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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: A MICROBIAL SURVEY OF THE INTERNATIONAL SPACE STATION (ISS) Review round: 1 Reviewer: 2

Basic reporting: No comment. Experimental design: No comment. Validity of the findings: No comment. Additional comments: Lang et al. present a manuscript that examines the microbiome of the ‘built environment’ of the International Space Station (ISS) using 16S rRNA sequencing on the Illumina MiSeq. Sample diversity did not cluster by sample surface. Comparisons of ISS swabs to data from homes and humans revealed that the surfaces of human homes are more similar to ISS surfaces than human body surfaces are, but that neither is significantly similar. The proportion of known human pathogens on the ISS surfaces was comparable to those in ventilated rooms in a health-care facility. Overall this is a clearly-worded, concise manuscript. The question of which microbes inhabit the surfaces of human-built environments is both important and interesting, especially in the case of the ISS. Many of the comments/edits that I have suggested are minor, with the exception of a few bigger concerns that I would like to address. Firstly, in the introduction where the authors mention studies that also used deep sequencing to examine ISS microbiome, it would be helpful to have a sentence or two expanding on the results that they obtained, in order to compare your results. You did have a comparison in the results section to the Venkateswaran study, so you could expand upon the Novikova and Castro studies there. At least a comparison of the surface samples that are relevant to your study. While I understand the necessity of closed OTU picking for the comparison of your data to HMP and Homes data, it would be good to include a statement about the limitations of this method. Also, you are losing a lot of your data by rarefying to 1000 or less sequences for your comparisons with HMP, Homes and mechanically ventilated areas. Could you please explain why it was necessary to rarefy to such a level? Finally, you should put the statistical numbers stated in the text in their appropriate figures too, for example the Adonis tests. Below are some smaller edits with reference to their location in the text: - Line 32 in the Abstract – “as plans for human exploration and colonization...come to fruition.” I think it was phrased better in line 53 of the introduction where it says “possibility of colonization”. It comes off a little strong when stated as a fact. - As stated above I think it would be worth expanding on the findings of previous studies in paragraphs 2 and 3 of the introduction - Line 97 This link leads to a site that directs you to a new link. So maybe just put the new link? - Line 165 – for Table 1 it might be good to have an explanation in the supplemental information on why you correlated particular Homes samples with particular ISS samples. Most of them are intuitive but for example the similarities between air vents and door trimming is not obvious - Line 181 – Should the EMP primers have a citation? - Line 192 – This is a little vague – do you mean there were equal concentrations of each product in the pooled sample? - Line 199 – Is there a link for the QIIME workflow? - Lines 201 and 209 – there are some ### and I’m not sure if some information is missing there. The sentence in 201 is definitely not complete. - Line 228 Describe limitations of closed OTU reference picking - Line 232 – you are missing a space after the bracket - Line 235 – Did you mean rarefy to “1000 sequences” - Line 243 – Why rarefy to 700 sequences here instead of using the 1000 cutoff from before - Line 299 – Missing a space before the bracket

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

Title: CELL WALL COMPOSITION AND LIGNIN BIOSYNTHETIC GENE EXPRESSION ALONG A DEVELOPMENTAL GRADIENT IN AN AUSTRALIAN SUGARCANE CULTIVAR Review round: 1 Reviewer: 1

Basic reporting: Authors should provide the comparative analysis of lignin biosynthesis pathway genes expression data and lignin content from different cultivars available till date. This will give support to this study that how it adds additional information with the preexisting data. Experimental design: In Materials and methods section, remove the para describing about HCT gene from line 71-82 and place it in discussion section. Validity of the findings: Expression analyses of the genes provided by the authors did not match with the lignin content across developmental gradient. Early step genes e.g., PAL, C4H, 4CL, and C3H are also involved in biosynthesis of variety of flavonoids so their expression can not be solely related with lignin production. Downstream pathway genes e.g., CCR and CAD are highly expressed in young tissue (A), which has lesser lignin content as compared to other tissues. Similarly, CCR and CAD showed higher expression in roots but no data for cell wall component is provided. F5H and CCoAOMT are not showing any specific expression pattern across developmental tissue stages. I suggest authors to add functional validation experiments of one or more genes e.g., CCR/CAD/COMT using gene silencing or gene editing approach to support data provided in this manuscript. Additional comments: Manuscript is written well.

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

Title: CELL WALL COMPOSITION AND LIGNIN BIOSYNTHETIC GENE EXPRESSION ALONG A DEVELOPMENTAL GRADIENT IN AN AUSTRALIAN SUGARCANE CULTIVAR Review round: 1 Reviewer: 2

Basic reporting: Figure 1 Should be improved as mentioned in general comments. Experimental design: Authors should explain better why they did chose the cultivar and also why 9 months. All these information should be added to the text. Validity of the findings: Work here analyzed did not represent any unprecedented data as commented by authors in the manuscript. By the other hand, they add some knowledge about lignin accumulation mainly in root which for me is the main result of this manuscript. Authors also elucidate the possibility to use some promoter in further genetic manipulation basing on their expression profile. Still, would be desirable if authors could add S/G ratio values in order to check if the expression of some key enzymes (such as F5H and COMT) could be related to S/G in different parts of sugarcane culm. Additional comments: Line 38-40: Authors should explain this sentence better Line 86: Authors should explain why they choose this stage (9 months) Line 98-99: Authors did not mention that RNA integrity was checked before proceeding to DNAse treatment Line 126: Authors must include in figure 1 a picture of sugarcane cultivar that they analyzed at 9 months showing details of five sections here analyzed and roots as well. This definitely will help readers to understand which part of sugar cane were studied here Line 132: sentence should be replaced by separated into two categories Line 135: word fall should be replaced all long the manuscript. Inadequate word Line 137: Change word ‘Of’ for ‘Among’ Line 139-143: Rewrite the sentence. CAD showed the lower expression difference among all five sections analyzed with different variation ranging from 25 to 40%. This example should also be used to rewrite previous sentences comparing results. Line 171: This explanation should be added to M&M section to explain why use this cultivar. Authors should also explain if they expect that this smut-resistant feature could have or not any relation with lignin biosynthesis/accumulation.

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

Title: CELL WALL COMPOSITION AND LIGNIN BIOSYNTHETIC GENE EXPRESSION ALONG A DEVELOPMENTAL GRADIENT IN AN AUSTRALIAN SUGARCANE CULTIVAR Review round: 1 Reviewer: 3

Basic reporting: The manuscript by Bewg and Coleman describes the patterns of gene expression related to lignin metabolism and the composition of stem and root materials of the Australian cultivar KQ228. The manuscript is well written in general and brings results that deserve to be published in my opinion. The discussion about gene expression is well balanced in itself. However, the authors failed in contextualizing the existent knowledge about cell walls. As a result, the manuscripts end up quite lignin centric. This won't be a problem if authors haven´t measured cell wall composition. In this part, the manuscript is quite weak, describing methodology poorly and not discussing it adequately in the face of the available literature. Because authors did not discuss the carbohydrate portion of the wall at the same level they discuss lignin gene expression; the manuscript fails to take most of what the results can give. My opinion is that the manuscript is publishable, but it needs major changes so that it reports the scientific context of sugarcane cell walls, describe methods accurately and discuss more profoundly the results in relation the available literature. Experimental design: Preparation of material: A picture or drawing of the plant and the positions sections were made could be useful for the reader and other authors that intend to repeat the experiment. Cell wall composition: 1) Please describe how acid hydrolysis was made instead of just citing another paper. Instead of giving the components of a solution only, please report time and temperature. Why was sulfuric acid chosen? It usually hydrolyzes cellulose and decreases the proportion of pectin and hemicellulose monosaccharides. This happened as can be seen in Table 1. What authors want to see by accessing mostly cellulose? As lignin is bound to hemicellulose – the arabinoxyl residues of arabinoxylans – wouldn´t it be better to look at the levels of arabinose and therefore use TFA instead of sulfuric acid for hydrolysis? 2) How was acid linin extracted? What acid lignin means. There are several different methods for lignin extraction. Explain why this method was chosen. The lack of adequate methodological description prevents interpretation of the results reported in Table 1. The reader would have to find literature that describes methodologies and guess what authors have used in this paper. 3) Usually, cell wall monosaccharides are analyzed on Dionex because other types of chromatography do not separate well some of them. Was the Waters chromatography efficient to separate them? It would be important to add a chromatogram as supplemental material to inform the reader about the accuracy of monosaccharide measurements. Validity of the findings: Results 1) Didn´t authors notice that there is a strong negative correlation (r2=0.89) between total lignin and arabinose? How authors interpret that? 2) The percentage of total lignin is quite high. Could this be an artifact of the method used? Authors should comment that. 3) Authors cannot call the composition “secondary cell wall”. Hydrolysis with sulphuric acid attacks all cell walls, including the primary ones. Discussion For discussion on sugarcane cell wall composition, authors failed to include De Souza et al., 2013, which would be a better comparison than maize, which is relatively distant taxonomically from sugarcane and miscanthus. Lignin (yield only) in sugarcane roots has been reported by Leite et al. 2017, Annals of Botany. The authors found rather low yields in roots of a Brazilian variety Authors missed an important trend that is the inverse correlation between lignin and arabinose. It is accepted that ferulic acid, the holder of lignin in the wall of grasses is esterified to arabinose residues of arabinoxylans (the main hemicellulose in cell walls). (see De Souza et al., 2013, Bioenergy Research for sugarcane and De Souza et al., 2015 Journal of Exp. Botany). This could be interesting for the discussion of the manuscript. Additional comments: The manuscript by Bewg and Coleman describes the patterns of gene expression related to lignin metabolism and the composition of stem and root materials of the Australian cultivar KQ228. The manuscript is well written in general and brings results that deserve to be published in my opinion. The discussion about gene expression is well balanced in itself. However, the authors failed in contextualizing the existent knowledge about cell walls. As a result, the manuscripts end up quite lignin centric. This won't be a problem if authors haven´t measured cell wall composition. In this part, the manuscript is quite weak, describing methodology poorly and not discussing it adequately in the face of the available literature. Because authors did not discuss the carbohydrate portion of the wall at the same level they discuss lignin gene expression; the manuscript fails to take most of what the results can give. My opinion is that the manuscript is publishable, but it needs major changes so that it reports the scientific context of sugarcane cell walls, describe methods accurately and discuss more profoundly the results in relation the available literature. Introduction The introduction does not have a single word about the composition and structure of sugarcane cell walls (e.g., De Souza et al., 2013 Bioenergy Research). It doesn´t mention either important work performed on sugarcane lignin (e.g., Bottcher et al. 2013 Plant Physiology; Vicentini et al. 2015 PloS one). Another example regarding lignin is the work of Jung et al. 2013, Plant Biotechnology Journal. Authors should contextualize better sugarcane cell walls in the introduction. Methods Preparation of material: A picture or drawing of the plant and the positions sections were made could be useful for the reader and other authors that intend to repeat the experiment. Cell wall composition: 1) Please describe how acid hydrolysis was made instead of just citing another paper. Instead of giving the components of a solution only, please report time and temperature. Why was sulfuric acid chosen? It usually hydrolyzes cellulose and decreases the proportion of pectin and hemicellulose monosaccharides. This happened as can be seen in Table 1. What authors want to see by accessing mostly cellulose? As lignin is bound to hemicellulose – the arabinoxyl residues of arabinoxylans – wouldn´t it be better to look at the levels of arabinose and therefore use TFA instead of sulfuric acid for hydrolysis? 2) How was acid linin extracted? What acid lignin means. There are several different methods for lignin extraction. Explain why this method was chosen. The lack of adequate methodological description prevents interpretation of the results reported in Table 1. The reader would have to find literature that describes methodologies and guess what authors have used in this paper. 3) Usually, cell wall monosaccharides are analyzed on Dionex because other types of chromatography do not separate well some of them. Was the Waters chromatography efficient to separate them? It would be important to add a chromatogram as supplemental material to inform the reader about the accuracy of monosaccharide measurements. Results 1) Didn´t authors notice that there is a strong negative correlation (r2=0.89) between total lignin and arabinose? How authors interpret that? 2) The percentage of total lignin is quite high. Could this be an artifact of the method used? Authors should comment that. 3) Authors cannot call the composition “secondary cell wall”. Hydrolysis with sulphuric acid attacks all cell walls, including the primary ones. Discussion For discussion on sugarcane cell wall composition, authors failed to include De Souza et al., 2013, which would be a better comparison than maize, which is relatively distant taxonomically from sugarcane and miscanthus. Lignin (yield only) in sugarcane roots has been reported by Leite et al. 2017, Annals of Botany. The authors found rather low yields in roots of a Brazilian variety Authors missed an important trend that is the inverse correlation between lignin and arabinose. It is accepted that ferulic acid, the holder of lignin in the wall of grasses is esterified to arabinose residues of arabinoxylans (the main hemicellulose in cell walls). (see De Souza et al., 2013, Bioenergy Research for sugarcane and De Souza et al., 2015 Journal of Exp. Botany). This could be interesting for the discussion of the manuscript.

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

Title: CELL WALL COMPOSITION AND LIGNIN BIOSYNTHETIC GENE EXPRESSION ALONG A DEVELOPMENTAL GRADIENT IN AN AUSTRALIAN SUGARCANE CULTIVAR Review round: 2 Reviewer: 1

Basic reporting: No comments Experimental design: No comments Validity of the findings: No comments Additional comments: In this revised manuscript, authors answered all the questions/comments raised by reviewers so in my opinion this manuscripts has sufficient data to publish. Authors improved this manuscript well, however; my only concern is about the English language/grammar which need to be improved for publication and it would be great if authors could provide the lignin content and cell wall composition data for roots.

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

Title: CELL WALL COMPOSITION AND LIGNIN BIOSYNTHETIC GENE EXPRESSION ALONG A DEVELOPMENTAL GRADIENT IN AN AUSTRALIAN SUGARCANE CULTIVAR Review round: 2 Reviewer: 2

Basic reporting: No comment Experimental design: No comments Validity of the findings: No comment Additional comments: The authors have improved the previous manuscript version and also asked all the question resulting in a better version. All the doubts regarding plant material used, developmental stages used for experiments were answered.

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

Title: CELL WALL COMPOSITION AND LIGNIN BIOSYNTHETIC GENE EXPRESSION ALONG A DEVELOPMENTAL GRADIENT IN AN AUSTRALIAN SUGARCANE CULTIVAR Review round: 2 Reviewer: 3

Basic reporting: Article is relevant for the area of bioenergy Experimental design: Authors corrected the problems pointed out accordingly Validity of the findings: Results are sound and valid. Deserve to be published Additional comments: Authors have changed the text and properly responded the queries. In my opinion the manuscript is now acceptable 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: MICROCOS MAGNIFICA (SPARRMANNIACEAE) A NEW SPECIES OF CLOUDFOREST TREE FROM CAMEROON Review round: 1 Reviewer: 1

Basic reporting: New species are significant scientific discoveries, and it is important that such findings be published (and for new species to be properly described and named). This reviewer finds that such work is within the stated scope of the journal PeerJ. The structure of the manuscript is consistent with that seen in short articles in the field of plant taxonomy. The structure also seems to be acceptable according to PeerJ standards. The author of the manuscript is a first-language English speaker, but this reviewer finds that the way the text is written may be difficult for non first-language English speakers to understand. It almost seems as if the text was written in a hurry. Let me recommend that long sentences should be shortened and the writing should be done in a more direct (telegraphic) style. The line drawing (Fig. 1) by Andrew Brown is fine work, but the version seen by this reviewer appears to be an early draft (not a finished plate). The version presented is not acceptable for publication. The distributional map (Map 1) is also fine, but it would help to orientate the readers (especially those unfamiliar with the geography of Cameroon) if some additional landmarks could be added, e.g., administrative boundaries (regional and international) as well as the locations of major cities and/or regional capitals. It would be really helpful if high-resolution scanned images of the specimens cited could be posted on the Kew Herbarium Catalogue (http://apps.kew.org/herbcat/gotoHomePage.do ) or a similar on-line database. This way the specimen-images and associated information would be publicly accessible. Below are references to some additional, seemingly relevant literature which the author may want to cite in the Introduction of the revised manuscript: Brunken U, & Muellner AH. 2012. A new tribal classification of Grewioideae (Malvaceae) based on morphological and molecular phylogenetic evidence. Systematic Botany 37: 699-711. [Grewia and Microcos as distinct genera, placed in different subclades of tribe Grewieae Endl. The ‘Microcos subclade’ also includes Colona Cav., Goethalsia Pittier, Luehea Willd. and Lueheopsis Burret.] Chattaway MM. 1934. Anatomical evidence that Grewia and Microcos are distinct genera. Tropical Woods 38: 9-11. Czarnecka E, Wiland-Szymańska J, Gawrońska K. 2006. Phytogeography of the genus Microcos L. (Malvaceae, Grewioidae) in Africa. Biodiversity Research and Conservation 3-4: 269-271. Nurul-Aini CAC, Noraini T, Latiff A, Chung RCK, Nurhanim MN, Ruzi M. 2013. Systematic significance of petiole anatomical characteristics in Microcos L. (Malvaceae: Grewioideae). Malayan Nature Journal 65 (2&3): 145-170. Shokefun EO, Ayodele,AE & Akinloye AJ. 2016. Systematic importance of leaf anatomical characters in some species of Microcos Linn. section Eumicrocos Burret in Nigeria. American Journal of Plant Sciences 7: 108-117. http://dx.doi.org/10.4236/ajps.2016.71012 Sprague TA. 1909. The section Microcos of Grewia in Africa. Bulletin of Miscellaneous Information (Royal Botanic Gardens Kew) 1909 (2): 66-68. Experimental design: Fine although it would be good for the reader to know approximately how many specimens of African Microcos were studied by the author in the K, P and WAG herbaria as part of the pattern-matching exercise leading to the conclusion that the plants from Mt Kupe (Cameroon) are a new species. This reviewer tested the hyperlink provided by the author for his search of African Microcos in the herbarium of MNHN Paris, and the resulting page was not of Microcos or Malvaceae sensu lato but was instead for a specimen-record of Ledermanniella schlechteri (Podostemaceae). This hyperlink thus needs to be double-checked and corrected if necessary. Given that the last worldwide treatment of Microcos was published by Burret (1926), that Burret was based at the Botanischer Garten und Botanisches Museum Berlin-Dahlem, and that the Berlin herbarium was largely destroyed during WW2, it would be interesting for the reader to know to what extent our knowledge & further study of African Microcos is hampered by the type specimens having been lost. To what extent are Burret's types still extant or duplicated in other herbaria? Why was the Berlin herbarium evidently not consulted in the course of this study? Validity of the findings: The author’s ‘Key to the species of Microcos tree species in Africa west of DRC & the Congo River’ includes only three species and is thus evidently incomplete. For example, the recent article by Shokefun et al. (2016) reported there are 8 species of Microcos in West Africa, and they reportedly sampled six of these species for their leaf anatomical study of Nigerian Microcos. The “missing” species in the present author’s key would thus seem to include at least M. africana, M. barombiensis, M. iodocarpa, M. malacocarpa, M. oligoneura and perhaps others as well. Although I have no reason to doubt the author’s taxonomic judgement, the apparent omission of several West African species from the author's key leads this reviewer to think that with further checking the plants from Mt Kupe (Cameroon), proposed by the author as a new species M. magnifica, might prove to be a species previously described by a different author and under a different species name. Additional comments: It is acknowledged there are differences of opinion on whether the major clades within the order Malvales should be treated as subfamilies within a broadly circumscribed Malvaceae or whether it is better to treat them as separate families. In fact the choice of rank for these taxa is more-or-less arbitrary. However, in the interest of avoiding any possible confusion, this reviewer recommends that just one family-name should be used in the TITLE of the manuscript, i.e. the family-name should be given as either Sparrmanniaceae or Malvaceae-Grewioideae, but not both. Of course it would still be appropriate to present some background information on these alternative family treatments in the INTRODUCTION.

