pests The relevance of species richness of all taxa collected to assessing treatment impacts need to be more clearly set out. Would like to see this analysis for “beneficials and “pests” separately. Is increased species richness desirable if it is caused by increased pest species or taxa? Clarification needed-in Fig 3 it appears carabid abundance declines Jan 2006-June 2006 but higher rate of chlorpyrifos was applied June 21st 2006-is this correct? L308 pest community L309 June 2005 and September 2005 sampling but Table 3 says July 2005 and October 2005 L312 insert date of increased chlorpyrifos referred to in text to Table 3 L315staphylinids marginally affected March 2006-is this correct? L330 and elsewhere X2 to 2 DP and P L395 insert ref L377 please check intent of Thomson and Hoffmann 2006 which is actually a field study not “laboratory bioassays” L564 insert para Figure 2 and other figures labels delete “none” and replace with “control” for consistency Insert “X taxa of beneficials and pests” following species richness Figure 3 caption insert units for abundance in text Table 1 should planting dates read 2005 etc? Table 3 insert to caption for the beneficial and pest communities Figure 4 caption L1 and L4 shows not dhows Label “beneficial community” not” natural enemy community” for consistency

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

Title: BROAD SPECTRUM PESTICIDE APPLICATION ALTERS NATURAL ENEMY COMMUNITIES AND MAY FACILITATE SECONDARY PEST OUTBREAKS Review round: 2 Reviewer: 1

Basic reporting: xxx Experimental design: xxx Validity of the findings: xxx Additional comments: The authors have successfully improved the manuscript. Their answer to my comment on the data analysis was also convincing, but I was disappointed that they did not add at least 2-3 sentences in the manuscript like: " We are aware of xxx and therefore we conducted preliminary analyses. Theses showed that ..." In addition a couple of minor issues should be fixed before publication: Line numbers refer to document with tracked changes 103 add comma after chemicals 164/165 can you provide any information what these default settings are? 185 should be: the effects of pesticides 191 Pascual not in reference list - check all references carefully (and is it really Pascual or Pascoal?) 283 should be „though“ not „thought“ 360 should be: „can interact“ 381 should be „that reported that“ ? 406 should probably be „Stäubli“ 411-413 statement of sentence is convoluted

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

Title: BROAD SPECTRUM PESTICIDE APPLICATION ALTERS NATURAL ENEMY COMMUNITIES AND MAY FACILITATE SECONDARY PEST OUTBREAKS Review round: 2 Reviewer: 2

Basic reporting: Clear and unambiguous, excellent English throughout with literature providing sufficient background and context Experimental design: Research question well defined, relevant and meaningful Validity of the findings: Data is robust statistically sound; conclusion well stated and linked to original research question Additional comments: The authors have prepared an exemplary response to reviewer comments and the revised manuscript reflects this attention to detail

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

Title: ASSOCIATION BETWEEN GENETIC POLYMORPHISMS OF THE IL28B GENE AND LEUKOMONOCYTE IN CHINESE HEPATITIS B VIRUS-INFECTED INDIVIDUALS Review round: 1 Reviewer: 1

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: Dear Authors, I have read with great interest Your manuscript entitled “Association between genetic polymorphisms of the IL28B gene and leukomonocyte in Chinese hepatitis B virus-infected persons”. The study showed the interesting association between IL28B polymorphisms and HBV infection and biochemical characteristics of HBV-infected individuals with different serological expressions. The influence of host-related genetic variability on HBV infection, HBV surface antigen seroclearance and differences in response rates to therapy in CHB patients is not well understood. Unlike many studies confirming that SNPs in the interleukin 28B (IL28B) gene play a primary role in IFN-based treatment outcomes in patients with chronic hepatitis C, the association between IL28B SNPs and the clinical course and serologic profiles of acute and chronic hepatitis B has been a subject of very few studies. Interestingly, the investigation was carried out within Chinese HBV-infected population and will be helpful for further research in this field among specific subpopulation. It is a study with relatively large amount of cases with chronic infected phase and patients undergoing convalescence but small for patients at acute infected phase to final conclusions. Considering the above, the authors skilfully and carefully draw conclusions, point out the need for further research. However, I have a few comments: 1. Please precise the criteria for patients undergoing convalescence in section "Subgrouping of HBV-infected persons". 2. Create a legend for the results in Table 3 (p-value, OR, 95%CI) 3. It is very interesting to compare patients who developed chronic HBV infection with patients who have undergone self-treatment – Please show the results of the comparison between the second and the third group in table 3. 4. Authors identified the relationship between IL28B genotypes and biochemical parameters, but these analyzes were performed within the entire group of patients enrolled in the study. These patients are in various phases of HBV infection (acute, chronic and convalescence) and may therefore have different biochemical parameters. Please explain why not have done these analyzes. Please provide biochemical results in three patient groups. If no significant differences between these three groups are found in terms of biochemical parameters then table 4 may remain. On the other hand, if the patient groups differ significantly in biochemical parameters, then additionally, please analyze the relationship between IL28B and biochemical parameters for each group separately for IL28B dominant models (body article or supplemental).