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

Title: MICROCOS MAGNIFICA (SPARRMANNIACEAE) A NEW SPECIES OF CLOUDFOREST TREE FROM CAMEROON Review round: 1 Reviewer: 2

Basic reporting: There are some inconsistencies in citations and in formatting in the references, but these are minor issues. Experimental design: The manuscript presents a description of a new species from West Africa. It appears to satisfy all of the requirements set out in the International Code of Nomenclature for Algae, Fungi, and Plants (2012). Validity of the findings: The author has made a convincing argument that this is indeed a new species and he has placed it in context of other taxonomic and conservation research in West Africa. 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: MICROCOS MAGNIFICA (SPARRMANNIACEAE) A NEW SPECIES OF CLOUDFOREST TREE FROM CAMEROON Review round: 1 Reviewer: 3

Basic reporting: The paper provided interesting information on the new species of an endangered tree discovered in Cameroon, Africa. The abstract of this paper was adequately summarised the contents and the keywords were well representative of the paper. The introduction, results, discussion and references were clear, adequately discussed and cited. Experimental design: In general the it was excellent and clearly written. The author appears to comply with the provisions of the ICN (McNeill et al., 2012) that are necessary for validly publishing a new species. Was this paragraph (Lines 132-142) necessary to be included in this section or could it be shortened the paragraph by just citing provisions in ICN. Validity of the findings: The results are reasonable given as there were no inflorescences and flowers material since 2004 from all the reported locations. Additional comments: *Lines 1 & 62: Delete this family (or Sparrmanniaceae) in the title and abstract. A synonym of Malvaceae sensu lato. Follow the Angiosperm Phylogeny Group IV (2016). *Line 88-91: Author should include the subfam. Dombeyoideae as the genus Schoutenia from the ‘former Tiliaceae’ was placed in this subfamily. The article of Cheek (2007) was written for the family Sparrmanniaceae only. Better to cite Heywood et al. (2007) which was included other families such as Brownlowiaceae, Tiliaceae s.s. and Pentapetaceae. *Line 108: Was this key to genera modified by the author based on his additional observations on both the genera. *Line 111: Yes, in some Grewia species but it is not in Microcos. *Line 150: Incomplete sentence, hanging. *Line 176: The word “Verrucate” was used in the introduction and species description. *Lines 297-298: Would the author will also consider to study the molecular phylogenetic of both Grewia and Microcos in Africa in order to establish the generic delimitation of these two genera for Africa. **Map: Author should have a general map of Africa to show the location of Cameroon, then enlarge with this map to indicate the distribution of the new species. ***Other comments and suggestions to improve the paper, please refer to the attached PDF 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: MICROCOS MAGNIFICA (SPARRMANNIACEAE) A NEW SPECIES OF CLOUDFOREST TREE FROM CAMEROON Review round: 2 Reviewer: 1

Basic reporting: New species are significant scientific discoveries, and it is important that such findings be published (and for new species to be properly described and named). This reviewer finds that such work is within the stated scope of the journal PeerJ. The structure of the manuscript is consistent with that seen in short articles in the field of plant taxonomy. The structure also seems to be acceptable according to PeerJ standards. This reviewer suggests that all generic and species names where first mentioned in the main text should include the relevant authority for that name. The authority for the genus Microcos should also be cited in the Abstract, unless this would go against the journal’s editorial policy. As it now stands, there is one generic name (Schoutenia) and several species names (e.g., the names listed under the subheading ‘Distribution and habitat’) that are provided without authority. The generic name Microcos is mentioned in the first sentence of the Introduction, but the authority for this name doesn’t appear until the second paragraph; this problem could be solved by moving the authority to the first mention of the name Microcos in the first paragraph, or possibly by rearranging the paragraphs of the Introduction so that the background info about the genus appears first, with the specifics about the discovery of the new species Microcos magnifica coming later. There are two species names (Microcos gossweileri and Microcos coriacea) that first appear in the key to species, but without authority; in the case of the name Microcos coriacea, the authority is provided where the name is mentioned again later in the text, but in the case of Microcos gossweileri the key is the only place where this name is mentioned. The main point is that authorities for names are important and that these authorities should be mentioned consistently in the revised manuscript. Experimental design: No comment. Validity of the findings: In a previous version of this manuscript, the present reviewer pointed out that the author’s ‘Key to the species of Microcos tree species in Africa west of DRC & the Congo River’ includes only three species and is thus evidently incomplete. For example, the recent article by Shokefun et al. (2016) reported there are 8 species of Microcos in West Africa, and they reportedly sampled six of these species for their leaf anatomical study of Nigerian Microcos. The ‘missing’ species in the present author’s key would thus seem to include at least M. africana, M. barombiensis, M. iodocarpa, M. malacocarpa, M. oligoneura and perhaps others as well. To this comment, the author has responded that ‘The key presented is to the tree species of Microcos in West-Central Africa, and not to the climbing species which include those mentioned by the paper referred to’. This response is accepted, but this reviewer further notes it would be good for the author to mention explicitly the fact that there are additional Microcos species in West & Central Africa that are climbers, not trees (& perhaps state approximately how many species). Additional comments: Once these minor, added comments have been addressed, then this reviewer recommends that the paper should be accepted 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: MICROCOS MAGNIFICA (SPARRMANNIACEAE) A NEW SPECIES OF CLOUDFOREST TREE FROM CAMEROON Review round: 2 Reviewer: 2

Basic reporting: The author addressed the concerns of the reviewers and the revised manuscript sontinues to satisfy the requirements of the International Code of Nomenclature for the publication of a new species name. Experimental design: The experimental design is appropriate for the subject. Validity of the findings: The conclusions seem appropriate. Additional comments: There are two unresolved bibliographic citation issues -- missing journal number and questionable publication date. The attached version includes copy editing of the References especially as these were checked carefully against 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: MICROCOS MAGNIFICA (SPARRMANNIACEAE) A NEW SPECIES OF CLOUDFOREST TREE FROM CAMEROON Review round: 2 Reviewer: 3

Basic reporting: This paper provides a new endangered tree species discovered from West Africa. The abstract, keywords, introduction, results, discussion and references are clear, adequately discussed and cited in the paper. This work is within the stated scope of the journal PeerJ. Experimental design: It is excellent and clearly written. It follows the provisions of the ICN (McNeill et al., 2012). Validity of the findings: The results are reasonable given by the author in the paper that this is indeed a new species in West Africa. Additional comments: The author should follow APG IV (2016 in Bot. J. Linn. Soc. Vol. 181: 1-20), Mabberley (2017 in Mabberley's Plant-Book) and Steven's APG website (http://www.mobot.org/MOBOT/research/APweb/) to use family Malvaceae-Grewioideae which is widely accepted by all botanical journals. Sparrmanniaceae is a synonym in Malvaceae s.l. now as mentioned in Mabberley (2017) and also in Steven's APG website. The novel dismemberment of Malvaceae by Cheek (2006) and Cheek in Heywood et al. (2007) was not well-accepted by the botanical community/journals. In addition, the Heywood et al (2007) publication was not widely distributed and had not updated since 2007 as compared to the Steven's APG website and Mabberley (2017). Other comments and suggestions to improve the paper, kindly refer to the attached PDF 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: SKIN TEMPERATURE AND REPRODUCTIVE CONDITION IN WILD FEMALE CHIMPANZEES Review round: 1 Reviewer: 1

Basic reporting: The authors investigated body temperature changes of pregnant and cycling chimpanzee females in the field. They have used a technology new to behavioral ecology to test two clearly stated hypotheses. The first is that surface body temperatures will increase throughout the swelling cycle and peaking at maximum tumescence. The second hypothesis is that the temperature patterns of pregnant females will mimic those of non-pregnant females. The manuscript is well-written, well-structured, and clearly organized raw data and Rmarkdown analyses details have been shared as supplemental information. I have the following minor comments regarding basic reporting: 1. If females have evolved a strategy to conceal temperature changes as part of a broader strategy to conceal pregnancy, one must assume that males can detect the slight temperature changes that would correspond with pregnancy. Is it logical to assume male chimpanzees would be able to detect such small differences in female skin temperature? Is there any literature that would suggest temperature to be a meaningful, detectable signal to male chimpanzees, or to other species? (The authors do mention that male elephants were more attracted to female elephants with increased skin temperature Hilsberg-Merz 2008 , 
but presumably the attraction could have been due to olfactory or other cues also associated with reproductive state. They also mention male primates responsive to skin color and tone, but not temperature in the Higham et al 2011 reference). I think it is fine to explore this hypothesis as an extension of the concealed pregnancy hypothesis, but wanted to encourage the authors to include more justification for their hypothesis that is based on temperature perception if it is available. If it is not available, this lack of knowledge should be explicitly included in the introduction. 2. Number of cycling and pregnant females should be included in the abstract. 3. Figure 2: Is there a reason why the y-axis does not have equally spaced intervals? These should be equally or clearly labeled on the y-axis as reflecting a specific scale. Experimental design: The research question is well defined, relevant and meaningful. I have the following minor comments regarding experimental design: 1. What transformations were made to the variables (lines 158-159)? This information should be included in the main text, or reference to the supplemental information should be made here. 2. Has IRT been used before on wild chimpanzees? If so, are the methods stated in paragraph beginning line 109 based on previous studies? 3. How much do the image analysis results depend on the polygon drawing? It seems there should be inter-rater reliability on average temps obtained due to the user-defined polygons. Validity of the findings: While the data are somewhat inconclusive, the authors have shared important new methodological approaches and been upfront about the limitations of their results. I have the following minor comment about validity of the findings: 1. The authors are very upfront about limitations in their design (paragraph beginning line 274). Should decisions about photographing distance/time/lighting also be included here as variables that may influence findings, given the rarity with which IRT is used in the field? Additional comments: Additional minor comments: Line 51: “cattle animals” – reword as cattle, livestock, or farm animals. Line 42: parenthesis missing around citation Line 74: “In a first step” – reword as “As a first step” Line 75: suggest deleting “seemingly strategic” Line 77: change “which” to “whom” Line 123: “sampled” refer to sampled with IRT? Line 125-6: could females with offspring <4 years of age been cycling, even at a low probability? If so, rephrase. Line 171: “this” effect and “the” interaction – it’s not clear which variables are being referred to Line 202: is “deflated” a common term for minimal swelling? If not, consider replacing. Figure 1: It is difficult to discern the content of the images, is there perhaps a standard image available not showing the infrared that could be shown side by side? Alternatively, could some labels be placed on the photos for reference?

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

Title: SKIN TEMPERATURE AND REPRODUCTIVE CONDITION IN WILD FEMALE CHIMPANZEES Review round: 1 Reviewer: 2

Basic reporting: This manuscript had several typos and poor English construction in some parts. I would encourage the authors to make sure they carefully review a manuscript before submitting it for publication. I was never convinced about the purpose of this study. There may well be temperature changes during the reproductive cycle – such as seen in humans and other animals – but it’s not clear why the authors have jumped to the conclusion that any temperature change would serve as a “signal” to males. Has anyone ever proposed that humans use genital temperature as a cue? Or do cattle researchers think bulls use vaginal temperature of cows as signals? I doubt it. I don’t get the significance of temperature in communication. Also, remember that most of what’s physically causing the swelling of female chimpanzee ano-genitalia skin is the interstitial fluid (mostly water). Some parts of the manuscript seem to imply the swelling is mostly blood. That’s not true. Experimental design: The authors admit there were several problems with the actual design. I wonder if it would have been best to first set up an experimental design that would test the validity of the technology used in that setting, at those distances, and in those environmental conditions. Validity of the findings: Mostly this represents negative results - which is OK in this case. Graphic details were well presented. I appreciate the fact that the authors went to great lengths to describe the shortcomings of the study, but it bordered on overkill - as though it underscored the lack of importance and planning for the study. Additional comments: Careful observation of female chimpanzee swelling patterns allow for prediction of pregnancy very early on. When observation is made daily (which is possible at Budongo), one can easily map the irregular patterns of early pregnancy and predict delivery date. See the Wallis and Goodall 1993 paper for details on this and further discussion about the value of swelling during pregnancy. I would recommend carefully assessing the goal of the study. Maybe I'm missing something, but it seems you are suggesting that skin temperature may be being used as a cue for males and there's no logical explanation given for this.

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

Title: SKIN TEMPERATURE AND REPRODUCTIVE CONDITION IN WILD FEMALE CHIMPANZEES Review round: 1 Reviewer: 3

Basic reporting: This manuscript was clearly divided into different sections: introduction, methods, results and discussion. Overall, I could follow the prose in each section. For me, the main drawback was the set up for the rationale of the study. I did follow the logic that based on prior research (40-50), we may expect female chimpanzees’ skin temperature to vary over the reproductive cycle. However, I did not understand the rationale for predicting that pregnant females at maximal tumescence will mimic the temperature profile of non-pregnant females at maximal tumescence in order to confuse paternity. I wonder, is there evidence of this in other species? Reading the authors’ description of a study on elephants and rhinos (41-44), it was not clear whether males were more attracted to females during oestrous specifically due to skin temperature changes, or other signals (I could not access the cited article). If there is evidence that certain species use skin temperature of potential mates to assess reproductive state, it should be discussed in more detail in the introduction. If there is not literature on this topic, why should we assume that male chimpanzees can and do attend to the skin temperature of females and that females evolved a temperature profile to deceive males and confuse paternity? Some language in the manuscript was ambiguous, causing me confusion: 201-204: “Generally, pregnant females had lower surface temperatures than cycling females when deflated and during smaller swelling stages (stages 0 – 2, Figure 2), of less than 1°C overall. This pattern changed later in the cycle, with pregnant females having higher skin temperature compared to cycling females” It is confusing to apply the words “cycle” and “cycling” to both pregnant and non-pregnant females. The authors differentiate two groups of females “cycling vs. pregnant females” (11-12, 201-202) but then in the same sentence, mention the cycles of both pregnant and cycling females (203-204). The authors should select unambiguous phrasing for the different female reproductive groups and use it consistently throughout the manuscript (e.g. perhaps non-pregnant females). Another example of confusing language is referring to pregnant females as non-reproductive: 94: “if pregnant females follow an evolved strategy to conceal their non-reproductive state” Grammatically speaking, throughout the manuscript there were areas of awkward language (e.g. 40: “strands of research”; 74: “In a first step”; 293: “which has not or seldom been used”). Cleaning up these phrases will allow the reader to concentrate on the research, rather than the grammar. 53: The authors state, “we are not aware of systematic use of this technology on wild animals” but see Thompson et al. 2017, Journal of Thermal Biology, for a study on wild howler monkeys: http://www.sciencedirect.com/science/article/pii/S0306456516302182 Experimental design: I was pleased to see research aimed at better understanding wild female chimpanzee physiology, as there is much discover in this area. In addition, I appreciate the authors highlighting how infrared thermal imaging is a tool that can be used to study animals noninvasively. I found the authors’ two study aims to be clearly conveyed: 1. Do non-pregnant females have higher skin temperatures during estrous compared to anoestral phases? 2. Do pregnant females at maximum tumescence have similar skin temperatures compared to non-pregnant females at maximum tumescence? The authors also included a lot of detail about the statistical methods, which was useful. On a critical note, I have a concern about study methodology. I question how the authors designated pregnant versus non-pregnant females using the presence or absence of offspring after the average gestation period of chimpanzees elapsed following thermal image collection (114-120). During my own experience conducting pregnancy tests on wild female chimpanzees, pregnancy loss ostensibly occurred during the early months of gestation (based on several consecutive positive pregnancy tests followed by negative tests). Evidence of early pregnancy loss is also mentioned in Emery Thompson (2005, pg 147-148). This means that images of females in early pregnancy could have been included in the non-pregnant female category. I know it is not possible to get around this issue, but at least mentioning it will allow for better assessment of the results by the reader. Validity of the findings: The authors state: “…we did not find any difference in skin temperature between measures taken from anoestral and oestral stages in cycling females. This is inconsistent with the existing literature, and casts some doubt on the possibility that fertility is associated with a general thermal signature in female chimpanzees” (242-245). I wonder about validity of infrared thermal imaging, especially in a wild environment. Although the authors control for ambient temperature, humidity, and recording distance in their statistical models (which is good), they do not mention potential drawbacks of infrared thermal imaging. Some captive studies indicate low reliability of infrared thermal imaging to assess temperatures in primates: http://www.jzar.org/jzar/article/view/132 http://onlinelibrary.wiley.com/doi/10.1111/j.1600-0684.2007.00214.x/full It may be useful for the authors to mention whether any steps were taken to assess efficacy of their thermal imaging data. For example, Pizzi et al. (2015) mention examining reliability coefficients. http://www.jzar.org/jzar/article/view/132 Regarding the discussion, I am not convinced that because skin temperatures for pregnant and non-pregnant females were most similar during maximal tumescence, that skin temperature is necessarily part of a female strategy to remain sexually attractive and confuse paternity. The authors do not explore any alternative explanations. For example, it is possible that skin temperature is not a deceptive signal in chimpanzees, but rather, something about being maximally tumescent increases body temperature regardless of pregnancy status. There is much research on wild chimpanzee females documenting changes that occur during maximal tumescence including increased: copulation rates (Watts 2007), male sexual competition over females (Emery Thompson and Wrangham 2008), and female resistance to male solicitation (Stumpf and Boesch 2005). Could increased activity and social upheaval lead to small temperature increases during maximal tumescence? It is certainly possible. Because the authors’ dataset is modest, and they do not present the reader with clear evidence that skin temperature is a signal in animals, the discussion is not very compelling. In particular, the use of the Hiramatsu et al. 2017 citation (285) as support seems like a reach, as it is about facial coloration in rhesus macaques, as assessed by humans. The manuscript could be improved by focusing the discussion more closely on the data presented and acknowledging competing explanations for the observed temperature patterns observed. 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: SKIN TEMPERATURE AND REPRODUCTIVE CONDITION IN WILD FEMALE CHIMPANZEES Review round: 2 Reviewer: 1

Basic reporting: No comment. Experimental design: No comment. Validity of the findings: No comment. Additional comments: In this revision, the authors have addressed the minor concerns that I had with the original submission.