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

Title: ASSOCIATION BETWEEN GENETIC POLYMORPHISMS OF THE IL28B GENE AND LEUKOMONOCYTE IN CHINESE HEPATITIS B VIRUS-INFECTED INDIVIDUALS Review round: 1 Reviewer: 2

Basic reporting: Background on IL28B polymorphisms is all right. Background on the role of leukomonocyte (LYM) levels in HBV patients is lacking. There are some grammar mistakes that should be revised. Experimental design: The design is ok as well as the technical standard. Validity of the findings: The findings are valid, however in my opinion the interpretation of the results are not completely right. Additional comments: The authors evaluated the role of the IL28B gene in HBV-infected individuals in China. They screened genotypes of three single nucleotide polymorphisms (SNPs, rs12979860, rs8099917, and rs12980275) in HBV infected persons and general controls by using SnapShot and sequencing. The results showed no significant differences in genotypes and alleles between the HBV infected persons and controls. Haplotype frequency was also similar between the HBV infected patients and controls. On the other hand genotype GT (P = 0.033) and allele G (P = 0.038) of rs8099917 showed statistically higher frequency in patients with acute hepatitis than compared to HBV patients undergoing convalescence. They also found that leukomonocyte (LYM) levels were associated with SNPs in the IL28B gene. In addition, the LYM levels were lower in the HBV-infected persons with genotype CC of rs12979860 and AA of rs12980275 than in the patients with other genotypes of these two SNPs. They concluded that genetic polymorphisms of the IL28B gene might be associated with the pathogenesis or treatment effect of HBV infection. Grammar has some errors that should be revised. I do not think the term “popular disease” should be used. Instead, the authors should say “important disease” or “relevant disease”. The abstract should specify the studied population (acute hepatitis B, chronic HBV and convalescence). Major issues I do not agree that differences in leukomonocyte (LYM) levels are striking results. If so, the authors should have reported the background on the issue. I do not see relevance on this finding. Conclusions “genetic polymorphisms of the IL28B gene might be associated with the pathogenesis or treatment effect of HBV infection” are not supported by the results.

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

Title: ASSOCIATION BETWEEN GENETIC POLYMORPHISMS OF THE IL28B GENE AND LEUKOMONOCYTE IN CHINESE HEPATITIS B VIRUS-INFECTED INDIVIDUALS Review round: 1 Reviewer: 3

Basic reporting: English language of the manuscript is very poor, starting from the abstract, with terms and expressions that are not correct and sometimes totally wrong (i.e. Abstract background:” Hepatitis B infection is the most popular hepatic disease in China”. Most popular ”??? This is an expression used for other issues). My suggestion is to have a native English speaker perform a deep correction of the text. The background is too superficial. The previous works are cited without a clear explanation of their findings and main messages: i.e. “However, a genome-wide association study (GWAS) among Asians found that this gene is not correlated with HBV (Kamatani Y. et al. 52 2009; Hu Z. et al. 2013).”: correlated to what? Viral clearance? Treatment outcome? HCC risk? Faster or slower evolution of progressive liver damage? Please explain. Also: “Recently, genetic variations of the IL28B gene have been identified to be associated with HCV infection, viral clearance, and response to therapy” : the first papers on this issue have been published in 2009… they are not recent. Then further, in the abstract: “ However, the single-nucleotide polymorphisms (SNPs) or the haplotypes constructed by SNPs in the IL28B gene can influence the HBV infection, HBV surface antigen sero-clearance, or treatment of HBV-infected persons (Seto W. K. et al. 2013; Domagalski K. et al. 2014).” This appears in contrast with the sentence above, but no explanation was given. Then, further in the background: “The present study investigated whether SNPs in the IL28B gene influence the HBV infection and biochemical characteristics of HBV-infected individuals with different serological expressions in Yunnan, China.” Reading the manuscript it is hard to understand which are the biochemical characteristics the authors are referring to. The authors reported some data about serological HBV parameters in the supplementary materials (the files are named “supplementary”, please change), but this is clearly not enough for a well designed study related to a hepatotropic virus. What about the other liver-related parameters (ALT, AST, bilirubin, albumin etc…)? My suggestion is to add a table with all the patient features, even if the same subjects have been analyzed in a previous study. In summary, intro & background are to superficial and the literature could be better explained to understand the novelty coming from this study. Raw data are supplied but they are not enough to perform a complete and solid analysis (all the main liver parameters are completely missing). A table reporting all the patients features is mandatory. Experimental design: The aim of the study is not completely clear as already stated. Methods are not described with sufficient detail & information; usually, even if the methods are described in a previous paper, a brief explanation of the techniques could be helpful, at least to understand the type of approach. Here this description is completely missing. Please, add more information about patients and methods and try to collect more data about the patients to make a exhaustive and maybe find some association with other liver/disease related parameters. Validity of the findings: Although the results could raise some interest the impact and the novelty of this study, in the present version, are not sufficient . I suggest to implement the data related to patients and also perform new analyses on them. This could make the paper suitable for publication. Additional comments: The paper needs a deep revision and many changes are needed: -English language editing is mandatory; - making the aim of the study clearer is also necessary; - more laboratory and clinical data of the studied patients could open the way to more interesting analyses (multivariate analysis); - Background should be more detailed and clearer to easily understand the novelty of this research; Some concepts should be corrected and expressed in a more scientific way: “HBV-infected persons (persons it is a very unusual word, please change) were also devoid of any liver disease”. It is very strange that no one patient had liver damage since the cohort is pretty wide. How did the authors assessed the liver damage? also:” Although no consistent result is obtained in different populations, HBV infection is reportedly associated with the IL28B gene “ how can an infectious disease be related to some particular host genotype? Maybe the pathogen clearance or the higher chance to be successfully treated could be related to a particular genetic pattern. The contagion is completely independent by the host genetics. “Subgrouping of HBV-infected persons” paragraph: “Group 3 comprised HBV-infected persons undergoing convalescence (N = 262).” What do the authors mean with the term “convalescence”? My suggestion is to provide all the missing infos and to make all the suggested changes in order to improve the manuscript.

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

Title: ASSOCIATION BETWEEN GENETIC POLYMORPHISMS OF THE IL28B GENE AND LEUKOMONOCYTE IN CHINESE HEPATITIS B VIRUS-INFECTED INDIVIDUALS Review round: 2 Reviewer: 1

Basic reporting: Background on IL28B polymorphisms and the role of leukomonocyte (LYM) levels in HBV patients is all right. The manuscript is at great length, well written, seems accurate and well organized and the abstract represent the content of the paper. The authors clearly presented any doubts concerning the investigation conducted by them. The manuscript is discusses the reported findings in the context of the present knowledge - literature review is adequate. Experimental design: The aim of the study is clear. Methods are described with sufficient details. Validity of the findings: The findings are valid. The paper makes original contribution and it is clinically exhaustive. There are some limitations regarding the small size of some subgroups; However, as the authors mentioned in the review division of HBV-infected individuals into three subgroups to analyze the biochemical characteristics, but no statistical difference was identified. The results of these analyzes should be included in the form of a table to the supplement. This could make the paper suitable for publication. Additional comments: The study showed the interesting association between IL28B polymorphisms and HBV infection and biochemical characteristics of HBV-infected individuals with different serological expressions. The influence of host-related genetic variability on HBV infection, HBV surface antigen seroclearance and differences in response rates to therapy in CHB patients is not well understood. Unlike many studies confirming that SNPs in the interleukin 28B (IL28B) gene play a primary role in IFN-based treatment outcomes in patients with chronic hepatitis C, the association between IL28B SNPs and the clinical course and serologic profiles of acute and chronic hepatitis B has been a subject of very few studies. Interestingly, the investigation was carried out within Chinese HBV-infected population and will be helpful for further research in this field among specific subpopulation. It is a study with relatively large amount of cases with chronic infected phase and patients undergoing convalescence but small for patients at acute infected phase to final conclusions. Considering the above, the authors skilfully and carefully draw conclusions, point out the need for further research. I have 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: ASSOCIATION BETWEEN GENETIC POLYMORPHISMS OF THE IL28B GENE AND LEUKOMONOCYTE IN CHINESE HEPATITIS B VIRUS-INFECTED INDIVIDUALS Review round: 2 Reviewer: 2

Basic reporting: The article meets the required standards. Experimental design: The article meets the required standards. Validity of the findings: The article meets the required standards. Additional comments: The authors have made the required changes.

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

Title: ASSOCIATION BETWEEN GENETIC POLYMORPHISMS OF THE IL28B GENE AND LEUKOMONOCYTE IN CHINESE HEPATITIS B VIRUS-INFECTED INDIVIDUALS Review round: 2 Reviewer: 3

Basic reporting: I think the authors' replies to the reviewer's questions are satisfying; the authors made the required changes in the text. There are still some minor typing mistakes to correct ("Untile" instead of until... etc.). Experimental design: The authors made the required changes Validity of the findings: See the review of the first version Additional comments: Please, have the minor typing mistakes corrected before to resubmit the final version 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: ASSOCIATION BETWEEN GENETIC POLYMORPHISMS OF THE IL28B GENE AND LEUKOMONOCYTE IN CHINESE HEPATITIS B VIRUS-INFECTED INDIVIDUALS Review round: 3 Reviewer: 1

Basic reporting: The article meets the required standards. Experimental design: The article meets the required standards. Validity of the findings: The article meets the required standards. Additional comments: The authors have made the required changes.

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

Title: ASSOCIATION BETWEEN GENETIC POLYMORPHISMS OF THE IL28B GENE AND LEUKOMONOCYTE IN CHINESE HEPATITIS B VIRUS-INFECTED INDIVIDUALS Review round: 3 Reviewer: 2

Basic reporting: No new comments regarding the current revised version. Experimental design: No comment Validity of the findings: No comment Additional comments: The authors fixed the minor mistakes present in the previous version. I think the manuscript is now suitable for publication.