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

Title: SKIN TEMPERATURE AND REPRODUCTIVE CONDITION IN WILD FEMALE CHIMPANZEES Review round: 2 Reviewer: 2

Basic reporting: This version is much improved. The basic reporting and article structure are fine - and it appears most of the relevant literature have been included. See below for more details. Experimental design: I find no problems with the experimental design, although it may be useful to carefully review the methods and results section with the editorial staff; some of the wording is awkward and less clear than could be achieved with some additional effort. Validity of the findings: I'm pleased with the new version. Many of my concerns about the initial version have been addressed. In particular, the authors have toned down their suggestions about the "signalling" potential of temperature of the genitalia. One thing that concerned me last time that I failed to bring up: If there is anything about temperature that helps communicate information to the males and influence their behavior, we should be just as interested in how the temperature change influences the FEMALES' behavior. To imply that something going on in a female is only important to study as it relates to males is short-sighted. In other words, this is akin to suggesting that the anogenital swelling of a female is only about communicating to the male. We wouldn't ignore the very obvious tactile stimulation being experienced by the female herself - and we have plenty of data showing very proceptive behavior by females during times of swelling. Another thing to consider is that, though anogenital tumescence occurring during pregnancy is very likely a phenomenon that helps with sociosexual relationships, we can't say it is ONLY about confusing paternity. There are studies from captive conditions that confirm sexual attractivity and receptivity during pregnant (while swollen) - but those same studies show a difference in males' grooming and genital inspection rates. So, actual masking of pregnancy may or may not be going on. In other words, they are being treated slightly differently from the non-pregnant females - and still having affiliative behaviors (including sex) shown to them. Additional comments: Though the manuscript is much improved and I support publication, there are still a fair number of typos and basic sentence structure problems throughout. Much of this will be caught by careful proofreading by the editorial staff. Some specific areas of concern (these are just some examples): line 37: extra parenthesis should be deleted line 57: re-word this bit. You refer to a "swelling cycle" before explaining what that is. for those unfamiliar with chimpanzees, they need an introduction. line 63: be careful when referring to swelling "size." It's not really about the physical size but the level of tumescence (and lack of "wrinkles"). line 87 (and elsewhere): You may want to avoid using "oestrus" - and just stick with fertile. Many researchers argue that the term oestrus should be limited to those animals that have an extremely discrete period of sexual receptivity that is directly related to the ovulatory period. Clearly, chimpanzees exhibit interest in sexual behavior outside the time of ovulation (though they DO show higher interest while the swelling is present). Thus, I would caution how you use the term and - if you're going to use it, at least include a short discussion/disclaimer about the term. line 97, 99: extra parenthesis line 111: missing end period line 279: You mean to write "during pregnancy" line 326: Period is missing after "fertility" --- and similar missing punctuation occurs throughout the manuscript. In other words, provide a careful proofreading by multiple people to catch these minor mistakes and address the other issues I've mentioned here. At that point, I think it will be 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: SKIN TEMPERATURE AND REPRODUCTIVE CONDITION IN WILD FEMALE CHIMPANZEES Review round: 2 Reviewer: 3

Basic reporting: Overall, the manuscript is improved compared to the previous submission. Upon the advice of the editor/reviewers, the authors have provided clarity in several areas, including the sample size of females (both pregnant and not), as well as some of the background literature cited. I also found that this version of the manuscript was easier to read throughout all sections, as the authors made the recommended changes regarding sentence clarity and grammar. There were some minor grammar/typographical errors, which I note under "General Comments" as they did not greatly affect the manuscript's readability. Experimental design: I appreciate the authors response to the critique about the limitations of designating pregnant versus non-pregnant females using presence/absence of offspring after the average gestation period of chimpanzees elapsed following thermal image collection. However, I do not think the possibility of pregnant females being included in the non-pregnant female dataset is explicitly stated (lines 127-136). The authors have mainly added a justification for not relying on pregnancy tests, which is not the core issue. A clear sentence would be something like, “As we relied upon the presence/absence of offspring after the average gestation period of chimpanzees elapsed following image collection, it is possible that some females we designated as 'not pregnant', may have been in the early stages of pregnancy when thermal images were recorded, and then subsequently did not carry the pregnancy to term.” Validity of the findings: While I do think the authors were more cautious about the discussion of their findings in light of the pregnancy concealment hypothesis in this version of the manuscript, my main critique stands. The authors do not explore any alternative hypotheses for why pregnant and non-pregnant maximally tumescent female chimpanzees have similar temperature profiles. I copy and paste what I stated in my initial review, as it still is applicable: “The authors do not explore any alternative explanations (for their findings). For example, it is possible that skin temperature is not a deceptive signal in chimpanzees, but rather, something about being maximally tumescent increases body temperature regardless of pregnancy status.” Considering that a) this study has a very small sample size of pregnant females b) the temperature changes recorded throughout the study are very small in magnitude, and c) there is no evidence that male chimpanzees can detect changes in skin temperature of females, it seems like a huge misstep to completely ignore alternative explanations for the authors’ findings. It is certainly more simplistic (Occam’s razor) to conclude there is something about being maximally tumescent that increases body temperature regardless of pregnancy status, rather than arguing that female chimpanzees have evolved a strategy to mimic tiny temperature changes that males can somehow attenuate to. Additional comments: The authors have done a good job responding to and addressing the previous critiques. Moving forward, the most important thing the authors can do is acknowledge alternative explanations for their results (other than the pregnancy concealment hypothesis). This can be done easily in the discussion. Secondly, minor changes include being explicit about the designation of pregnancy status, and finally, cleaning up the grammatical and typographical errors. 34: or dogs -> and dogs 35: deployed -> employed or utilized 56: may be useful to add “wild” before “female chimpanzees” 150: fur -> hair 226: “surface temperatures” -> skin surface? 235: body temperature -> skin surface temperature 288-294: Very confusing section. What are these studies about? How were they conducted? Needs more explanation. 314: The line about difficulty getting hormonal samples is a bit confusing to me. There’s plenty of hormonal work from Emery Thompson, Deschner, Crockford and others (at Budongo and elsewhere). Perhaps it was not feasible for this study’s timeframe or budget? 326: "skin" is capitalized and should not be

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

Title: COMPARATIVE ANALYSES OF PUTATIVE TOXIN GENE HOMOLOGS FROM AN OLD WORLD VIPER, DABOIA RUSSELII Review round: 1 Reviewer: 1

Basic reporting: Although the English has been improved from the previous version of the same paper, which I also reviewed, there is still some repetition between sections. Zoological details are also frequently inaccurately given: eg Protobothrops mucrosquamatus is not a New World pitviper; on line 101, the word “crotalin” should be “crotaline” while on line 230, the word “crotalinae” should be capitalised. Line 331: the correct spelling of the Latin name of Russell’s viper is Daboia russelii. Line 345: correct spelling is Viperidae. There are too many figures of dubious value (even if the analysis was considered sound, for which see following sections). Experimental design: In a previous review of this study for another journal, I pointed out that while a major comparison made in the paper was between "Old World vipers" and "New World vipers", crotalines are present in the Old World too, and it was unclear whether they were being included in the OWV category, or whether the comparison was in fact between the Viperinae and the Crotalinae subfamilies (since all the NWV are crotalines). The authors have amended the analysis to apparently compare crotalines and viperines instead, but they now describe Protobothrops mucrosquamatus as a New World pitviper, which it is not (note also that I also doubt that any re-analysis has actually be done since the results reported for the comparative analysis are identical (word for word) to the previous version. Validity of the findings: Although the units being compared have apparently been improved, compared to the previous version, and the wording slightly changed so that they no longer claim the “fished” sequences to be “venom-associated” as they did in the former version, there are still some unjustified assumptions being made which invalidate the analysis. Foremost among these is that the genes fished out of genomic DNA extracted from the skin have anything to do with expression in the skin…they do not. Without transcriptomic studies, we have no idea where they are expressed. They may be expressed in the venom gland and they may be expressed in the skin, but could also be unexpressed or expressed all over the body. This invalidates all the conclusions being drawn and I therefore will not comment on them further. Additional comments: Although the authors have deleted a comment suggesting that conventional sampling can adversely affect the snakes from this version as requested by me in my previous review, I have recently become aware that according to Indian Wildlife Protection Act, 1972, and its subsequent amendments, even the shed skin (not exuviate, which is an incorrect term as I have already pointed out) is the property of the Government. Hence, it does not really circumvent the need to obtain permits. Nevertheless the ability to recover high quality DNA from the shed skin is certainly worthy of a note in a journal focusing on methodological studies. However, additional details will be needed such quality of the assemblies (N50, % coverage etc.). As I have already suggested in my previous review, it would perhaps be of interest to do a phylogenetic analysis to see if those encoding proteins that have been found in the venom of Russell's viper or related spp in the past (ie from EST or transcriptomic studies) for which the tag "venom- associated" can be justified, are more closely related to each other than to non-toxic housekeeping genes (e.g. Jacobo Reyes-Velasco, Daren C. Card, Audra L. Andrew, Kyle J. Shaney, Richard H. Adams, Drew R. Schield, Nicholas R. Casewell, Stephen P. Mackessy, Todd A. Castoe; Expression of Venom Gene Homologs in Diverse Python Tissues Suggests a New Model for the Evolution of Snake Venom. Mol Biol Evol 2015; 32 (1): 173-183. doi: 10.1093/molbev/msu294; Casewell NR, Huttley GA, Wuster W. Dynamic evolution of venom proteins in squamate reptiles, Nat Commun. 2012, vol. 3 pg. 1066). However, you would need to bear in mind that you just have coding sequence data, which will be confounded by strong selection pressure (see Malhotra A, Creer S, Harris JB, Thorpe RS. 2015. Toxicon 107(Pt B):344-58). The noteworthy finding in this work is that DNA suitable for NGS can be obtained from shed skin and the report ought to focus on this aspect, but probably additional analyses would be needed to compare the quality with other more standard sources.

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

Title: COMPARATIVE ANALYSES OF PUTATIVE TOXIN GENE HOMOLOGS FROM AN OLD WORLD VIPER, DABOIA RUSSELII Review round: 1 Reviewer: 2

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: When the residue identity was compared and discussed, the authors seemed to use hydrophobic vs polar as the only criteria, which is useful but not complete. For instance, in figure 2 and page 16, F changing to I removes the bulky aromatic ring, whereas S could be a phosphorylated site as opposite to the N in the same position. This could bring bigger difference in the protein structure and function than just changing the polarity. The authors need to address this to improve the argument. In figure 2, residues 67 and 68 don’t align with the original sequence (above figure 2B). It is hard to figure out the corresponding counterpart residue. The author can either change the font of the sequence or point out simply by adding arrow. Same issue in figure 6A, it is not clear to pinpoint residue 117, 18 and 19. Adding an arrow to point to the secondary structure would be helpful. In figure 4 and page 18, ’ Of the residues that are evolved in the members of crotalinae, the second residue, a positively charged one, alanine (A) is present only in the members of viperinae, which is replaced by a hydrophobic residue, proline (P), in the crotalines and elapids’, which is the positively charged residue? Ala is not charged. Is this supposed to be Arg (R)? In figure 6 (B and D) and page 19 ’The pockets appeared small in all cases for the elapids, and largest in the case of viperines’. It would be useful and straightforward to quantitatively describe the size of pocket. In figure 6 E and page 19, ’ Similarly, residue 46 of the CAP domain and residues 4 and 31 of the CRISP domain showed rotamer conflict for the viperines’. Residue 4 is in the loop region, which could experience highly dynamics motions and might be different rotamer configuration. How significant of this? Does different rotamer configurations imply anything in the protein function or it is purely due to the dynamics in the loop. It would be useful to show the side chain in the figure to clearly display the rotamer conflict between different speicies. Several references are missing in the reference section. For instance, in page 23, ‘..When we compared the 4 published studies (Kalita et al. 2017; Mukherjee et al, 2016; Sharma et al. 2015; Tan et al. 2015)…’ Kalita et al. 2017; Mukherjee et al, 2016 and Tan et al. 2015 are not listed in the literature cited. This happened in other places too and the literature cited is very short considering the reference talked in the maintext. The authors need to read full text carefully and add the corresponding references accordingly in the literature cited 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: SDMTOOLBOX 2.0: THE NEXT GENERATION PYTHON-BASED GIS TOOLKIT FOR LANDSCAPE GENETIC, BIOGEOGRAPHIC AND SPECIES DISTRIBUTION MODEL ANALYSES Review round: 1 Reviewer: 1

Basic reporting: This manuscript provides detailed documentation concerning the release of a new version of SDMtoolbox: 2.0. As the use of geospatial analyses is continually increasing in many ecological fields, the development and introduction of new tools such as this that can be used for robust study design is highly important. Generally, this manuscript and the associated “Getting Started” and “ User Guide” are clear and well written. However, I have provided detailed edits in the attached document that can improve organization and clarity of the manuscript, getting started, and user guide documents. The User Guide needs page #s to match the Table of Contents. Additionally, some of the language is unprofessional and needs to be changed as I noted in the detailed edits. Overall, the User Guide was straight-forward, however I received some toolbox errors when following the User Guide as closely as possible, and I have included the error logs for reference. I think that if a better description of the files that are being used for each step and their relative location are added, these errors may be avoided. Finally, there is a lot of detail in the User Guide concerning Maxent, however a description of the new Clog Log function is absent; this should be added. Experimental design: N/A Validity of the findings: N/A 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: SDMTOOLBOX 2.0: THE NEXT GENERATION PYTHON-BASED GIS TOOLKIT FOR LANDSCAPE GENETIC, BIOGEOGRAPHIC AND SPECIES DISTRIBUTION MODEL ANALYSES Review round: 1 Reviewer: 2

Basic reporting: Please follow correct citation style (sometimes publication year is within brackets and other times it is not) Experimental design: Ok Validity of the findings: Ok Additional comments: Reviewer 1: SDMtoolbox 2.0: The next generation python-based GIS toolkit for landscape genetic, biogeographic and species distribution model analyses. I congratulate the authors for creating and updating SDMtoolbox 2.0. The toolbox aids the automation of rigorous spatial analyses; thus, it is highly desirable among geospatial modelers and conservation professionals. My recommendation is to accept with minor revision. I encourage the authors to make suggested changes and resubmit for publication. Here are my comments. Detailed Comments Line 36, a total of 79 scripts Line 40, future climates, Line 55, a series of Line 56, complicated spatial analyses in ArcMap (ESRI) and python Line 55, line 108, and others, please write abbreviations in full at first use. Line 74, Rewrite the last sentence. Line 89, e.g., Line 101, importance of Bias files in Maxent Line 117, delete brackets, ..tools batch project or define projections of input rasters…. Line 118, delete ‘aims to’, another set of tools facilitate batch exportation of images or aid in RGB band visualization of multiband rasters Line 156, rewrite. Sentence starting As following is not grammatically correct. Line 163, make sure all figures are mentioned in the text. Line 164,insert ‘in’, it can be done in several ways Line 191, “lower levels”, did you mean resulting from lower regularization multiplier values Line 196, delete “(i.e. Bio1)” Line 205, that is more sensitive details of ? sentence appears incomplete Line 207, “which subsequently is implement in the”, sentence appears incorrect Line 217, tests and evaluates? Line 241, if the journal allows this should be written as footnote or in the acknowledgment section. Please follow correct citation style (sometimes publication year is within brackets and other times it is not) I installed the tool using the software installation guide provided in the supplemental material. The user guide needs some editing as well. Example, first sentence needs the preposition “to”. It should read like “designed to automate”. The second paragraph needs a comma immediately after SDMtoolbox 2.0. Table of contents lists page 215 while the document is only 95 pages. General comments: Line 213, the latest maxent default combination is: L, Q, P, H which appears to be more appropriate than the previous maxent versions that included all of them (i.e, L,Q,P,H and T). Are the users able to use this new maxent default combination in SDMtoolbox 2.0? The methods section is very brief, can the authors expand the methods section a little bit. ESRI is an institution that frequently changes its software versions. Given this fact, what is the authors long-term plan to keep SDMtoolbox running on new ArcGIS software versions? Can SDMtoolbox become a standalone tool?