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

Title: UNDERREPRESENTATION OF WOMEN IN THE SENIOR LEVELS OF BRAZILIAN SCIENCE Review round: 1 Reviewer: 1

Basic reporting: This is an interesting submission that highlights the gender imbalance in representation of women in science in Brazil. Minor suggestions include avoiding the use of "hard" as a descriptor of disciplines or sub-disciplines of science. Hard versus soft science? Hard versus easy science? There are some typos or corrections not incorporated (line 322-323). Discussion could be expanded and strengthened to relate loss or exclusion of women from "higher" levels of science (but not humanities) to cultural context. Institutional, federal, local policies that support women, parents, minorities etc. Parental leave policies for instance, which differ significantly between European countries and the US leading to different stressors on women at specific stages in their scientific careers - what is the situation in Brazil? Why is the impact felt more acutely in science?. Are there recommendations that would come out of this study (along the lines of an Athena Swan or SAGE Pilot type of program for instance). The paragraph starting at 312 acknowledges some attempts at systemic change but there appears to be little effect or impact. Can the work presented here inform and guide policy-making? If so, how? Experimental design: No comments Validity of the findings: Data are provided and made available as per journal instructions I would encourage the authors to be more speculative about the underlying mechanisms that lead to their observation (making it clear they are speculating as per the journal instructions). And looking to their data and findings for any potential policy recommendations (what would they be? who would be responsible?) 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: UNDERREPRESENTATION OF WOMEN IN THE SENIOR LEVELS OF BRAZILIAN SCIENCE Review round: 1 Reviewer: 2

Basic reporting: THIS IS MY FULL REVIEW OF THE PAPER ----------------------------- This paper presents a simple summary of gender imbalance in Brazilian science. It is not an experimental study, but rather summarises available data. If this is a reasonable topic for PeerJ, then I provide some comments to improve the paper: Abstract - please remove or spell out acronyms (PQ, CNPq). What is meant by 'Exact Sciences' - is that mathematics? It is not a term I have come across. Intro I imagine the majority of readers, like me, have no familiarity with the Brazilian funding system. It would be helpful to have clearer description of the categories and the grants that can be applied for. When I first read line 87, I thought these categories were the scores applicants were given for their grant, not their career stage. It was also only later that I worked out how the level 1A/1B/1C/1D categories relates to career stage, and the level of funding. And it is not stated how this relates to doctoral or postdoctoral scholarships. It would help to set this out explicitly (maybe in a table) at an early point in the introduction. It seems that the 1A/1B etc categories depend on years post PhD - does this calculation take into account career breaks / maternity leave etc? can the authors comment on how this might influence the gender distribution? p131 - what does the 58% mean? Is this 58% female or 58% of the sample are category 2? the latter does not seem very relevant here. p5 - why is this analysis divided into these particular subfields? are these categories used by the CNPq? p6 - as far as I can tell, PeerJ does not require a 'methods at the end' format, so I strongly advise putting the methods section betwen the intro and results - it makes the paper much more coherent and easier to read. p6 = what are the standardised residuals? how are these calculated? p7 - the authors say there are more women than expected in some fields and less in others. but this seems to be based on the overall proportion of 25% women. Presumably, if the default expectation were 50% women, then all the subfields would be less than expected? It would be worth spelling out what the expected values mean here, so readers can make sense of the deviation from expectd. p9 - is the analysis of level of funding confounded with career stage? It wasn't quite clear if only researchers at some career stages could apply for some funding levels, but if that is the case, then this analysis might be redundant. Discussion Reviewing the studies of Nosek & Moss-Racusina et al feels a bit out of place here - these are very different studies and don't really fit with the flow of this paper. It would be better to consider these studies in relation to things that could be done to reduce the gender imbalance. It might be worth adding a limitations section - in particular, the present data doesn't show if lower numbers of women with grants are because fewer women apply, or because those who do apply are less likely to be funded. Without data on the gender balance in applications, it is impossible to determine this, but it is a key factor for developing effective solutions to gender imbalance. It could also be worth discussing what kind of research could be done in future to better understand & reduce gender imbalances in science. Tables 1-4 Why do the % columns sum to 100% when you read downwards? surely it would be much more helpful if they summed to 100% along the rows so it is easy to see the % women in each category? Fig 2B seems redundant as it is just the opposite of Fig2A. Minor grammatical issues line 51 - "Although some progress HAS BEEN MADE, ..." line 58-61 - rewrite this sentence line 322-323 - rephrase Experimental design: this is not an experimental paper so I leave it to the editor to determine if it is in scope. the analysis could be described better. Validity of the findings: The findings seem reasonable. Additional comments: no further comments

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

Title: UNDERREPRESENTATION OF WOMEN IN THE SENIOR LEVELS OF BRAZILIAN SCIENCE Review round: 2 Reviewer: 1

Basic reporting: The language has been improved where necessary and additional supporting information included as recommended by the reviewers. The article is professional and self-contained as per requirements of the journal. Experimental design: Rigorous as far as I can assess. Validity of the findings: No comment. Additional comments: The manuscript has benefited from very useful feedback from the reviewers and is strengthened. It is a useful addition to the overall field.

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

Title: UNDERREPRESENTATION OF WOMEN IN THE SENIOR LEVELS OF BRAZILIAN SCIENCE Review round: 2 Reviewer: 2

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: The paper is much improved and I have just a couple of minor comments. First, I still find the terminology confusing in places. In particular, why do you suggest that STEM is the same as 'Exact Sciences'. STEM is a term that applies to ALL science & technology (including life sciences etc). It would be more helpful to introduce the 3 domains distinguished in Brazil (Physical & exact sciences: ETEC; Life Sciences VC and Humanities and Applied Social Sciences (HASS) and then stick to those three acronyms throughout. there is no need to try to re-define STEM to mean just some sciences. Second, I recommend caution in the sections on cognitive differences between men and women. The phrase 'cognitive differences between men and women are quite small' implies that there are still differences, when in fact many studies suggest zero difference. It is important to make this clear.