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

Title: SDMTOOLBOX 2.0: THE NEXT GENERATION PYTHON-BASED GIS TOOLKIT FOR LANDSCAPE GENETIC, BIOGEOGRAPHIC AND SPECIES DISTRIBUTION MODEL ANALYSES Review round: 2 Reviewer: 1

Basic reporting: This manuscript provides detailed documentation concerning the release of a new version of SDMtoolbox: 2.0, and will be an excellent contribution to PeerJ. Following the edits and suggestions of the reviewers, the authors have greatly improved the manuscript and the associated “Getting Started” and “ User Guide” for the toolbox. They also made changes to the script to correct previous errors. I fundamentally disagree on the continued use of "The 10 commandments of SDMtoolbox 2.0", especially by beginning each line with "Thou shall". This is, in my opinion, the only unprofessional part of the document. I have no further edits or suggestions. Experimental design: No comment Validity of the findings: No comment Additional comments: No additional comments

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

Title: SDMTOOLBOX 2.0: THE NEXT GENERATION PYTHON-BASED GIS TOOLKIT FOR LANDSCAPE GENETIC, BIOGEOGRAPHIC AND SPECIES DISTRIBUTION MODEL ANALYSES Review round: 2 Reviewer: 2

Basic reporting: Ok Experimental design: Ok Validity of the findings: Ok Additional comments: I have minor comments. The discussion section reads well but other sections need additional work. Line 111, sentence not clear, rewrite. Line 122, sentence not clear, particularly use of the verb visualizing Line 130, incorrect use of punctuation marks. Delete the colon and all semi-colons, and replace the last phrase with "to name a few". Or rewrite. Line 155, a coma is need after OR. Or rewrite Line 203, delete (i.e mean annual temperature) 79 scripts in the main document vs 78 in the user guide. Which is correct? User guide table of content still refers to page 215

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

Title: SDMTOOLBOX 2.0: THE NEXT GENERATION PYTHON-BASED GIS TOOLKIT FOR LANDSCAPE GENETIC, BIOGEOGRAPHIC AND SPECIES DISTRIBUTION MODEL ANALYSES Review round: 3 Reviewer: 1

Basic reporting: Ok Experimental design: Ok 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: CONSTRUCTING A COMPREHENSIVE GENE CO-EXPRESSION BASED INTERACTOME IN BOS TAURUS Review round: 1 Reviewer: 1

Basic reporting: The article is acceptable in this area Experimental design: 1- The authors claimed that they constructed the first multi-tissue gene co-expression network in cattle (Lines 1,23,68,162,289,231 and 323), which is not true. The first large-scale multi-tissue gene co-expression network in cattle has been already constructed by Beiki et al (2016). 2- Combining a large number of experiments into a single robust analysis will minimize the effects of variables that can plague individual experiments. In contrast, the authors just used gene expression information from single animal (one sample per tissue) to construct the cattle multi-tissue network. Why not use all available gene expression data for this species? 3- It is well known that unwanted noise and un-modeled artifacts such as batch effects can dramatically reduce the accuracy of statistical inferences in high-throughput experiments. Unfortunately, the authors did not adjust the data for these sources of noise, which may have biased all the results of this study. 4- The version of the reference genome used in this study need to be added in the method section (line 80). 5- What’s the rationale behind the selection of the most variable genes? Filtering genes based on their variability will result in information loss during network construction. For example, while transcription factors may have very subtle expression changes between different biological conditions, the may have a very large phenotypic impact. Variation filtering can easily lose this useful information. Also, line 141-143, the authors mentioned that because the large number of genes were not differentially expressed between samples (tissues?), they decided to filter genes based on their variability. Differential expression analysis results are highly dependent on the number of samples used in the study. How many samples used per tissue in this study? If only one, how can differential expression analysis be performed based on a single sample per tissue? 6- Eigen value definition needs to be corrected on line 110. In WGCNA, Eigenvalues are calculated based on Singular Value Decomposition (SVD) not PCA and defined as the first left singular vector (referred to as the eigenvector) that explains the maximum amount of variation in the expression matrix. 7- Instead of the description of network topology based of different network statistics specifically developed for WGCNA networks (network heterogeneity, network density, network centralization…) why did the authors use topological centrality analysis in Cytoscape (line 119)? 8- What is the benefit of the shortest path analysis (line 121) while TOM based neighbor analysis can be easily conducted on the data? 9- the Results and methods sections are not fully separated in the manuscript. For example, lines 128-177 described the method used in the study while included in the results section. 10- The reference gene set used in the functional enrichment analysis (KEGG pathway and GO term analysis) was not included in the method section. 11- The significant level of connection between genes needs to be selected based on the TOM values distribution. What basis did the authors use - 0.01 as the significant TOM connection strength (line 215)? 12- The sentence at line 130-131 needs to be re-written as it unclear. Validity of the findings: I have enough concerns with the methods used that I am not confident in the validity of the finding of this project. Additional comments: No additional comments

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

Title: CONSTRUCTING A COMPREHENSIVE GENE CO-EXPRESSION BASED INTERACTOME IN BOS TAURUS Review round: 1 Reviewer: 2

Basic reporting: The paper from Chen et al. is overall written well with however some typos and/or unclear sentences (e.g., L105-107, confusing the “as more gene as possible”; L115: “interested genes” should be “gene of interest”) throughout the whole manuscript. The authors are right in pointing out the importance of having an interactome in bovine. The author’s hypothesis was “that the complex genetic traits related to cattle production are reflected by the perturbation of gene-gene co-expressing networks”. The method used (Weighted Gene Co-Expression Network Analysis) in the transcriptome of 92 tissues of bovine without any type of animal production data associated with them does not seem to be able to attempt to demonstrate the hypothesis. I suggest the authors to change the hypothesis and, maybe, provide a statement of need instead of a hypothesis, based on the fact that such large co-expression database is missing. What provided in this paper is a co-expression dataset using transcripts. The transcripts are, in general, mature mRNA (or other types of RNA); thus, the coexpression analysis using the transcriptome somewhat disregards post-transcriptional regulations and, therefore, do not really reflect the real co-regulation at DNA level. I would like the authors to consider this point. It is unclear to the reviewer why the authors had selected the genes with the highest variability between tissues considering that the purpose was to study co-expressed genes. Experimental design: No comments Validity of the findings: The authors did not provide any validation of their findings. Because they purpose was to find co-expressed genes I suggest to check if any of the networks detected present enrichment of up-stream regulators. If the genes are really co-expressed they should have commons up-stream regulators. Additional comments: L119: unclear what is “degree”. Please, clarify L134: unclear the word “mature” L142: how the statistical analysis was run to identify differentially expressed genes? L171: unclear the word “friends” L210: the method used can aid in identify co-expressed genes but cannot really identify communication between genes. I suggest changing in “coexpression” L290-292: unclear how the method used by the authors can characterize subcellular localization and complex formation of genes products.

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

Title: CONSTRUCTING A COMPREHENSIVE GENE CO-EXPRESSION BASED INTERACTOME IN BOS TAURUS Review round: 2 Reviewer: 1

Basic reporting: Acceptable Experimental design: Acceptable Validity of the findings: acceptable Additional comments: This statement in the abstract “By using robust WGCNA (Weighted Gene Correlation Network Analysis), we described the gene co-expression network of 13,405 protein-coding genes from the cattle genome.” Is not correct given this statement “Using 19,064 genes with expression values, we ran a quality control step and removed those genes without expression values in more than half of 92 tissues. This provided a list of 13,405 genes with expression across 92 tissues. However, a large number of these genes were not differentially expressed between samples. Therefore, the data set with 13,405 gene expression was processed further by focusing on the 5,000 most variant genes (Table S1). The remaining 8,405 genes, which showed no or very low changes in expression between samples, were not used for WGCNA analysis.” In the materials and methods section. If I am understanding correctly, the network is for only 5000 genes. If this is correct, the abstract needs to be corrected. I am confused by this statement “However, a large number of these genes were not differentially expressed between samples”. Based on the authors response, no differential gene expression analysis was conducted. Given that how can this statement be made?

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

Title: PATTERNS IN ARTISANAL CORAL REEF FISHERIES REVEALED THROUGH LOCAL MONITORING EFFORTS Review round: 1 Reviewer: 1

Basic reporting: The authors did a great job in reporting on the disparate data of creel surveys. The MS is clearly written although could benefit from an additional read-through to streamline the text as some is duplicated. Sufficient background is included to provide context and understand the usefulness of non-commercial data to sustainably manage near-shore fisheries. It appears that the reported data is not the raw creel survey data but the estimated data by each of the literature sources. The results could benefit from adding SEs especially the CPUE data to improve the spatial comparisons. Of course this would mean that the raw data is accessible from the 18 literature resources used in this review. Experimental design: The title indicates guidelines of ‘best practices’ but the MS seems more a compilation of the creel survey data from the various literature sources and misses in-depth analyses. ‘Best practices’ seem to be, reduced effort on those gear types that are most effective overall (highest CPUE) while low in total gear-hours. However, as the introduction and discussion mention, knowing the human use (e.g. a recreational fisherman fishing with hook and line for fun versus a fisherman putting out his laynet to catch a living) is of course more important than establishing the general overall gear-hours per gear type. Furthermore, gear types do not always target the same species e,g, catching parrotfish with spearfishing versus catching octopus with the same gear type has very different ecological consequences. The MS would benefit from an evaluation of the species caught and how a reduction on those species might benefit the ecosystem. It is a great and laudable effort to compile all these data in one place. However, these data could benefit from standardization to make use of them more efficient. For example, In reporting effort, gill nets and surround nets were not included while total catch does include the catches of these methods (e.g. for Kane’ohe 58% of the total catch was from surround/gill nets and in Hanalei these gear types comprised 69% of the total catch). The MS would further benefit from separating out the invertebrates (e.g. the catches in Kane’ohe were largely comprised of invertebrates (octopus) caught by spear fishing – hence high spear fishing effort for that location), akelu/opelu (e.g. Hanalei catches comprised of 69% akule/opelu) and coral reef fish. This would benefit managers trying to regulate the fishery based on gear types/ species / species complexes as regulations for octopus could be very different from e.g. opelu. Moreover, it would facilitate comparing just reef fish (or invertebrates or opelu/akule) spatial patterns which I would argue is more interesting to reef managers and the fishing community then the overall total catch or effort where this information is lost. The data set comprises a temporal time span from 1980 – 2015 with most data sets from the latest 10 year. The Puako data showed that there can be very large temporal changes in the fishery over a 30 year time span as the authors mention as well in the discussion. The authors acknowledge that over a time span of 20 or 10 years similar changes could have occurred at other reefs. Maybe results can be binned by survey year to see if temporal patterns emerge which might be more important than the spatial patterns? Even if no additional analyses are done, it would be better to include the year of the survey in the results for clarity. Validity of the findings: In general, some of the conclusions are not well backed up by arguments/findings. For example, the statement that "The findings can directly improve the design of more cost-effective and standardized creel survey approaches that can be instituted to facilitate more informed fisheries management in Hawai‘i and beyond" is not well argumented. It would be very interesting if the authors include the "How". Knowing that fishing effort, catch and CPUE estimates varies across space could be partly due to the differences in target species, the inclusion of gill net & surround net in some places, or the temporal differences in survey years, it is unclear to me how these findings can improve a more cost-effective creel survey. As I think the authors are aware of, there is a movement to make reporting of recreational/non-commercial fisheries obligatory (through license fees) which of course would also give us information on the non-commercial catches and efforts once implemented. The MS would improve from including these efforts and maybe comparing the pros and cons of both. Additionally, NMFS-PIFSC has recently published a technical memorandum with stock assessments of 27 reef fish species. These could help inform the setting of ACLs. It is unclear and not elaborated on why the authors think that ACLs are 'most effective' for high valued invertebrates. Lastly, I am surprised that the authors find the results from the fish flow (i.e. that most of the catch is not sold) 'surprising' as this has been reported in pretty much all literature on non-commercial fishing. Additional comments: Some more specific comments: Ln 42 “Surprisingly’? In almost all articles related to non-commercial fishing it is reported that a great majority of the catch is not sold e.g. Friedlander and Parrish 1997, Friedlander et al 2012. Suggest to re-word. Ln 81-83: This sentence is of course true but some head-way has been made with the stock assessment of 27 reef fishes and is worth mentioning. (Nadon et al 2017 NMFS-PIFSC Tech Memo #60) Ln 96-97. This sentence seems to imply that all creel surveys are designed by community members which is not the case for most creel surveys mentioned in the tables. Suggest to delete last part of the sentence (everything after the second comma) Ln 117-119: is it not the lack of obligatory reporting of non-commercial fishing that hinders the data collection more so than that this fishery is non-commercial? In other words, the fisherman do not have to report their catches, (unlike the commercial fisherman) so don’t do it unless they participate in the MRIP data collection by DAR, and therefore data is limited. Ln 197 and ln 198 seem to say the same? Ln 197, 198. From table 3 it seems that line fishing is 79% (not 94%), spear 14% (not 0%) and net 7%? Please clarify discrepancy. Ln 208 Table 4. Without having checked all refs, it seems that for Kaneohe, the CPUE is reported for shore and boat based line fishing whereas the methods say that boat-based is not included. Not sure about others but suggest to review your methods/results so they are in line with the original literature. I understand the info given in the creel survey papers might be not sufficient to separate them out but if that is the case this should be mentioned so we know the table does not compare like with like. Ln 227. Figure 3. These catches include gill net and surround net for some locations (e.g. Kaneohe Bay and Hanalei) which makes them not comparable with other locations that do not include the catches of those gear types. Suggest to adjust catches to make them comparable. Ln 259 “Surprisingly” see comment earlier Ln 282-283. Some of this variation is because of inclusion/exclusion of gear types, targeted species (e.g. opelu v octopus) and would be more interesting if the authors can specify their results separately for reef fish, invertebrates, and akule/opelu. Ln 283-285. Can the authors expand on how the findings can improve cost-effect survey design? Ln 287. Can the authors explain how the define “health”. Ln 302. Isn’t a spatial measure also an input control as it restricts effort? Ln 307. What is meant by “inputs” (other than fishing technology and effective gear types)? Ln 308. What is meant by “outputs”, catches? Ln 343. Why are ACLs ‘most effective’ for a high value invertebrates? Pls expand. Ln 383-385. Suggest to include Jokiel et al 2011 Journal of Marine Biology where the authors compare traditional with western management. Ln 399. There is a movement to make reporting of recreational/non-commercial fisheries obligatory which of course would also give us information on the non-commercial catches and efforts once implemented. Might be worth mentioning.

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

Title: PATTERNS IN ARTISANAL CORAL REEF FISHERIES REVEALED THROUGH LOCAL MONITORING EFFORTS Review round: 1 Reviewer: 2

Basic reporting: L1-2: The title seems to imply some kind of comparison between different monitoring/monitoring approaches. The paper focused mostly on characterizing fishery and perhaps this should be reflected in title instead? L34: This “collaborative” dimension isn't very clear from methods/results. How does the approach mentioned in the study promotes/facilitates collaboration? For example, aren’t fishers simply asked questions (and if so, is that a collaborative process?)? L41: The word “rampant” seems to imply some kind of magnitude or direction has been assessed. Perhaps reword? L42: Why “surprisingly”? Is this different from most similar fisheries? Clarify or reword. L53: Perhaps you mean “services” instead of “values” here? L95: How is this participatory? Clarify as it currently seems information is simply requested of them. L124-125: I think this should be clarified in abstract as well as mentioning how this study differs from previous studies (e.g. by putting everything together, study is able to draw comparisons, identify regional patterns, etc). L126: What do you mean by “intercept” survey? Clarify. L147: Can't read full label in first column. L152: how do creel surveys differ from the interview-based surveys mentioned below then? Unclear. L162: Redundant? Interviews can only be undertaken with participants… rephrase. L168: What's this reference meant to support here? Also, if in review, perhaps it's not available to readers anyway? L259: See my comment above regarding term “surprisingly”. Experimental design: L129-137: What's rationale for inclusion of each different area? How were these selected? Are there any key differences among them that might be useful for interpreting results? L180: what about potential temporal effects? Does it make sense putting everything together? Some consideration or discussion required. L217 and others: I think these differences among study areas need to be stated much earlier on manuscript. Validity of the findings: L253: How was this information obtained? Clarify in methods. Also, examples don’t provide specific time periods or magnitude of problem so difficult to use them to assess situation (although every useful for illustrating potential threats). Examples should be more specific (for example, “33% of the fishing activity recorded was illegal” refers to how many fishers? When?) Additional comments: The manuscript “Patterns in artisanal coral reef fisheries reveal best practices for monitoring and management” by Delaney et al. provides useful insights about small-scale fisheries in Hawai‘i with clear implications for natural resource management. However, several key issues should be addressed before further consideration for publication. Firstly, the contribution of the study should be made clearer. To do so, I think the authors should: (1) explicitly mention their general approach in terms of data gathering from previously published/unpublished studies much earlier on the manuscript (e.g. in the abstract) and explain how this study differs/builds upon those (see specific comment); and (2) clarify how this study aims to contribute in terms of data collection/monitoring protocols. For example, are creel surveys widespread in fisheries studies or only occasionally used (and if so, are the authors suggesting this should be elsewhere?). Either way, this should be clearer so that readers understand how this study fits within wider literature and fisheries assessments and how it contributes to advancing any knowledge gaps. Secondly, in the introduction, more focus should be given to the types of challenges and barriers involved in small scale fisheries monitoring and managing in data-poor contexts as well as different approaches used to address this issue. I think that would be useful for putting this study into wider context. I also felt the introduction was a bit repetitive (e.g. the livelihoods/social/economic/etc. issue was mentioned several times throughout introduction) and occasionally jumped between general and specific issues with somehow unclear flow. I recommend restructuring and rewriting some sections so that it reads better in terms of narrative and is more focused around key management/monitoring challenges. Finally, while I commend the authors for the work involved in bringing all the survey information together, I think the potential caveats in doing so haven’t been properly mentioned and discussed. For example, it’s clear from reading appendix material that a lot of different assumptions and steps were involved in producing estimates from different data sources. I thank the authors for providing a detailed description in the appendix. Nevertheless, some discussion of potential biases due to different survey approaches, sampling period (e.g. the authors pooled together data from different decades), etc., is required.

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

Title: PATTERNS IN ARTISANAL CORAL REEF FISHERIES REVEALED THROUGH LOCAL MONITORING EFFORTS Review round: 2 Reviewer: 1

Basic reporting: Well written some minor comments: Ln 329 Define “health” (health of reef fisheries and ‘health of associated ecological community” – maybe replace by “productivity of reef fisheries” and “status of associated ecological community” although I am not sure why this sentence is added as the rest of this section focused on fishing and nothing is mentioned about “numerous ways to assess the health”. Suggest to delete or add a sentence that includes refs for the latter part of this sentence. Ln 331-336 This paragraph comes a bit out of the blue – the results show that the catches are hardly sold (fig 5) and there is no mention about the composition of commercial and non-commercial catches so the reader has no idea how much of the reef fish is actually sold. Maybe the authors could start this paragraph with a comparison of commercial and noncommercial fisheries? And back up these claims by references. Ln 378, 379 The refs used to back up this sentence seem not appropriate. Donovan et al 2016 has references therein that are appropriate and I would recommend to use them. DAR 2017 has regulations but no justifications (or at least not that I could find on the linked website). Ln 398 a scale or scales; against impacts of climate change Editorial comments Ln 86 “remains” should be “remain” Ln 225 add “they” compose Table 5, Pupukae “illegally fish” Ln 468 at at least Experimental design: no comment Validity of the findings: no comment Additional comments: The authors have done an excellent job in addressing my previous concerns and I look forward to seeing this MS being published.

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

Title: THE PHT4;1-3 MUTANT LINE CONTAINS A LOSS OF FUNCTION ALLELE IN THE FATTY ACID DESATURASE 7 GENE CAUSED BY A REMNANT INACTIVATED SELECTION MARKER—A CAUTIONARY TALE Review round: 1 Reviewer: 1

Basic reporting: The authors use of English is clear and professional, and there are some few minor recommended corrections that will further improve the clarity of the manuscript. However, I think that the introduction is insufficient and more background needs to be provided to establish which is the gap in research that the authors aim to fill. The question being answered needs to be stated clearly in order to set up an appropriate context for the reader. Experimental design: Please clearly state the question being addressed with your experiments. Validity of the findings: No comment. Additional comments: The authors address a problem often overlooked in reverse genetics. However, in my opinion the manuscript needs to state the question more specifically, as I found the introduction a little vague in that regard. Perhaps the authors would be better able to guide their readers by discussing the problem with establishing phenotypes of loss-of-function mutants, the need for researchers to ensure no other mutations in the background that might be causing them, and that having two or more alleles is preferable to define a Aside from this, I recommend some minor corrections for clarity before resubmitting. 1. The major issue is that the introduction needs to be more specific about the aim of the study. One paragraph of introduction does not seem sufficient to lay out the background and the gap in research that this study means to fill. 2. Correct nomenclature of the genes and mutant alleles should be used consistently and clearly throughout the manuscript. For example, in line 36, PHT4 refers to the entire gene family (the correct gene name is PHT4;1). So it would be simpler to refer to “the transposon in pht4;1-3” 3. Line 34, change “were” to “was”. 4. Line 50, since the pht4;1-1 mutant allele was not mentioned elsewhere in the manuscript, I assume this is a typo and the authors meant to write “pht4;1-2 and pht4;1-3”. Please clarify. 5. Line 79, add a comma after fad7 and “was” after “conducted”. 6. Line 83, change to “F2 plants were”, instead of “F2 plants was”.

You are one of the reviewers, your task is to write a review for the article. You will be 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 PHT4;1-3 MUTANT LINE CONTAINS A LOSS OF FUNCTION ALLELE IN THE FATTY ACID DESATURASE 7 GENE CAUSED BY A REMNANT INACTIVATED SELECTION MARKER—A CAUTIONARY TALE Review round: 1 Reviewer: 2

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: In this paper, the authors discribed a cautionary tale that the mutant line pht4;1-3 carried a remnant insertion in the FAD7 gene. And a series of biochemical and genetic evidences were supplied. The English laguage are professional and clear to understand. However, from this study, the authors concluded that every mutant should be confirmed by at least two independent lines. This is a well-known conclusion. Is it necessary to confirm again? Also, there are some major concerns as follows: 1. Introduction: 1) The introduction is too simple. Only PHT4;1 was discribed. More details about the phenotypes of pht4;1 mutants, FADs, as well as lipids and fatty acids should be introduced. Also, some mutagenesis methods should also be mentioned. 2. Materials and methods: 1) The mutant genotyping and the lipid analysis should be divided into two parts with two separate subtitles. 2) How did the authors get these mutant lines? From ABRC stock or given by the others? The stock numbers for these mutants? 3) How the Arabdopsis DNA was extracted? How did PCR and electrophoresis run? 4) Line 38: the tables should be listed in order. Table 1 is first, then folloed with Table 2, Table 3… 5) How many biological and technical repeats were carried? How many samples were included in each repeat? 3. Results and discussion: 1) Lines 50-51: the data about the lipid compositions are not shown? 2) Lines 53-55: in Fig 1, both DGDG and MGDG showed the quite different fatty acid compositions, why only MGDG was mentioned? 3) For all of the mutant lines mentioned in this paper, the authors should carry out the experiments such as Real-time PCR to detect the expression levels of PHT4 and FAD7. 4) The transposon was inserted into two separate positions (PHT4;1 and FAD7). This means more than one Ds copies in the mutant genome, maybe some other experiments such as southern blotting would be needed to detect the transposon insertion copies. 5. Tables and figures: 1) For the data in Fig1 and Table1, the significance analysis is required. 2) In Fig2, the insertion site in FAD7 should be marked in the gene model. 6. Supplemental data: 1) What did the capital letters and the lowercases mean in the FAD7 sequence? The blue letters are the coding sequence of FAD7 or not? If yes, the authors should indicate the triple coden, and the amino acid sequence should also be listed.

You are one of the reviewers, your task is to write a review for the article. You will be 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 PHT4;1-3 MUTANT LINE CONTAINS A LOSS OF FUNCTION ALLELE IN THE FATTY ACID DESATURASE 7 GENE CAUSED BY A REMNANT INACTIVATED SELECTION MARKER—A CAUTIONARY TALE Review round: 2 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: The authors have satisfactorily expanded the introduction and added sufficient background as to aid in better understanding of the reasoning behind the experiments. I recommend only some minor changes to the manuscript for clarity: 1. Line 22: Reverse genetics 2. Line 52: phosphoglycerolipids 3. Line 77: change “were” to “was” 4. Line 100: “leading us to suspect” (delete “e” and “d”) 5. Line 105: revealed 6. Line 107: same as #5

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

Title: UNANTICIPATED DISCOVERY OF TWO RARE GASTROPOD MOLLUSCS FROM RECENTLY LOCATED HYDROTHERMALLY INFLUENCED AREAS IN THE OKINAWA TROUGH Review round: 1 Reviewer: 1

Basic reporting: See attached report. Experimental design: See attached report. Validity of the findings: See attached report. Additional comments: See attached report.

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

Title: UNANTICIPATED DISCOVERY OF TWO RARE GASTROPOD MOLLUSCS FROM RECENTLY LOCATED HYDROTHERMALLY INFLUENCED AREAS IN THE OKINAWA TROUGH Review round: 1 Reviewer: 2

Basic reporting: The manuscript language is good both in overall structure and specific use. Literature citation is mostly adequate although limited (suggestions below). Figures are fine. Fasta sequences were provided but I don’t see them in DDBJ. Experimental design: It’s an observational study. Methods are adequate. Validity of the findings: It is fine to draw attention to these records at chemosynthetic systems. However, as neither two species themselves nor the presence of the family at vents is new, I would suggest the authors make a larger contribution by: i) Drawing a better picture of the likely role these animals play in this ecosystem (grazer? predator?) ii) Comment on the intersection of vent and non-vent species. There is a fairly robust literature that details the peripheral vent environment – this record is an important contribution so set it in that context. The most recent review, I believe, is Levin et al. 2016. 2016. Hydrothermal vents and methane seeps: Rethinking the sphere of influence. Frontiers in Marine Science, 3, dx.doi.org/10.3389/fmars.2016.00072 There are quite a few instances of deep-water gastropods on the periphery of vents and seeps – why are they successful in this niche? (I see Buccinum viridum frequently among tubeworms in the northern Juan de Fuca Ridge). Additional comments: Do you think they are competing with species that use the resources in a similar fashion? Or are these available niches? Why are they not found at the other vents in the region? Suggest that the last sentence of the Introduction is part of the habitat characterization and should move to the Discussion to explain the presence of these taxa. …. and when I get to end of Discussion, I do not understand this “maybe a seep” comment. The best indication of whether it is a cross-over habitat is your list of species – how do they compare with Watanabe et al’s list of vent fauna in the region? Are there seep fauna here, too? Are there any supporting temperature data to decide if there is a thermal anomaly here? Other points L 62: “drastically”? Are you talking about end-member fluids? pH in waters around animals is often no different from seawater. But you can justify if the Okinawa region is notably different in diffuse fluids. l 69: Lutz and Kennish do not really test this idea – but this paper does: Van Dover CL, Trask JL. 2000. Diversity at deep-sea hydrothermal vent and intertidal mussel beds. Marine Ecology Progress Series 195:169-178. l 90: in English, “Neither” takes singular subject and verb. l. 104: remove ‘by the junior author.’ Are there any temperature or chemical measurements made? Good imagery of T. ikukoae. Is the size within the range originally reported for the species? Figure 1: Add N and E to lat/long; attach two or three island names to orient the reader. Also, I think it useful to indicate the other vent sites from Watanabe and Kojima 2015.

You are one of the reviewers, your task is to write a review for the article. You will be 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 SHINBUTO AND NINJINTO ON PROSTAGLANDIN E2 PRODUCTION IN LIPOPOLYSACCHARIDE-TREATED HUMAN GINGIVAL FIBROBLASTS Review round: 1 Reviewer: 1

Basic reporting: no comment Experimental design: The methods are appropriate and the experiments are well performed. Validity of the findings: no comment Additional comments: In this manuscript, the author described the effect of shinbuto and ninjinto on prostaglandin E2 production and potential mechanism. The manuscript has potential interest to the field. The methods are appropriate and the experiments are well performed. Understanding more about the effect of kampo drugs is an essential step for medication usage. The manuscript have potential interest in the field. The methods are appropriate and the experiments are well performed. The manuscript is also acceptable to me except a few questions. 1. Line 182-185. I think “Shokyo and kankyo” here should be “shinbuto and ninjinto”. 2. Figure2 legend title is wrong. cPLA, annexin1 and cox-2 is not mentioned in this figure. 3. For the westernblot data, it is better if the statistical data is provided. Especially for fig.7. I think shoky and kankyo have effect for cPLA expression.

You are one of the reviewers, your task is to write a review for the article. You will be 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 SHINBUTO AND NINJINTO ON PROSTAGLANDIN E2 PRODUCTION IN LIPOPOLYSACCHARIDE-TREATED HUMAN GINGIVAL FIBROBLASTS Review round: 1 Reviewer: 2

Basic reporting: The authors investigate the anti-inflammatory effect of two kampo medicines (shinbuto and ninjinto) on human gingival fibroblasts (HGF) derived from patients affected by periodontal disease with deficiency pattern. They show these two compounds able to decrease PGE2 production while increasing IL-6 and not affecting IL-8 levels. They exclude the modulatory effect on PGE2 effect as a consequence of COX-2 inhibition, impact on ERK activation or modulation of expression of factors cPLA2, ammexin1, and COX-2. Finally, they tested the effect on PGE2 of single herbal components composing the two formulations and identified shokyo and kankyo as inhibitors of PGE2 production without affecting the above mention factors. The authors conclude about a potential impact of the two campo medicines on PLA2 activity. The ms is clear; however, english proofreading would be recommended due to the presence of several mistakes. Relevant literature is adequately provided. Figures are clear. Experimental design: The experimental procedures are clear as reported in Materials and Methods with ethical approval indicated regarding the use of specimens from patients . The aims of the present investigation are clearly indicated in abstract as well as in the introduction section. Validity of the findings: In its present form the ms, raises some questions to solve. The two kampo medicines decrease PGE2 levels while increasing IL-6 and not affecting IL-8 production. The authors show the ability of single components shokyo and kankyo to similarly affect PGE2 levels while not affecting the expression of PLA-related factors. 1) It would be interesting to verify if shokyo and kankyo are able to produce the same pattern of modulation of interleukin-6/8 as shinbuto and ninjinto. This phenomena might correspond to a kind of combinational effect of different components contained in shinbuto and ninjinto. 2) The authors hypothesize the impact of shinbuto and ninjinto on cPLA2 activity. It would be interesting to provide this additional experiment to make robust their hypothesis. 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 SHINBUTO AND NINJINTO ON PROSTAGLANDIN E2 PRODUCTION IN LIPOPOLYSACCHARIDE-TREATED HUMAN GINGIVAL FIBROBLASTS Review round: 2 Reviewer: 1

Basic reporting: Authors considered all remarks and addressed the points raised. Experimental design: The experimental procedures are clear as reported in Materials and Methods with ethical approval indicated regarding the use of specimens from patients. Original files for consent and ethics have been provided (in their original language). Validity of the findings: The ms has been implemented with additional experiments as suggested or strategies anyway have been considered and tempted. Additional comments: The ms has been implemented in its experimental part. Language has been revised.

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

Title: PLANTCV V2: IMAGE ANALYSIS SOFTWARE FOR HIGH-THROUGHPUT PLANT PHENOTYPING Review round: 1 Reviewer: 1

Basic reporting: Nothing to add Experimental design: The paper includes details of the listing of the available methods through PlantCV. This is fitting as the paper is about this new software. The main impact of the paper would be enabling the potential users to leverage this software. In that regard, adding a few "how to" could be helpful. For example, what is the general guideline of which method to use for segmenting? And how to tune many parameters? How to create a series of steps to work for some common tasks? How to fix any errors from the algorithm? What is the expected speed of each method? Validity of the findings: Nothing to add Additional comments: The paper is a good introduction to the PlantCV software. Overall the paper and proposed method seem to address much need of the field. The future steps are insightful and should further increase impact. Main comments are to maximize the utilization of the tool by answering typical struggle that non-technical users would face.

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

Title: PLANTCV V2: IMAGE ANALYSIS SOFTWARE FOR HIGH-THROUGHPUT PLANT PHENOTYPING Review round: 1 Reviewer: 2

Basic reporting: Language used is clear throughout and the relevant phenotyping literature is well cited. One of the sections I am direct to comment on here is whether the results are relevant to the hypothesis proposed, however this is a methods/resource paper and does not include hypothesis driven research. Experimental design: No comment. Validity of the findings: No comment. Additional comments: Lines 52-54: The point about floral structures being different colors in different plants (and the need for fixable analysis approaches as a result) is a good one, but the specific example used opens up a big can of worms. The floral organs (ie flowers) of Camelina are yellow and the inflorescence/glumes of brachypodium are green. The floral organs of brachypodium (such as anthers) would also be yellow and since anthesis is often used as the milestone for "flowering" in grasses, it would often be the case that folks would be looking for yellow in photos of grass inflorescences. What about yellow Camelina flowers vs white arabidopsis flowers? Line 85-86: I am afraid it wasn't clear what picture "The image of Arabidopsis thaliana" was in context here as there are several Arabidopsis images in different figures. Maybe include "used in Figure X or analysis Y" here. and for the wheat image listed a little later in this section. In the discussion of unit tests it is not clear if the development and maintence of unit tests is an expectation of contributors who write new modules for incorporation into Plant CV or of the core management team. 155-158: I would encourage the authors to include a copy of the SQLite database schema as it exists with the writing of this paper as a figure or supplementary figure. I assume the schema on github will continue to be updates at PlantCV matures, which may make the reference to github less relevant to the version of the software described in this manuscript. This could potentially replace the current Figure 1. Page 16: Could the authors add one to two sentences on the circumstances where a researcher might want to trade a reduction in image detail for a reduction in image noise? Page 19: "The ‘watershed_segmentation’ function can be used to estimate the number of leaves for certain plant architecture types and imaging platforms (Fig. 3)" It might be useful to add a little more detail on the characteristics of plant architectures where the watershed method is likely to work or likely to fail at this point. Pages 23-24: If the Bayesian method provides approximately the same output as a theshold-based analysis, but the Bayesian method requires fewer total steps that is a clear advantage to the Bayesian method. However, it wasn't clear what the other advantage(s) were to the Bayesian method since the sentence describing that point starts with "An additional advantage to using the naive Bayes..." (Lines 421-423).

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

Title: PLANTCV V2: IMAGE ANALYSIS SOFTWARE FOR HIGH-THROUGHPUT PLANT PHENOTYPING Review round: 1 Reviewer: 3

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

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

Title: EXAMINING PUBLICATION BIAS—A SIMULATION-BASED EVALUATION OF STATISTICAL TESTS ON PUBLICATION BIAS Review round: 1 Reviewer: 1

Basic reporting: Please see report. Experimental design: Please see report. Validity of the findings: Please see report. Additional comments: General Comments: This paper uses Monte Carlo simulations to test four different methods to identify publication bias: (i) Egger’s Test/Funnel Asymmetry Test (FAT), (ii) the p-uniform test (PU), (iii) the test for excess significance (TES), and (iv) the caliper test (CT – where 4 different versions of the CT are compared). The Monte Carlo experiments vary over a number of dimensions: (i) existence and type of publication bias (“p-hacking”; “file drawer”), (ii) number of observations in primary studies (N); (iii) number of studies included in simulated meta-analyses (K); and (iv) population effect size (both fixed at different values, and heterogeneous). Tests were compared on two dimensions, Type I error rates and power. As the author notes, there are a wide variety of results, making it difficult to come up with a single conclusion. But given the interest in identifying “best practice,” the author concludes: “The FAT is recommended as a test for publication bias in standard meta-analyses with no or only small effect heterogeneity. If no clear direction of publication bias is suspected the TES is the first alternative to the FAT. The 5%-caliper tests is recommended under conditions of effect heterogeneity, which may be found if publication bias is examined in a discipline-wide setting when primary studies cover different research problems” (cf. “Discussion”). Overall, the paper is clear and well-constructed. It makes a valuable contribution to an important topic about which relatively little is known. In this reviewer’s opinion, there is insufficient evidence to support the recommendations. However, the paper begins to fill in a blank sheet and hopefully moves the discipline closer to a better understanding of this issue. Specific comments follow. Specific Comments: 1. The paper attempts to construct realistic situations by which to support recommendations for meta-analysis researchers. In the opinion of this reviewer, the paper is too narrow to do that. Most of the emphasis in the paper is based on fixed effects models. There is only one variation of a heterogeneous effect, the case identified as “Het.” As this latter case surely most matches realistic situations, there is an insufficient database of realistic situations on which to support recommendations. This reviewer is not recommending that the author change her Monte Carlo experiments. The fixed effect cases are of interest as a benchmark. Instead, this reviewer recommends that the conclusion be rewritten to downplay recommendations and focus more on what we can learn from this narrow set of experiments. 2. One important issue that is overlooked in this and related studies is the relationship between effect size and publication bias. The author seems to think that the 50% and 100% publication bias regimes are holding constant the degree of publication selection bias. They are not. As effect sizes increase, the degree of publication selection bias changes. This is evident in the power results in Tables 7-10. Again, this reviewer does not suggest that the author change her Monte Carlo experiments. However, it would be useful if the “percent positive and significant studies” was reported as a separate column for each of the “N/K/Effect size” experiments. 3. Relatively little analysis is done of the results. This author needs to be more thoughtful about explaining why results differ across N/K/Effect Size (and “percent positive and significant studies”/incidence of publication selection bias). In this reviewer’s opinion, the major contribution of this study relies on the insights that can be learned about how these parameters affect the Type I error rates and power results. 4. Related to the previous point, more effort needs to be paid to how the publication selection process is likely to affect the results across different parameter settings. For example, in the Reed (2015) and Alinaghi and Reed (2016) papers cited by the author, it is noted how effect size is related to the incidence of publication selection bias, and how this generates different kinds of estimation bias depending on the type of publication selection bias (bias against insignificance, bias against negative signs). The author has chosen to model a publication selection process that simultaneously targets both the sign of the estimate and its statistical significance. This is common across simulation studies (such as any of the many Stanley and Doucouliagos studies), so this reviewer has no problem with this approach. However, it does complicate the relationship between the incidence of publication selection and the degree of estimation bias, and hence Type I error rates and power and thus it will be that much more challenging, and important, that the author explain this relationship. 5. The perils of implementing these tests with heterogeneous effects should be emphasized. In that context, the following references should be discussed and cited: -- Lau, J., Ioannidis J.P.A., Terrin, N., Schmid, C.H., and Olkin, I. (2006). The case of the misleading funnel plot. British Medical Journal 333: 597-600. -- Sterne, J.A.C., Sutton, A.J., Ioannidis, J.P.A., Terrin, N., Jones, D.R., Lau, J., Carpenter, J., Rucker, G., Harbord, R.M., Schmid, C.H., Tetzlaff, J., Deeks, J., Peters, J., Macaskill, P., Schwarzer, G., Duval, S., Altman, D.G., Moher, D., and Higgins, J.P. (2011). Recommendations for examining and interpreting funnel plot asymmetry in meta-analyses of randomised controlled trials. British Medical Journal, 343: d4002. -- Terrin, N., Schmid, C.H., Lau, J. and Olkin, I., (2003). Adjusting for publication bias in the presence of heterogeneity. Statistics in Medicine, 22: 2113-2126.