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

Title: THREE-DIMENSIONAL VARIATIONS IN THE LOWER LIMB CAUSED BY THE WINDLASS MECHANISM Review round: 1 Reviewer: 1

Basic reporting: This article meet the standards of the journal However, is necessary to check some expressions in English Experimental design: 1. It would be necessary to indicate the system of measurement of the anthropometric measurements (height, weight) 2. It would be helpful to indicate what of image was used to calculate the STJ with the Autocad system 3. Please, tell us something about the results of the test test of wilcoxon for the test with the independent sensors 4. What happened with the test in which the sensors were related ? Validity of the findings: I think that the findings are accurate, valid and very helpful for the scientific community Additional comments: This is a very good research and paper, I think it could be very cited in the podiatric medical community

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

Title: THREE-DIMENSIONAL VARIATIONS IN THE LOWER LIMB CAUSED BY THE WINDLASS MECHANISM Review round: 1 Reviewer: 2

Basic reporting: In general, edit for word tense and word choice. A native English spoken should review the whole paper for clarity before publication. The manuscript is full of grammar and spelling mistakes that should be corrected. Experimental design: There are major issues regarding design reporting in the manuscritp. Please, see the general comments for authors section. Validity of the findings: Conclusions are well stated. Additional comments: The paper is quite interesting but needs some modifications in order to be published. So, Some mayor issues and a few minor ones have to be addressed: MAJOR The major point of the paper is that the Materials and Methods are not clearly described in the paper which the manuscript not very understandable. This should be corrected before publication. 1. Kinematic description should be more clearly stated in the manuscript. The description of kinematics is usually quite complex and in this case needs a more detailed description in this paper. The starting point were the measurements were taken in the Bioval system should be clearly stated. You should define which was the zero position from the measurements taken. Did the system take a reference position from the relaxing standing position of the patient? Or did the system take the reference position from other choice?. For example: you say that metatarsophalangeal joint dorsiflexion was 8.83º with dependent sensors. Which was the reference position: standing position or 0º position?. The same can be applied to the all variables. At the same time, from the tables, it seems that variables measured were extension of 1st MTP joint, plantarflexion of 1st metatarsal, prono-supination of first metatarsal, shank rotation, femur rotation and STJ. It should de define how this variables were measured with the Bioval system. Please, specify what STJ means and how was measured. 2. In the manuscript there is a lack of explanation of the variables measured and compared. The authors used a significance test but we do not know which variables were compared. In lines 152-153, authors stated “to determine whether statistical significance was attained we used the Wilcoxon test for two related samples and to evaluate the correlation between variables Spearman’s Rho test was used.” However, it is not known what comparisons did the authors. You used a Wilcoxon rank sum test but you need to specify what exact variables did you compare and why and, also, this should match with the objectives of the study stated in the introduction. Lines 165 -168, please specify the values. Without this information, the results section is not understandable. 3. Why do you use the dependent sensors with the 45º wedge and the independent sensors with the maximum extension? From reading the manuscript it is not clear why you chose that methodology. If there is a reason for that, it should be clearly stated in the manuscript. Which is the difference between dependent and independent sensors? 4. Subtalar Joint Axis Location technique is highly subjective and you should state which investigator/investigators performed the test along with other information such us years of experience. Furthermore, How do you define normal? There is no clear agreement regarding what can be defined as a normal STJ axis using this test. I would recommend to state what would be considered abnormal and used it as a exclusion criteria. MINOR: In general, edit for word tense and word choice. A native English spoken should review the whole paper for clarity before publication. ABSTRACT: The methods section of the abstract should be more clearly define. It it not possible to have an understanding of the results of this interesting paper if you do not specify the method section better. INTRODUCTION: Regarding the introduction, authors give a huge explanation of the windlass mechanism. That description is so large and for easy reading I would recommend to shorten it. Furthermore, from the introduction I am not sure what gap(s) in the literature you hope this study can address. Please briefly tell the reader this before you declare a study purpose. MATERIALS AND METHODS Is it the statement in 106 right? (Oviedo Agreement and the Declaration of Helsinki? (Sure is Oviedo?) In the exclusión criteria you stated, “inability to understand instructions related to the study”. As participants of the study were students from your university it seems hard to believe this exclusion criteria, please, modify it. I recommend to change “variables measurement” instead of the “Methodology” subheading in the Materials and Methods section. In order for reproducibility of the study you should say which investigator or investigators did the measurements. Was just one or more than one? These aspects should be clearly stated in the test so anyone can reply the study. As Bioval system is not widely used or known, I would recommend a brief (1 or 2 sentences) description of the system. I would recommend to add a subheading “Statistical Methods” in line 144. Line 147, you stated “pronosupination of the first metatarsal”. I would recommend to clarify this concept as considerable confusion still exists regarding axis of movement and movement of first meatatarsal-ray in the literature. It is recommended to say “Wilcoxon Rank sum test” instead of just “wilcoxon” DISCUSSION The discussion is interesting and well developed. There are several studies that that have also found less than 60º of 1st MTP dorsiflexion for normal walking in normal subjects. AS a suggestion I would recommend the authors to use these studies in 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: THREE-DIMENSIONAL VARIATIONS IN THE LOWER LIMB CAUSED BY THE WINDLASS MECHANISM Review round: 1 Reviewer: 3