You are one of the reviewers, your task is to write a review for the article. You will be 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 PUBLICATION BIAS—A SIMULATION-BASED EVALUATION OF STATISTICAL TESTS ON PUBLICATION BIAS Review round: 1 Reviewer: 2

Basic reporting: I was able to load raw data from the supplement using R's haven::read_dta(). There's no codebook so I'm not sure what all the columns represent, however. I can also confirm that the enclosed .do files appear to contain code; however, I do not know STATA and cannot comment on the quality of the code. Tables would benefit from more thorough captions. I often did not understand what the tables represented (particularly Table 5). Experimental design: The author compares the performance of four tests or publication bias / p-hacking: Egger's test, TES, p-uniform, and the caliper test. I think that this is a worthwhile endeavor. Among these four tools, it would be useful to know which ones perform well under which conditions. I was confused by the use of effect size Beta. I am familiar with Cohen's d, odds ratios, and Pearson's r. Isn't Beta equivalent to a Pearson r? It must not be because r cannot exceed zero. Please clarify what the effect metric is. I was a little surprised by the sizes selected for N and K. Are these parameters intended to be representative of a particular research area? These Ns are larger than I would expect in my area of experimental psychology. Also, I think it is unusual for a substantive meta-analysis to have k = 100. Perhaps it is your intent to test an entire research literature for publication bias (e.g. 1000 studies from some journal) -- it is generally my opinion that these samples are too heterogeneous to be informative. Please justify the decision to use these Ns and Ks. It is worth considering that the implemented form of p-hacking is only one form of p-hacking. P-hacking may come in many forms (selection among several dvs, selection among several treatment groups, optional stopping, outlier exclusion), all of which increase the Type I error rate to various degrees and may influence these tests in different ways. Thus, it is sensible to caution that the results may not generalize to all forms of p-hacking. Validity of the findings: See general comments. Additional comments: Unfortunately, I had a lot of trouble understanding the manuscript. Reorganization, use of subheadings, and pruning of unnecessary text would be very helpful. 1. Organization The manuscript is very long and dense. It would be helpful to give it a fresh read and evaluate what is most important. Organization by subheadings, especially in the results section, would be very helpful. Some technical details, such as how long the code took to run, are technically impressive but not of scientific importance. I'm also not sure that it's necessary to fully explicate the math behind each test when they are described in their respective publications. Footnotes are also frequent and not all seem necessary. Some footnotes contain factual inaccuracies; for example, footnote 4 cites Nuijten et al. 2016 as an estimate of the prevalence of fraud, but this paper only checks the prevalence of p-values that don't match their test statistics (which can happen for benign reasons). I am also unsure about whether "publication bias is heavily punished" in any area except clinical trials. 2. Results The results are confusing to me because they seem to describe many results in terms of unconditional probabilities, e.g. averaged across all conditions. This is confusing because many of these probabilities will depend heavily on the condition. For example, the frequency with which null results are censored will depend heavily on the true effect and the number of observations. The regression model might help to address this concern, but I remained too confused about what data and conditions were in the model to learn anything from this section. Additionally, I find the emphasis on dichotomous significance tests a little dissatisfying. A better question might be, how badly has the true effect been misestimated? You mention that "PU was only able to discover file-drawer behaviour under low underling true effects." (line 449). Perhaps at this N and this K, file-drawing does little to influence the results of a meta-analysis? Just how bad is the bias? I don't understand Table 5. 3. Interpretation. The Discussion and Conclusion seems very brief compared to the rest of the manuscript. I found this disappointing because there is a lot that is done in the Results section that was not clear to me. I was hoping that the Discussion would establish the meaning and importance of all these analyses and tables. The author recommends the use of TES when publication bias selects for results of either sign. It is not clear to me that two-tailed significance selection is a hazard in the same way that one-tailed significance selection is. Consider that when publication selects for significant results of either sign, this doesn't always lead to bias in the meta-analytic effect size estimate. There is a loss of efficiency due to the censored studies in the middle, but the positive and negative results often cancel each other out. With this in mind, what is learned by a significant TES result? Does one learn that the naive meta-analytic result is necessarily wrong? Or is the idea that somebody has to be punished for file-drawering, even if the meta-analytic estimate is roughly unbiased? It doesn't seem very informative to scientific progress. Some other corrections to the introduction may influence the interpretation of your results. Simonsohn and van Aert disagree (line 196) because they are considering different estimands and different meanings of heterogeneity. van Aert attempts to recover the overall mean of all studies whereas Simonsohn attempts to recover the true mean of only the significant studies. If it's Beta or Delta or Rho you intend to recapture, then van Aert is correct: p-uniform will overestimate the true mean under heterogeneity. You may need to reinterpret your p-uniform results accordingly. Second, there are studies that have considered the FAT under heterogeneity. See https://www.ncbi.nlm.nih.gov/pubmed/12205693 and http://onlinelibrary.wiley.com/doi/10.1002/sim.1461/full. I apologize that I must sound a bit slow given how much I did not quite understand. I assure you that I tried my best. I always sign my reviews, Joseph Hilgard

You are one of the reviewers, your task is to write a review for the article. You will be 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 PUBLICATION BIAS—A SIMULATION-BASED EVALUATION OF STATISTICAL TESTS ON PUBLICATION BIAS Review round: 2 Reviewer: 1

Basic reporting: No comment. Experimental design: No comment. Validity of the findings: No comment. Additional comments: The revised manuscript successfully addresses my previous 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 PUBLICATION BIAS—A SIMULATION-BASED EVALUATION OF STATISTICAL TESTS ON PUBLICATION BIAS Review round: 2 Reviewer: 2

Basic reporting: It's fine. I don’t mean to be picky, but I found myself struggling with the language at points. Line 316, commas are misplaced. If I understand it correctly, it should read as, “For β = 0.5 in the 50% publication bias condition only 35%; for β = 1, 15%; for β = 1.5, only 9%; and in the heterogeneous condition, 22% of the studies employed publication bias practices.” Minor points. Line 383: Don’t you mean compared to the file-drawering condition? You may find it helpful to make explicit what is the predicted quantity in Table 3’s OLS regression. It was not clear to me. The number of analyses conducted? The mean p-value? Graphs may also be useful – in general, I found it difficult to interpret these many tables and their many numbers. Experimental design: I find myself wishing there was more attention to the bias in the actual effect size estimate. I think the attention on the p-value and the p-value alone is rather shortsighted. I understand that this suggestion would potentially lead to a rather different paper, however. This unusual emphasis on the p-value alone leads to unusual statements such as line 306, “Because the heterogeneous effect condition does not allow an absolute bias measure, the p-value deflation factor was used for all decisions.” I find this strange – why not conduct random-effects meta-analysis and compare the meta-analytic mean (and tau) against the true mean (and tau)? Consideration of the p-value deflation factor alone seems odd to me. If there is some true effect, I don’t particularly care whether 5/10 studies find it or 10/10 studies find it so long as the resulting meta-analytic estimate and inference is correct. Validity of the findings: You may also consider the nature of your file-drawer condition. This is a relatively mild form of publication bias in that each agent has only a (1-(.95^10)) = 40% chance of getting a significant result, and if they get no significant result, they publish a nonsignificant result. More commonly, simulations like this generate k studies and publish only some percentage of the nonsignificant results. Compared to social psychology, where almost every published result is significant, having 60% of published results be nonsignificant may be pretty modest bias. Also, does it really make sense to compare the power of tests under file-drawering vs. p-hacking given that they inflict different amounts of bias in the meta-analytic result? I can see the utility of comparing these methods against each other, but to make inferences about the power of the method under one scenario, as compared to another scenario, seems inappropriate. To say that methods gained or lost power in going from p-hacking to file-drawering seems to overlook that the two practices inflict different amounts of bias. The noted weakness of the caliper test is interesting, namely that under less-than-huge K, few studies fall within the caliper. But at Line 357, discussing high false-positive rates of caliper tests, can you provide evidence that the underlying true effect yielded an overrepresentation of just-significant values? That doesn’t sound like something that should happen, if I understand how p-values work. It is also curious that this happens at what appear to be specific levels of Beta, N, and K, rather than behaving in some predictable relationship. I am a little wary of endorsement of the TES given the numerous criticisms of it that have been published —see https://alexanderetz.com/tag/test-of-excess-significance/ for a compendium. Additional comments: The revised paper is rather easier to read, and the conclusions seem sensible. FAT works well under homogeneity and one-sided selection, but the CT and TES may work under heterogeneity and two-sided selection. I have a litany of suggestions, which you are free to accept or to reject. I do not want to be the reviewer who asks for a completely different paper, but I think consideration of these points would make for a clearer, more informative 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: A TYRANNOSAUROID METATARSUS FROM THE MERCHANTVILLE FORMATION OF DELAWARE INCREASES THE DIVERSITY OF NON-TYRANNOSAURID TYRANNOSAUROIDS ON APPALACHIA Review round: 1 Reviewer: 1

Basic reporting: see below Experimental design: see below Validity of the findings: see below Additional comments: This paper describes two new dinosaur specimens from the Late Cretaceous of New Jersey. Dinosaur material remains very rare in the eastern United States, so any new specimens are noteworthy and deserve publication, particularly as many of them are latest Cretaceous in age, so have relevance for understanding dinosaur diversity and biogeography during the run-up to the mass extinction. I have worked on several of the east coast theropod specimens, particularly the tyrannosauroids, and so I am happy to see new material discovered and described. There is no doubt that these specimens are worthy of publication in the scientific literature. However, I cannot yet recommend publication of this paper. The presentation and content require major revisions. Here are my main issues. I hope you can address them, and at that point, the paper will be ready for publication in PeerJ: 1) More information is needed on how the specimens were found. Were they found associated? How close together were they, and in what orientation? What else was found with them? How do we know they belong to the same individual? Particularly as the metatarsal IV has marks indicative of shark feeding traces, and the specimens are found in a marine unit, meaning they must have been transported far from the site of death. 2) You need to make very clear which features support a tyrannosauroid affinity, rather than, say, an ornithomimosaur affinity. The best way to do this is to include them in a broad phylogenetic analysis of coelurosaurian theropods, like the Theropod Working Group matrix (most recent version: Brusatte et al. 2014, Current Biology). 3) Is the metatarsal II actually a metatarsal II? In proximal view there are notches on both lateral and medial surfaces, which is not the case of metatarsal II in tyrannosauroids (there is only a notch on the lateral surface, where metatarsal III articulates). What would be articulating with the medial notch if this is a metatarsal II? There is nothing there in tyrannosauroids. Also, the proximal end is much more bulbous compared to the shaft than in other tyrannosauroids. Are you sure this is not a metatarsal III of a non-arctometatarsalian animal? 4) On first glance, the specimens (especially the metatarsal II) do not strike me as obviously tyrannosauroid. They seem to be a little smaller and more gracile than the metatarsals of Appalachiosaurus/Dryptosaurus-like tyrannosauroids, not to mention the larger and more robust Tyrannosaurus-like species. One possibility is that they belong to an ornithomimosaur (see above). The other is that they belong to a juvenile. Therefore, comparisons with juvenile tyrannosauroids are needed. If the specimens belong to juveniles, ontogenetic variation may explain some of the differences with Dryptosaurus and Appalachiosaurus (both of which are known from larger, presumably more mature, holotypes). 5) The specimens seem somewhat deformed. Could this have caused some of the subtle shape differences compared to Appalachiosaurus and Dryptosaurus? I agree that metatarsal IV does not have the autapomorphically ‘flattened’ condition of Dryptosaurus (in which the proximal articular surface is much wider mediolaterally than long anteroposteriorly). However, deformation must be ruled out as the reason for this difference. The same is true of other observed differences. 6) You simply assert that the few differences between your new specimens and Appalachiosaurus and Dryptosaurus cannot be the result of individual variation. You have to back this up with evidence. What type of individual variation is present in the metatarsals of well-sampled tyrannosauroids, such as Tyrannosaurus, Tarbosaurus, Albertosaurus, or Gorgosaurus? And more importantly: what type of ontogenetic variation is present in these taxa? 7) The treatment of the ‘unnamed specimen from the Maastrichtian of New Jersey’ is problematic. This specimen is alluded to throughout the text without mention of its specimen number, until that number is finally given in the discussion. This is AMNH 2550-2553. It is not a tyrannosauroid, but rather, an ornithomimosaur. I suggest that you simply ignore this specimen for the time being and leave it out of your paper. It isn’t very relevant to the material you are describing. On that note, you say that you are currently writing up a description of this specimen. You may not be aware, but my colleagues and I (Brusatte, Benson, Choiniere, Norell) began studying this material in 2012 and wrote an SVP abstract about it, which was peer-reviewed and presented at the 2012 Raleigh meeting. I respectfully ask you to respect the priority we have established in describing and naming this material. We will be writing a paper on it; we already have a draft manuscript. 8) I agree that the distal end of metatarsal IV is different in shape to that of Appalachiosaurus, as you describe. However, you also need to describe how it is different in shape to that of Dryptosaurus. You simply assert this without delimiting the specific anatomical features that differ. 9) The figures need to be labelled—point out the most important anatomical features. 10) What would really beef up this paper, and drive home the differences with Appalachiosaurus and Dryptosaurus, are comparative figures and tables. Show photographs of the new specimens alongside the same bones of the Appalachiosaurus and Dryptosaurus holotypes, and also present a table of measurements for the specimens. 11) You should include the new specimens in a phylogenetic analysis. There are very detailed, comprehensive phylogenetic studies of tyrannosauroids in the literature, which use vast datasets of anatomical characters including many features of the metatarsus. The most comprehensive is the recent Brusatte & Carr analysis (2016, Scientific Reports), which is a follow-up of an earlier study (Brusatte et al. 2010). You could simply score the new specimens for this dataset, run the analysis, and see where it is placed on the cladogram. In that vein, I was very surprised to see that these two studies weren’t even cited to support the following statement: ‘The tyrannosauroid taxa of this landmass, Dryptosaurus and Appalachiosaurus, have repeatedly been found outside Tyrannosauridae in phylogenetic analyses’. They need to be read, considered, and cited. 12) You consider Dryptosaurus as present in both the Campanian and Maastrichtian, and even as present in the same formation as the new specimens. Be careful here. Yes, several specimens from across eastern North America have been referred to Dryptosaurus, but this has not been based on autapomorphies, but rather on general similarity. The holotype of Dryptosaurus is Maastrichtian in age. Do any of these other specimens share unique features with the holotype? If so, then you can confidently refer them to Dryptosaurus. If not, then you should probably consider them Tyrannosauroidea indet. This is important, because the identification of these specimens factors into your biogeographic discussion and conclusions. In fact, your biogeographic discussion hinges, in a sense, on whether or not these specimens are truly Dryptosaurus or belong to another taxon. 13) I think it is way, way too premature to argue that Appalachian tyrannosauroids had a similar diversity to Laramidian tyrannosauroids. I am not convinced that what you have here is a ‘new morphotype’ rather than an individual variant of Appalachiosaurus, or possibly Dryptosaurus. But even if it is a new taxon, that means there are three known tyrannosauroids from the entirety of the Campanian and Maastrichtian of Appalachia. That is far, far fewer than the known number from the entire Campanian and Maastrichtian of Laramidia. Of course, there are huge sampling biases—much less is known about Appalachian dinosaurs, because much less outcrop is available for study. But, at this point in time, there is simply no positive data to suggest that Appalachia ‘probably enjoyed similar (tyrannosauroid) diversity’ to Laramidia, as you conclude in the discussion. It is an hypothesis that can be tested by future discoveries, but we certainly cannot conclude that it is correct at this point! 14) Finally, there are many misspellings and grammatical issues throughout—e.g., autopomorphy instead of autapomorphy. The references are presented in a random, non-alphabetical order. There are also some basic anatomical errors in this paper. Metatarsal II is said to have an articular surface for metatarsal III in medial view; however, it would be the lateral surface of metatarsal II that articulates with metatarsal III. The manuscript needs a careful edit to reach the standard needed for the peer-reviewed literature. Steve Brusatte, Univ of Edinburgh, July 23, 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: A TYRANNOSAUROID METATARSUS FROM THE MERCHANTVILLE FORMATION OF DELAWARE INCREASES THE DIVERSITY OF NON-TYRANNOSAURID TYRANNOSAUROIDS ON APPALACHIA Review round: 1 Reviewer: 2

Basic reporting: OK Experimental design: OK Validity of the findings: Identification of the bones as belonging to Tyrannosauroidea is not well supported. Alternative identifications are not considered. Additional comments: The paper describes partial theropod metatarsus (proximal fragments of metatarsals II and IV) from the Campanian Merchantville Formation of New Jersey, USA. Because of extreme rarity of theropod dinosaurs from Appalachia, this funding represents a considerable interest. The author referred the specimen to the Tyrannosauroidea based on metatarsal III (reconstructed) of crescentic shape and restricted to the plantar surface of proximal end of metatarsus. These characters are considered by author following Holtz (2004) as autapomorphies for the Tyrannosauroidea. However, these characters are found also in the Ornithomimidae. The author did not discuss the alternative identification of the described specimen as an ornithomimid and did not rule out this possibility. However, described bones show considerable similarity with some ornithomimids (see, for example, a well described metatarsus of Qiupalong henanensis; Xu et al., 2011). By gracile nature I think that the described metatarsals from the Merchantville Formation are more referable to an ornithomimid rather than to the tyrannosauroids, which had massive and robust metatarsals. The author violates the International code of Zoological Nomenclature (Article 36). Some other comments are introduced in the attached pdf.