Basic reporting: I have a quite a few concerns with this study. - The background work needs to be thorough and talk about what has already been done and what information is lacking with proper references. - The author needs to focus on what's novel about this study and the significance of it. - The methods described are not clear and need to be more detailed. - Last but not the least, the language used throughout the paper is very hard to understand. The manuscript should be reviewed by an English language expert before any further consideration. Experimental design: The experimental design is not clear and needs to be more detailed. Validity of the findings: It is difficult to comment on the validity since the design of the study is not clear and the language used is ordinary. Additional comments: I have a quite a few concerns with this study. - The background work needs to be thorough and talk about what has already been done and what information is lacking with proper references. - The author needs to focus on what's novel about this study and the significance of it. - The methods described are not clear and need to be more detailed. - Last but not the least, the language used throughout the paper is very hard to understand. The manuscript should be reviewed by an English language expert before any further consideration. Thank you for submitting your 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: THREE-DIMENSIONAL VARIATIONS IN THE LOWER LIMB CAUSED BY THE WINDLASS MECHANISM Review round: 2 Reviewer: 1

Basic reporting: Clear an unambiguous, professional Experimental design: Original Validity of the findings: OK 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: INFORMATION FROM DYNAMIC LENGTH CHANGES IMPROVES RELIABILITY OF STATIC ULTRASOUND FASCICLE LENGTH MEASUREMENTS Review round: 1 Reviewer: 1

Basic reporting: In general, the description of the protocol is difficult to follow. For example, how can five subjects times two legs time three raters equal 56 sets of images (L200)? Please consider including a sketch summarising the protocol or use a more streamlined description. Experimental design: See general comments Validity of the findings: See general comments Additional comments: The present article compared different strategies to digitise muscle fascicle length from ultrasound images. The main findings are that informed digitisation (via analysis of preceding or consecutive frames) yielded more reliable results. The results also show that estimating changes in fascicle length in a series of images is more reliable than the assessment of single images when considering inter-rater differences. The manuscript is globally well written and the study is interesting to the increasing number of scientists measuring fascicle length from ultrasound images. The conclusions are consistent with motor control theories highlighting the importance of optic flow in visuomotor tasks. My main question would be regarding the execution of the analysis and, by extension, the possibility of a missing experimental condition: In the sequence conditions, were the raters instructed to refrain from displaying the preceding images, playing the sequence back and forth? This strategy would be different than the strict, direct, digitisation of each image without playback, providing the rater with an optic flow, instead of discrete cues. In relation to this, it would have been interesting to propose another DIT condition, in which the frame of interest would have been in the middle of a sequence. Minor comments: Why taking 5 subjects out of 28? The increased fascicle tension induced by maximal dorsiflexion considerably improves the echo and contrast from these structures. This aspect should be included in the factors making fascicle segmentation easier in these images (i.e. it is not the sole effect of fascicle orientation).

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

Title: INFORMATION FROM DYNAMIC LENGTH CHANGES IMPROVES RELIABILITY OF STATIC ULTRASOUND FASCICLE LENGTH MEASUREMENTS Review round: 1 Reviewer: 2

Basic reporting: The language is clear throughout the whole manuscript. References provided are appropriate and sufficient. The state of the figure is more than acceptable (although I will suggest to insert a new figure to better explain the methodology used). Experimental design: The experimental design is clear but sometimes it requires a lot of focus by the reader to actually visualise how the study was conducted. I suggest the authors to provide a schematic representation of the scan acquisition between the two groups. The rest of the method section is good. Please specify the probe length and how the operator were sure that the probe was place on fascicle plane (I'm sure it was, just better specify in the methods you actually took care of this, especially during the passive movement). Validity of the findings: The novelty of findings is appreciated, especially for a measure that we know being subjected to a lot of variability between many operators and also investigations. I have just a couple of points of discussion. 1- This was conducted on GM muscle, a muscle-tendon unit (MTU) that present a long and compliant tendon with a relatively short fascicle if compared to other muscle groups. For example, configuration of Quads (VL in the specific ) MTU may be different, as curvature of fascicle could differ along the length of the whole muscle, especially close to the myotendineous junction. While I appreciate and not question the validity data presented on GM, what would the authors think it could happen in a MTU with different configuration? This should be addressed in the discussion 2- What do the authors think of the influence of exercise adaptations in a scenario with pre-to-post changes in fascicle length? There is evidence for contraction dependent adaptations are found for example between eccentric vs. concentric loading type (done with single snapshot ultrasound without the use of DIT methodology as this paper advocates) (see Franchi 2014 and 2017, and I hope, then, that those data (mine) are actually saying the truth, then! ha ha) and we know that the length of fascicle can increase also because of greater fascicle curvature that can be found close to the superficial aponeourosis . Can this new methodology implement also to better identify where the real end of the fascicle presenting curvature is on the superficial aponeurosis? Maybe this could be a suggestion for a comment in the discussion or an actual study. And if so, you know who I am and I like to see if something can be done on VL too! 3- practical applications is then to see dynamic behaviour of muscle fascicle first. Can this be obtained with just a flash isometric contraction too, without moving the muscle throughout the whole range of motion? Additional comments: I enjoyed this paper and the advance in the methodology that this can bring. I thank the authors for a nice read and an entertaining explanation of a new methodology advice that could actually make fascicle length assessment more reliable.