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

Title: A TYRANNOSAUROID METATARSUS FROM THE MERCHANTVILLE FORMATION OF DELAWARE INCREASES THE DIVERSITY OF NON-TYRANNOSAURID TYRANNOSAUROIDS ON APPALACHIA Review round: 1 Reviewer: 3

Basic reporting: The prose could really be cleaned up, especially with regard to passive voice and redundancy. Literature references are incomplete and insufficiently used. Professional article structure is present. More measurements could be included. Self-contained and the results are relevant to hypotheses, although overstated and incomplete in places. See general comments and confidential note for details. Experimental design: original primary research is present, although arguably incomplete. Research question is defined, but more thorough comparisons could be made. The knowledge gap is incompletely filled. Ethical, but not rigorous. Methods are described and replicable. See general comments and confidential note for details. Validity of the findings: Impact and novelty could be improved with additional study of specimens and literature. Data are incomplete. Conclusions are overstated in places. See general comments and confidential note for details. Additional comments: Dear Editor, I submit to you my review of “A tyrannosauroid metatarsus from the Merchantville Formation of New Jersey increases the diversity of non-tyrannosaurid tyrannosauroids on Appalachia” by Brownstein (ms #19258). The article describes the partial metatarsus of a basal tyrannosauroid in comparison with other taxa and isolated bones, all from the late Cretaceous of Appalachia. My opinions on the ms are mixed, as I am happy to see an additional specimen added to the roster of tyrannosauroids from the Appalachia, but the description strikes me as incomplete. In particular, the comparisons of the new metatarsus with other Appalachian taxa appear to be fairly superficial, perhaps owing to the fact that only two museum collections were visited. To really nail down the comparisons, all of the relevant specimens must be examined first hand and, ideally, side by side. For example, it appears that the holotype of Appalachiosaurus was not view first-hand, and comparisons were only made from the published literature. I am not convinced that the author could really determine that “the spectrum of individual variation” of Appalachian tyrannosaurids was truly identified, since the relevant taxa are represented by only one specimen. Also, the author did not take advantage of the recent phylogenetic work on tyrannosauroids (e.g., Brusatte and Carr, 2016; Carr et al., 2017), where character states of the metatarsus are listed that could have helped flesh out the comparisons and characterizations of the new material and the other species. The figures could be more fully labeled so that they correspond to the observations made in the text. The captions are incomplete for metatarsal IV. I think a table that summarizes all of the major similarities and differences between the new metatarsus and other Appalachian taxa would greatly benefit the manuscript. The prose could really be cleaned up, especially with regard to passive voice and redundancy. The biogeographic conclusions are somewhat overstated and can be simplified to just the facts. In conclusion, I think the author could satisfactorily make the necessary revisions and so I recommend “major revision”. The author may know my identity: Thomas D. Carr. My specific comments are below. Sincerely, Thomas D. Carr, PhD Associate Professor of Biology Carthage College Specific comments: Lines 7-10: overuse of the word “obscure” and its variants. Lines 9-10: “do seem to be”; passive voice – fix. Lines 10-11: “significant”; this word has statistical meaning and should not be used informally here. Lines 18-20: the spectrum of variation cannot be known for taxa that are represented by only one specimen – please fix. Lines 19-20: replace “as well as by factoring in” with “and”. Lines 19-20: replace “The new specimen thus has significance for representing” with “Therefore, the new specimen represents”. Lines 21-25: convoluted – just state the result. Last sentence of abstract: change to “Appalachian non-tyrannosaurid tyrannosauroids had a diversity that was comparable to tyrannosaurids in Laramidia during the Campanian”. Line 31: in what way is Appalachia “very poorly known”? Clarify. Line 31: replace “comparison” with “contrast”. Line 40: delete “repeatedly”. Lines 42-44: how is all of that unfortunate? Clarify. Line 52: delete “described herein thus”. Line 53: insert “a new,” between “possibly” and “unnamed”. Line 53: delete “dinosaur”. Line 54: replace “. Thus, the specimen adds to the diversity of the clade Tyrannosauroids” with “, adding to the diversity of the the clade” Line 55: replace the comma with a period. Line 55: replace ”suggesting” with “The specimen is evidence that” Lines 56-57: replace “the aforementioned” with “that”. Line 61: replace “the described” with “this”. Line 67: in my experience, the full AMNH specimen number abbreviation is “AMNH FARB”. Line 77: delete “described herein”. Line 77: replace “retrieved” with “collected”. Lines 77-81: were the two bones found together? State explicitly. Line 79: replace the ampersand with “and”. Lines 81-85: any radiometric dates? If so, please include here. Lines 98-104: what are the sizes of the specimens? Line 107: replace “of metatarsal II with” with “for”, and label the feature in the figure. Lines 110: explain what is meant by “a more prominent articular facet”; this is vague – be exact. Line 111: delete “exists”. Line 111: insert “Therefore,” before “The”. Line 112: delete “thus”; “not as distinct” is vague – be exact; delete “in medial view”; “and,….metatarsal II.” is convoluted – fix. Line 115: replace “slight” with “shallow” and label it in the figure. Line 118: delete “as measured along the proximal surface”. Line 119: replace “better” with “best”. Line 120: replace “metatarsls” woth “bones”; insert “that are” after “scrapes”. Line 124: replace “confirms” with “is evidence”; replace “being” with “it was”; delete “and preserved”. Line 125: “a sharp ventral edge” is vague – clarify and label in the figure; replace “rounded” with “convex”. Line 126: is the ventral surface flat? Clarify. Line 128: “just above the diaphysis” is vague – clarify. Line 133: replace “suggests the presence of” with “is evidence for”. Line 134: delete “having”. Line 135: “plantar” – be consistent with terminology throughout the ms. Line 136: what is the specimen number of the unnamed specimen? Mention it here. Line 136: replace “also…specimen” with “less pronounced than in YPM…obs.); delete “distinguishing the two specimens”. Line 138: delete “unnamed New Jersey specimen’s”. Line 139: insert “of New Jersey” after “IV”; “a much thinner shaft” is vague, provide measurements; delete “(pers. obs.)”. Line 140: deete “notably”. Line 143: replace “further be” with “also be”, delete “in…latter”. Lines 149-152: Delete “In…2011).” Line 154: replace “an even less noticeable” with “more aubtle”. Line 161: give the estimated total length here. Line 167: “temporslly differentiated” is vague – be exact. Line 171: give the measurements instead of giving “pers. obs.” Lines 172-173: list all of the differences in a new paragraph here or provide a table of comparisons and differences. Line 173: delete “morphological” from here and elsewhere. Line 175: replace “element” with “bone”. Line 178: “less robust” is vague – be exact. Line 182: delete “the assignment of”, insert “is assigned to” after “21795”; delete “is considered best”. Line 184: list the comparisons! Line 184: delete “and this indeterminate taxon,”; delete “dinosaur”. Line 186-187: fix the sentence that starts with “The identification”. Line 188: replace “from” with “in”; replace “corresponding to this landmass” with “of Appalachia”. Line 196: replace “a possibly” with “an”, replace “animal” with “taxon”. Line 201” replace “tyrannosaur” with “tyrannosauroid”. Lines 205-206: delete “especially…account”. Line 207: replace “enjoyed” with “had a”. Lines 210-212: convoluted – fix. Line 217: what are the differences? List them here. Line 218: replace “currently…author” with “in progress”. Lines 218-219: what are they? Be exact. Lines 221-237: doesn’t all of this boil down to a sampling issue? Delete. Line 246: delete “from the lower 48”. Line 247: replace “tyrannosaur” with “tyrannosauroid”. Lines 248-250: Delete. Line 254-255: replace “. Additionally thank” with “, and”. Lines 255-256: delete “in…History”. Figure 2 caption: the caption is incomplete – what is the arrow pointing to? The distal condylar region is not included.

You are one of the reviewers, your task is to write a review for the article. You will be 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 TYRANNOSAUROID METATARSUS FROM THE MERCHANTVILLE FORMATION OF DELAWARE INCREASES THE DIVERSITY OF NON-TYRANNOSAURID TYRANNOSAUROIDS ON APPALACHIA Review round: 3 Reviewer: 1

Basic reporting: see below Experimental design: see below Validity of the findings: see below Additional comments: I thank the author for taking into account all of my suggestions and carefully revising the manuscript. I may have a few quibbles about some of the anatomical details, but at this point, I think the author has been very rigorous and deserves to have this manuscript published so others can assess the specimen. One thing I would ask the author to do: explain in more detail the phylogenetic analysis and how it was conducted. It is said that a ‘strict consensus analysis’ was run in TNT. This doesn’t make sense. I presume the author means that the analysis was used to generate a strict consensus phylogeny of the most parsimonious trees, after the analysis was conducted. So the author needs to be clear on what protocols were used in the analysis: New Technology search (and the specs)?, TBR branch swapping (and the specs?) etc. The author should try to follow the exact protocol that we outlined in the Brusatte & Carr (2016) paper, to make it maximally comparable with our analysis.

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

Title: A TYRANNOSAUROID METATARSUS FROM THE MERCHANTVILLE FORMATION OF DELAWARE INCREASES THE DIVERSITY OF NON-TYRANNOSAURID TYRANNOSAUROIDS ON APPALACHIA Review round: 3 Reviewer: 2

Basic reporting: In many places the prose is convoluted and compromised by redundancy and passive voice. The literature references are sufficient. Sufficient background and context is given. A professional article structure is followed, although subheadings for comparisons with other taxa might be helpful. The article is self-contained, although I think that the issues of individual and ontogenetic variation can be carried out more rigorously and concisely (see comments to author). The author may know my identity: Thomas D. Carr, PhD Experimental design: The article is within the aims and scope of the journal insofar as evidence for a new taxon of tyrannosauroid is presented, although based on a poorly preserved specimen. The research question is straightforward, where the author presents evidence for a new taxon of Campanian tyrannosauroid; given the limited nature of the fossil, it fills a very small gap in our knowledge. However, I am concerned that it hasn't been compared side-by-side with Appalachiosaurus, especially given that some differences between them are the result of damage to the Merchantville specimen (see ms Table 2). I don't think the comparisons made are rigorous (see comments to author), but these can be improved. The methods are adequately described and replicable, although I would run the Merchantville specimen in the latest TWiG matrix, not the Loewen et al. (2013) matrix. The author may know my identity: Thomas D. Carr, PhD Validity of the findings: Given the nature of the Merchantville fossil, and the limited scope of the paper, the impact is not great beyond the addition of a new tyrannosauroid from Appalachia. Not much can be said beyond that. The novelty is limited to the possibility of a sympatric tyrannosauroid with Appalachiosaurus, but given the wide range of the age of the Merchantville Formation, they might not really have lived at the same time. The Merchantville specimen does not reveal anything new about tyrannosauroid evolution, aside from increasing its diversity by one taxon. The data are robust, but the RI for the Merchanville taxon is compromised by deformation (table 2), which reduces my confidence in its difference from Appalachiosaurus. The conclusions are fine, but the closing statements regarding incomplete specimens isn't needed since the limited amount of information they contain is a given. Speculation is thankfully avoided. The author may know my identity: Thomas D. Carr, PhD Additional comments: October 9, 2017 Dear Dr. Farke, I submit to you my review of the second draft of “A tyrannosauroid metatarsus from the Merchantville Formation of Delaware increases the diversity of non-tyrannosaurid tyrannosauroids on Appalachia” #19258, by C. D. Brownstein. The author has taken care of many of the issues that I raised in the first draft, but some nagging problems remain: the prose is compromised by passive voice and excess verbiage, the introduction needs to be almost entirely redrafted, first-hand comparisons have not been made with Appalachiosaurus, and the discussions of individual and ontogenetic variation should be more rigorous. The bottom line is that the sample sizes for the three taxa of concern are, effectively, one each; therefore, the only resort to clarifying individual and ontogenetic variation is for the author to examine a growth series of metatarsals of a known tyrannosaurid. Such a growth series is available at the AMNH, based on the Albertosaurus bonebed material. Using that, the author can parse out the features that do change with size (as a proxy for maturity, size is not the best option - I know - but the gross patterns should be clear) and those that don’t, and then compare the corresponding features with the other Appalachian fossils to identify which ones are taxonomically informative and which aren’t. If this was my project, that’s what I’d do. If this procedure is followed, then the author would arrive at defensible, empirically based statements such as: “The RI does not change in the growth series of A. sarcophagus, where it is 0.33 in all specimens. The RI of the Merchantville specimen is less than this and that of Appalachiosaurus; therefore, it is a different taxon from them. The RI of Dryptosaurus is 0.15; therefore, the Merchantville specimen is not referable to Dryptosaurus. Given these differences, the Merchantville metatarsus represents a new morphotype, and hence a new taxon.” Or, “In end view, the distal end of metatarsal IV is triangular and rectangular in the growth series of A. sarcophagus, without any pattern. Therefore, the triangular shape of the joint surface in the Merchantville specimen is taxonomically and ontogenetically uninformative, and so it is not a reliable point of comparison with Dryptosaurus and Appalachiosaurus.” And so on. Unfortunately, I still think substantial revisions must be made. Please see my specific comments below for more areas of improvement. Sincerely, Thomas D. Carr, PhD Associate Professor of Biology Carthage College Kenosha, WI 53140 Specific comments Abstract Line 11: replace “this latter landmass” with “Appalachia”. Line 12: replace “of….apparently” with “were”. Lines 12-13: replace “Laramidian ones” with “Laramidia”. Lines 13-15: these sentences can be shortened and combined into one. Lines 17-19: The justification for giving the metatarsus such attention must be stated here: is it well-preserved enough to bring new insights into Appalachian tyrannosauroid diversity and evolution? Line 19: replace “may” with “can”. Lines 21-23: please list the defining features here; otherwise this sentence is just indicative. Line 24: replace “morphotype” with “taxon”. Lines 25-26: adding a new taxon is not a test of the hypothesis that Appalachia was a refuge of relict taxa – it is just evidence of a new taxon. The evidence for a relict taxon is its phylogenetic position; so, if it falls out in a cladistic analysis outside of Bistahieversor, then you have evidence for a basal, relict tyrannosauroids. Otherwise, you don’t. I.e., please state the results of your phylogenetic analysis in the abstract. Introduction Lines 29-32: this first sentence is unnecessary – just get to the point, which is in the second sentence. Line 46: keep in mind that we didn’t run a Bayesian analysis in Carr et al. (2017); the results published there were from a parsimony analysis. Lines 46-49: redraft this sentence as “The fossil record of tyrannosauroids from Appalachia is limited to the holotypes of Dryptosaurus and Appalachiosaurus, undescribed partial specimens, and isolated bones and teeth.” Lines: 50-88: This section is written like the discussion and conclusions, not a proper introduction that justifies the project, defines the research question and its importance and novelty, describes the methods and the main objectives of the project. Redraft. Methods Lines 92-95: As with the earlier draft, the author has not seen the holotype of Appalachiosaurus first hand. Material Lines 136-157: please restructure this by first stating the arctometatarsalian features that do not help to identify the taxon, followed by the unambiguous tyrannosauroid characters. Lines 158-180: it isn’t at all clear if these features are seen in the specimen or not; for each feature, describe what is seen in the purported tyrannosauroid so that the reader can clearly see what features you are eliminating and why. Lines 181-184: this is the biggest blunder of the article, given that Brusatte and Carr (2016) combined the data sets of Loewen et al. (2013) and Brusatte et al. (2010) to resolve the huge differences in results obtained from those data sets. Therefore, scoring the specimen into the Loewen et al. matrix makes no sense on theoretical or practical grounds – it is just irrelevant. In fact, Carr et al. (2017) updated the Brusatte and Carr (2016) data matrix with additional characters (and taxa), so that should be the data matrix used to assess the affinities of the metatarsus, if it is a tyrannosauroid. Please redo the phylogenetic analysis using the most complete and up-to-date data set. I also recommend scoring the specimen in the latest version of the TWiG (Theropod Working Group) data matrix to assess the identity of the metatarsus among Theropoda as a whole. Line 181: delete the underscore in the specimen number. Line 184: insert a space between the parentheses. Description Line 187: delete “noticeably”. Line 189: replace “may be found” with “are”. Line 190: is it deformed by the erosion or by something else? Clarify. Lines 187-275: this part of the manuscript is rife with passive voice; please fix, which will help to shorten the text somewhat. Lines 207-234: these sections are a return to assessing the taxonomic identity of the specimen, which is confusing. Please move the relevant parts up to where the identification as a tyrannosauroid is justified and referral to Ornithomimosauria is rejected. Line 235: perhaps each metatarsal should get their own subheadings in the description to aid the reader. Line 250: a comma usually precedes the words “but” and “which”. Line 274: insert “long” after “mm”. Discussion Line 295: it is said here that the specimen was coded into the matrix of Brusatte and Carr (2016), whereas earlier it was stated that it was scored for the Loewen et al. (2013) data matrix – which is it? Please fix. Lines 302-304: this description of the topology is ambiguous – how can the metatarsus and Bistahieversor both be the sister taxon of Tyrannosauridae? Please clarify and include a figure of the results. Lines 304-306: analysis in the Loewen et al. matrix is unnecessary, as explained above. Line 307: state the characters that support the position of the metatarsus on the topology before mentioning features that were not included in the data matrix. Lines 310-314; please state how the RI was obtained, state the result, and give some indication of how it is different from tyrannosaurids. Lines 315-317: this introductory sentence is convoluted; please rephrase concisely. Line 330: the phrase “Regardless of the ontogeny” is confusing, as what is meant by that is not explained. Please simplify this sentence to refocus it on the comparison with Dryptosaurus. Lines 333-335: make sure that this sentence is in reference to the Brusatte and Carr (2016) data matrix, because as stated here you could be talking about that analysis or the Loewen et al. results. Please fix. Lines 336-341: both of these sentences say the exact same thing – get rid of one of them! Line 349: the sentence “Description…author.” adds nothing; please delete. Lines 347-360: I’d consider discarding this section, since you do not unambiguously assess the taxonomic identity of the AMNH specimens. Lines 365-370: I’d rephrase this to say that the Merchantville metatarsus is approximately the same size as the Dryptosaurus and Appalachiosaurus fossils, and the differences between them are almost certainly not ontogenetic since the bones in the same taxon of the same ontogenetic stage would converge upon the same morphology. Lines 361-412: this is an unacceptably long paragraph; I recommend breaks at lines 371, 378, 384, 393, 403. Lines 371-373: the sample size for these taxa is effectively one each – please be exact to convey the point that individual variation is simply impossible to document for them. Lines 376-378: size is a poor proxy for relative maturity, so the real limit on assessing ontogeny is the same for that of individual variation – you’d don’t have a adequate sample to work with. Fix. Lines 382-384: You should compare the Merchantville specimen side-by-side with Appalachiosaurus in person. Briefly remind the reader here of what the difference are between the two. Lines 384-390: I don’t find the argument of geographic separation to be convincing, especially when both taxa are on the one and the same landmass. Lines 387-390: this sentence is too convoluted; be concise. Lines 384-413: I think that all of this can be boiled down to one paragraph that summarizes the salient differences between the Merchantville specimen and Appalachiosaurus, excluding the biogeographic argument. What would potentially seal it is comparisons of the RI of metatarsals of the same species of tyrannosaurid that are the same length. Line 403: please define what the RI is, and what measurements are used. Line 407: please include the RIs here for T. rex and A. sarcophagus. Please include the RI for Dryptosaurus. Line 408: delete “the author regards”. Lines 408-411: the only relevant line of evidence to separate the two is the morphological differences. Revise. Biogeography Line 417: replace “morphotype” with “taxon”. Lines 421-423: this sentence is redundant; delete and modify the following sentence accordingly. Lines 429-430: delete this sentence; it does not contribute any meaningful information, especially since there are no overlapping bones with the Merchantville metatarsus. Line 433: you have provided evidence for increased diversity; please delete. Line 438: this could be changed to “cf. D. sp.”. Line 451: insert a space between the parentheses. Lines 457-460: restate this as a specific, defensible hypothesis or delete. Lines 461-465: convoluted and excessively long sentence – please trim down and clarify. Lines 469-471: there isn’t a clade – the Appalachian taxa form a grade, so there’s nothing to name. Conclusions Line 475: I think it is acceptable to say the morphotype is a new taxon, which you have wisely not named. Line 477, and above biogeography discussion: I recommend that you avoid discussing the affinities of the other Appalachian material unless they are metatarsals. I think that teeth and postcranial bones that are not metatarsals are beyond the reach of what you actually have described. It is sufficient to mention those fossils, but it is important to point out that their identity cannot be assessed based on the Merchantville metatarsals, since none of them are associated with a metatarsus. Lines 481: I’d restate this in terms of the challenge of identifying isolated bones that that autapomorphies. Acknowledgements Lines 487-493: make sure that you thank your reviewers by name. Figures The font size of the labels are different; please standardize. I recommend adding a comparative figure of the Merchantville specimen side-by-side with the corresponding bones from the holotypes of Dryptosaurus and Appalachiosaurus. Table 1. Insert a space between adjacent sets of parentheses. Insert a + next to “112” if that number is artificially low. Table 2. Insert a space between the parentheses and the preceding text. “(this paper)” is unnecessary; delete. Supplemental material I recommend that the analysis of placing the Merchantville specimen within theropod (as a test of the Ornithomimosaurian hypothesis) be done using the latest version of the TWiG data matrix, not the Loewen et al. (2013) data set.