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

Title: INFORMATION FROM DYNAMIC LENGTH CHANGES IMPROVES RELIABILITY OF STATIC ULTRASOUND FASCICLE LENGTH MEASUREMENTS Review round: 1 Reviewer: 3

Basic reporting: The authors provide a clear and unambiguous manuscript with sufficient literature and background context. Ultrasound imaging of skeletal muscle is an emerging technique to understand dynamic muscle behaviour and is of general interest to the biomechanics, muscle mechanics fields. The article structure is sound and the hypotheses are well laid out. Experimental design: The experimental design is adequate and the research question is well defined. The techniques and ethical standards are indeed met. There are some additional details of the methods utilized that I have suggested in the general comments section which the authors should clarify in the manuscript. Validity of the findings: The data is statistically sound, however I would like to see some additional analysis performed (as outlined in the general comments). I have some suggestions regarding the conclusions for the authors which, in my opinion, would strengthen the manuscript. Additional comments: Information from dynamic length changes improves reliability of static ultrasound fascicle length measurements SUMMARY OF WORK: In this study, the authors examine different strategies for manually tracking medial gastrocnemius fascicle length from B-mode ultrasound images during passive ankle rotations. Specifically, they compare fascicle lengths determined from a single static image, termed uninformed fascicle identification (UFI), to fascicle lengths determined from tracking a series of images allowing the identifier to adjust the initial fascicle length, termed data-informed tracking (DIT). They compared results from 3 different raters as well as from conditions where the fascicle was at an initial short length versus an initial long length. Their results show that DIT improves the reliability of fascicle length measurements and suggest caution when measuring fascicle lengths from one single ultrasound image as well as when comparing absolute fascicle lengths across different studies. The manuscript is well-written and would be of interest to ultrasound researchers. I suggest a number of things below that the authors should address in order improve the clarity/accuracy/completeness of the manuscript. --------------------------------------------------------------------------------------------------------------------- COMMENTS: ABSTRACT: 1. Line 40: It is unclear what the authors are referring to by “four strategies.” Do you mean UFI, DIT, initial long and initial short? I am under the impression that UFI was compared to DIT only for a single frame (ie: the initial frame) whereas DIT initial long was compared to DIT initial short for a passive cycle (many frames). This sentence needs clarifying as the four strategies are not for the same condition. INTRODUCTION: 2. Line 65: I’m not sure that ‘seriously erroneous’ is the correct word choice. Inferences about fascicle behaviour from kinematics alone is challenging in MTUs that contain considerable SEE, but may provide information about fascicle behaviour in MTUs with negligible external tendon. I suggest rephrasing. 3. Please clarify in the first paragraph of the introduction that you are referring to B-mode ultrasound. 4. Line 72: I suggest replacing “in dynamic settings” with “during dynamic tasks” 5. Line 96: I suggest using “optical flow algorithms” rather than “…modelling” 6. Line 117: Please clarify to the reader what you mean by “in practice” 7. Line 136-126: Please include why the medial gastrocnemius is popular for ultrasound studies. METHODS: 8. Line 145: why was 5 out of the total 28 participants chosen for the analysis? Indeed a larger sample would improve the statistical power. This requires some clarifying. 9. Line 167: how was the ultrasound probe secured to the participant during the experiments? 10. Line 170-174: To my knowledge the ultrasound images were not “processed using a semi-automated algorithm” as is stated in the manuscript. This is partly misleading. I am under the impression that the authors used the Ultratrak GUI to manually track all images (both single frame and image series). Was Ultratrak in fact used to initially track the image sequences and then the observer then clicked through tracking results for each frame to update the locations of the upper and lower fascicle insertion sites? If so, this needs to be clarified. If not, it is unclear why Ultratrak was used rather than ImageJ or another image processing software. Please clarify. 11. Line 180-184: Did the raters watch the ultrasound sequences first before tracking (for both the uninformed and the informed tracking)? From my experiences this is a very helpful way to understand fascicle behaviour before tracking manually during cyclical tasks. Further, did the raters always come back to the initial frame and adjust the fascicle end points in the DIT? This needs to be clarified. 12. Line 230: I would like to see a brief description of the “offset-corrected condition” within the analyses portion of the methods. Please include this. 13. The major concern I have with this analysis (and the accompanied statistical results) is that the authors present absolute lengths only. Indeed (as the authors note in the discussion), the differences in results from DIT for the initial short versus the initial long conditions are an artifact of the absolute fascicle length (and the fact that the fascicle may have been out of the field of view for the initial long condition) - specifically the increased likelihood for differences in identifying total fascicle length between raters. I would like to see further analysis comparing initial short and initial long but using % length change or strain. RESULTS: 14. Line 244-261: It is my preference to leave out p values if they are not significant. 15. Please include results from analysis of % fascicle length change or strain as noted previously. These could be added to Figure A with a dual Y axis (left side absolute length change and right side % length change from initial or rest). DISCUSSION: 16. Line 290: I believe this is a typo and should be “this type of error” or “these types of errors” 17. Line 309-318: To my knowledge, the joint movements you are tracking fascicles throughout are not cyclical but rather start at either a plantarflexed position (initial short) and move to dorsiflexed or vice-versa and do not repeat (ie: are not cyclical since they do not come back to initial position). This paragraph begins with discussing cyclical movements- which your results can provide some insight for, but do not assess. Please re-work this paragraph. 18. Line 312: Out of curiosity, how many researchers did you personally communicate with in regards to their preference to start tracking on lengthened fascicles? Were all of these researchers trained in ultrasound by the same group(s)? In my opinion, lengthened typically means that the fascicle end points are out of (or nearly out of) the field of view of the telemed linear transducer, so I prefer shorter lengths. 19. One of my major suggestions for the discussion would be for the authors to include some discussion of comparing these methods to the key frames adjustment that is implemented within Ultratrak. The key frames adjustment would qualify as a type of ‘informed tracking’ and seems to be the gold standard for many research groups tracking fascicles during dynamic tasks or dynamometer contractions. In particular, the first author of this manuscript has used Ultratrak (so presumably the key frames correction) in his previous studies. 20. Line 354-355: As previously mentioned, I would like to see this analysis added to the manuscript. The statement “ this effect may be cancelled when calculating differences relatively to the absolute fascicle length” is, in my opinion, the major limitation to this study which the authors have the data to perform the analysis on. 21. Line 360: I am not sure what you mean by “in a relative sense.” I believe that you mean that normalized length changes or strains are more consistent between raters but absolute length changes differ. Please clarify this sentence in the discussion. 22. Line 365: “both static and active” should be rephrased to either “static and dynamic” or “passive and active”. 23. Line 369-371: The statement “As such, we recommend caution when drawing conclusions from fascicle length comparisons with differences below 10% of absolute fascicle length” seems a bit bold to me. Numerous studies (including some by co-authors of this manuscript) do report fascicle length changes that are less than 10%, for example in the plantarflexors during normal walking. Comparing normalized length changes or strains across studies is, in my opinion a valuable metric. 24. Line 373: “…when comparing absolute fascicle lengths” across what tasks? Please clarify FIGURES: 25. Figure 1: I would like to see Figure 1 also include the initial short conditions (either on the same plots or as C/D panels. Of interest to readers would be whether the fascicle follows a similar length change trajectory when shortening and lengthening. In addition, as previously mentioned, some methodological details regarding the “initial frame offset” (Fig 1B) is necessary within the manuscript. OTHER COMMENTS: 26. What was the rationale for passive ankle rotations rather that active (as a % of Fmax or max ankle moment)? From my own ultrasound tracking experiences, I do believe that tracking fascicle length changes in active muscle to be (slightly) more straightforward than passive muscle. If anything, I would lie to see this discussed as a limitation of the current 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: INFORMATION FROM DYNAMIC LENGTH CHANGES IMPROVES RELIABILITY OF STATIC ULTRASOUND FASCICLE LENGTH MEASUREMENTS Review round: 2 Reviewer: 1

Basic reporting: I don't have any further comment. Experimental design: I don't have any further comment. Validity of the findings: I don't have any further comment. Additional comments: I don't have any further comment.

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

Title: INFORMATION FROM DYNAMIC LENGTH CHANGES IMPROVES RELIABILITY OF STATIC ULTRASOUND FASCICLE LENGTH MEASUREMENTS Review round: 2 Reviewer: 2

Basic reporting: All is good. Experimental design: All is good. Validity of the findings: All is good. Additional comments: I commend the authors for a nice manuscript. This has now further improve.

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

Title: INFORMATION FROM DYNAMIC LENGTH CHANGES IMPROVES RELIABILITY OF STATIC ULTRASOUND FASCICLE LENGTH MEASUREMENTS Review round: 2 Reviewer: 3

Basic reporting: Text: Line 95-95 The sentence “...because it is a superficial muscle with relatively short muscle fascicles and it has important contributions during tasks such as walking, running and jumping” should include some reference(s). Line 117: Authors state "From these 28 participants, 5 were randomly selected and single-image ultrasound recordings were taken for an intra-rater reliability test and the UFI analyses" If I correctly understand the newly added Figure 1, then I believe that following the above copied sentence you need to add one more sentence that says. “For the DIT comparisons, all 28 subjects were included” If this is not correct then I am not sure Figure 1 matches the methods text, as from the left side of the figure it appears that all 28 subjects were included in at least the DIT portion of the analysis. If I had not looked at this figure but only read the text, then I would be under the impression that only 5 of the 28 were included for all analysis. Line 280-281: Please include the specific figure number in Seymore paper in which you are referring to here Line 321-323: The added sentence “However, other muscle-tendon units often present different fascicle geometries, for example the knee extensors have much longer fascicles and more curvature of the fascicles” requires a reference. Line 399 Please add “the” to sentence “…by a single rater reliability of the analysis can be increased” should read “…by a single rater the reliability of the analysis can be increased” Supplemental Files: What do the data examples AVI videos correspond to? The videos appear to be of a failed auto tracking algorithm? These files need a description bit of text or title to accompany them. Figure 1 caption: I suggest “schematic” rather than graphical summary (this is not really a graph). Experimental design: No comment Validity of the findings: No comment Additional comments: No comment