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

Title: ACUTE ISCHEMIC PRECONDITIONING DOES NOT INFLUENCE HIGH-INTENSITY INTERMITTENT EXERCISE PERFORMANCE Review round: 1 Reviewer: 1

Basic reporting: Ischemic preconditioning (IPC) has been suggested as a novel treatment that has the potential to improve exercise performance. The current study is aimed to examine the acute effect of IPC on intense intermittent exercise performance and associated physiological markers in amateur soccer players. Subjects were divided into three groups receiving: a) IPC (high pressure exerted on each thigh during blood occlusion phase); b) IPC SHAM (low pressure exerted on each thigh during occlusion phase); and c) control (no treatment). Following the treatment, YoYo intermittent Endurance Test level 2 (YoYoIE2) was performed to evaluate intermittent maximal exercise performance in each group. The results showed that IPC has no significant effect on players’ performance of YoYoIE2. In addition, there was no marked difference in lactate concentration, rate of perceived exertion, and heart rate between the three groups. These results show that IPC does not have any influence on vigorous intermittent exercise duration in amateur soccer players or related biomarkers. Experimental design: see below Validity of the findings: see below Additional comments: Points: 1. The following sentence is shown in lines 138-139, “Mean HR and peak HR presented no difference (p=0.17) among the three trials (Table 1).” However, in table 1, the p value of HR peak is presented as 0.06 instead of 0.17. Could the authors explain which the correct p value for HR peak is? 2. It is concluded in this article that “IPC does not influence high-intensity intermittent exercise performance in amateur soccer players”. However, the study only tested IPC protocol consisting of four cycles of 5-min occlusion. Could the authors justify why they use this IPC protocol? Both the number of cycles and the duration of occlusion may have significant effects on exercise performance. To confirm their conclusion, different IPC protocols (such as increasing or decreasing the IPC cycles) should be tested for their effects on exercise performance. Furthermore, the current study should clarify in their conclusion that only acute IPC did not have effect on their exercise performance, because the study has not tested the effectiveness of chronic IPC protocols (for several days or weeks), which could be a future direction. 3. “HR” shown in line 29 in abstract should be defined first time shown in text; IPC in figure 2 should be defined in its figure legend. 4. The authors only measured 13 players, whose sample size is too small to justify their conclusion. Why did the authors say this suffices for statistical points? Please discuss the limitation of this with convincing details. 5. The discussion on the molecular levels of such study is not in-depth. Reactive oxygen species play an important role in both IPC and exercise. The authors need to discuss this aspect by adding a few sentences like this: “Exercise can strengthen the body against ROS attack through redox-associated IPC preconditioning. This exercise-induced adaptation involves the enhancement of cellular antioxidant capacity to reduce ROS levels. Increased ROS generation in striated muscles plays a critical role in exercise adaptation as it can trigger exercise-mediated adaptive responses through redox regulations…..” Here for above the authors should cite the following recent key papers by experts in the fields: 1) Front Physiol. 2016; 7: 486. 2016. doi: 10.3389/fphys.2016.00486; 2) Cell death & disease 4 (9), e787, 2013. 6. In lines 41 and 42, change “its supposed exercise enhancement is still to be clarified” to “its supposed exercise enhancement has yet to be clarified” 7. In lines 42-44, change the sentence “Among potential mechanisms of IPC to exercise enhancement, are highlighted…” to “Potential mechanisms of IPC exercise enhancement are hyperemia (5-6 fold increase in muscle blood flow) during reperfusion (Libonati et al. 2001), attenuated ATP depletion, and increased phosphocreative production/oxygen uptake during the reperfusion phase (Andreas et al. 2011).” 8. In lines 51-53, change the sentence “In this context, the YoYo intermittent endurance test…” to “The YoYo intermittent endurance test level 2 (YoYoIE2) is useful to simulate high-intensity intermittent exercise similar to the activity seen in team sports, and is effective due to its simplicity, reproducibility, and its association with both V ́ O2 and citrate synthase activity in untrained men” 9. Please clarify what team sports are being simulated by the YoYo test. It is too vague to attribute high-intensity intermittent exercise to team sports in general as every team sport is very different from one another. 10. In lines 55-59, it is stated that “aerobic energy production plays a more important role in fatigue resistance in the untrained during the YoYoIE2 compared with trained soccer players” however, high-intensity intermittent exercise implies predominantly anaerobic energy production. What is the role of aerobic energy production in the YoYoIE2 test? 11. In lines 170-171, change “These same authors have been argued that there are IPC responders and non-responders” to “These same authors have argued that there are IPC responders and non-responders”. Also, please clarify what is meant by IPC responders and non-responders and what differs between the two.

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

Title: ACUTE ISCHEMIC PRECONDITIONING DOES NOT INFLUENCE HIGH-INTENSITY INTERMITTENT EXERCISE PERFORMANCE Review round: 1 Reviewer: 2

Basic reporting: The study investigated the acute effects of ischemia preconditioning on hign intensity intermittent exercise performance in amateur soccer players. The authors found out that IPS had no influence on the exercise performance. Experimental design: It's good. Validity of the findings: OK. Additional comments: Please confirm the IPS protocol did have a effective stimulation on the subjects. Any marker from the blood immediately can be used to evaluate the response of subjects to IPS before the exercise?

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

Title: ACUTE ISCHEMIC PRECONDITIONING DOES NOT INFLUENCE HIGH-INTENSITY INTERMITTENT EXERCISE PERFORMANCE Review round: 1 Reviewer: 3

Basic reporting: The purpose of this study is to exam the acute effect of ischemic preconditioning (IPC) on a high-intensity intermittent exercise performance and physiological indicators in amateur soccer players. Paper is poorly written and lack of clarity in some sentences (please see below for the details). In addition, there are a lack of details in methodology (for example, what is the sampling time for lactate?). Lastly, there is a lack of depth in discussion. Experimental design: According to the previous studies (Patterson et al. 2015 & Incognito et al. 2016), IPC does demonstrate that there is an improved peak and mean power output during the early stage of repeated sprint event as well increase the maximal oxygen consumption during incremental bicycle exercise. My understanding is that the time interval between IPC and exercise testing should be essential in the impact of the IPC. However, there is a lack of information of the time interval between IPC and Yo-Yo intermittent endurance test. Although this study included three groups (IPC, SHAM, Control) with single-blinded design, it is still very different to rule out the psychological effect of the participant. The significant finding form the previous study could be partially explained by the highly motivated participant following IPC. Since there was IPC non-responder and responder individual difference in soccer players, having a larger sample size might make more sense. Incorporating EKG, ventilation, R , VO2, peak power, and maximal oxygen measurements in this study will strengthen the quality of this paper. Validity of the findings: no comment Additional comments: Introduction Line 51-59: there is a lack of clarity in these sentences. For example “ being associated with both VO2 and citrate synthase activity in untrained men” I am not sure what this means? Based on the logic flow, author assumed that untrained is similar to amateur which I am not completely agree with. Discussion: There is a lack of discussion in depth in physiological explanation for the absence of significant finding during YoYoIE2 test following IPC in comparison to other studies, although author briefly mentioned that this difference is probably due to the type of exercise. It seems like that IPC had a certain acute effect in exercise performance from previous study, however how long this acute effect could last was not clear. The nature of the intermittent maximal test might counterbalance the acute effect following IPC.

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

Title: ACUTE ISCHEMIC PRECONDITIONING DOES NOT INFLUENCE HIGH-INTENSITY INTERMITTENT EXERCISE PERFORMANCE Review round: 2 Reviewer: 1

Basic reporting: The authors have addressed my major concerns. Experimental design: See below Validity of the findings: See below Additional comments: The authors have addressed my major concerns. I accept it in the latest 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: ACUTE ISCHEMIC PRECONDITIONING DOES NOT INFLUENCE HIGH-INTENSITY INTERMITTENT EXERCISE PERFORMANCE Review round: 2 Reviewer: 2

Basic reporting: The study investigated the acute effects of ischemia preconditioning on high intensity intermittent exercise performance in amateur soccer players. The authors found out that IPS had no influence on the exercise performance. Experimental design: The ischemic preconditioning protocol is under the steady state where muscle was not working and in the rest condition. Please consider to include some level of exercise in the ischemic preconditioning protocol that may generate better preconditioning effects. Validity of the findings: OK Additional comments: NO

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

Title: ABILITY TO PREDICT REPETITIONS TO MOMENTARY FAILURE IS NOT PERFECTLY ACCURATE, THOUGH IMPROVES WITH RESISTANCE TRAINING EXPERIENCE Review round: 1 Reviewer: 1

Basic reporting: Please see below Experimental design: Please see below Validity of the findings: I believe the statistics need to be re-run. Additional comments: The authors have investigated an interesting idea, however, I believe the authors may need to re-calculate all of their values. For example, it’s hard to believe that a mean difference of 4 repetitions corresponds to a standard error of 1. I downloaded the spreadsheet and used your data and I believe that you put the “predicted” in the criterion instead of putting the “actual reps” as the criterion. Please see below. Also, you may consider implementing the minimal difference (SD of difference score x 1.96) in addition to what you have. I believe that these changes will likely change some of the interpretation of the manuscript. Just a couple of examples, Beginner Chest Press You have 1.94 (1.48, 2.84) I believe it should be 4.0 (3.04, 5.84) Leg Press Expert You have 1.27 (1.05, 1.63) I believe it should be 1.73 (1.42, 2.21) I would also delete the figure, it adds nothing to the manuscript. A few other things to consider: Introduction: Resistance training is associated, at best, with a wide range of health benefits…this should be made quite clear. The Dankel study does not actually offer much support for “resistance training” as it found the strongest associations with the outcome of strength as opposed to the behavior. I would tone this down some Was this IRB approved? This information should be put into 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: ABILITY TO PREDICT REPETITIONS TO MOMENTARY FAILURE IS NOT PERFECTLY ACCURATE, THOUGH IMPROVES WITH RESISTANCE TRAINING EXPERIENCE Review round: 1 Reviewer: 2

Basic reporting: Quite a negative tone concerning the accuracy and use of repetitions in reserve (RIR) during resistance training. Therefore, the writing style does not seem to present a balanced argument of RIR for resistance training. Experimental design: The investigation performed is not adequate for the aim of the study. The investigators did not have participants estimate repetitions in reserve (RIR) during the actual exercise used to assess accuracy in RIR.Therefore the results presented are highly questionable. Validity of the findings: There are a lot of unknowns that affect the results reported in this study, but the major one relates to the flawed methodological design. Additional comments: Overall this paper is written in quite a negative tone concerning the accuracy of using repetitions in reserve (RIR) for monitoring of resistance training effort. An error of approximately 1 repetition is excellent for training purposes and RIR is best method that can be used to monitor resistance training effort. For example, RPE during resistance training is poorly correlated with RIR (soon to be published paper) and the information derived from RIR is impossible to fully decipher. So RIR is the obvious best method to provide coaches and trainers with added information, but RIR should not be used to prescribe resistance training. The RIR is most useful for assessing progression (adaptations) and fatigue (predictor of overtraining and maladaptation). The sample size used for this study is great but there is a major methodological flaw. The accuracy of the RIR can only be assessed with a trainer reporting their rating during the exercise in which they will then performed repetitions to momentary failure. This is because you need the trainer to be able to utilise the many physiological and psychological cues to make their estimation. It makes no sense to have trainers make an estimation based on their previous training experience. The number of repetitions performed to failure with a specific relative load is likely to change on a day to day basis. Factors such as sleep, diet, previous training, time of day, other stressors, motivation etc. will impact upon the repeatability of performing repetitions to failure at a set load. The participants in your study may not have been motivated by the assessor but they were under research conditions and probably confounded your results. Also, it is no surprise that you found more experienced trainers would be more accurate with RIR. I might even hypothesise that this was due to the experience RT greater training frequency. Did you assess when your participants performed their last training sessions prior to your study? Potentially this could have impacted some of the results. Specific concerns: Line 65: ‘applications of RT to maximise these outcomes …..’ does not make sense? The ‘application’ refers to putting RT into practice. I think you mean the RT prescription? Line 106-108: Trained participants are good at predicting repetitions to momentary failure. What criteria are you using to make your statement? You could not be possibly thinking that trainers can develop an ability to predict RIR with 100% accuracy. It will always be dependent on the loads used and proximity to momentary failure. Line 194: Never end sentence with a prepositional primer (also line 316). Lines 232-233: Anchoring of perception…… interesting because the study participants were making estimates based on previous exercise experience. Seems inappropriate to have this 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: ABILITY TO PREDICT REPETITIONS TO MOMENTARY FAILURE IS NOT PERFECTLY ACCURATE, THOUGH IMPROVES WITH RESISTANCE TRAINING EXPERIENCE Review round: 1 Reviewer: 3

Basic reporting: No comment, all areas of basic reporting meet standards. Experimental design: No comment, all areas of experimental design meet standards. Validity of the findings: Data is robust and statistically sound and controlled. Interpretation is the only area where revision is recommended; see general comments. Additional comments: First I wish to thank the authors for their contributions to this field of study. This is an important body of evidence to inform this area of research, the writing is clear, the findings are well presented, and the methods are sound. Additionally, the authors are correct that the work by Hackett and colleagues is influenced by the fact that multiple sets to failure were performed in both their 2012 and 2016 studies. Thus, as correctly pointed out by the authors, previous sets would help to improve accuracy of subsequent sets, thus potentially inflating the statistical representations of accuracy in both studies. Thus, the approach of estimating how many repetitions the lifter believes they can accomplish before ever beginning a set, and then comparing that estimation to the actual performance is novel. That said, there are some revisions needed to ensure an accurate comparison of this data set is made to that of the existing literature. In the title, and lines 41 (abstract), 107, 197, 202, 235, 261, 264, 276, 278, and the 318, the use of the word “poor” should be amended to a less subjective term. I would advise “not perfectly accurate” as this isn’t a debatable description, given the subjects in this study were unable to predict with complete accuracy how many repetitions they could perform to failure with a given load. The reason I believe this is important is the authors cite Hackett 2012 and 2016, and refer to the accuracy as “poor”, while Hackett and colleagues themselves in 2012 referred to the same accuracy as “high” and then as “quite good” in 2016 in the discussion sections of both papers. Specifically in their 2012 article, they state the following “Despite the differences between the estimated- and actual- repetitions-to-failure for the earlier sets of exercise, the difference was approximately only one repetition, indicating that participants only slightly underestimated this.” The use of “only slightly underestimated” contrasts the authors’ use of the word “poor” to highlight my point. Both Hackett and colleagues and the present authors use a subjective description for the objective distance to failure achieved by their subjects. Neither can be said to be “correct” as it is a subjective determination, however, the authors can use less subjective terminology. Thus, please change all instances of “poor” that I highlighted above to “not perfectly accurate”, or similar, or when in doubt, simply state the actual distance from failure quantitatively as the number of repetitions. The limitations the authors refer to when citing Hackett 2016 in lines 111-116 need to be revised as they contradict the methods of Hackett 2016. As it is stated in the methods of Hackett 2016 “Subjects aimed to perform 5 sets per exercise, but if concentric failure occurred prior to reaching 10 repetitions for a set, the testing for the exercise ceased.“ Additionally, in the beginning of the results section of Hackett 2012 it is stated “For both the bench press and squat, the estimated- and actual-repetitions-to-failure for set 5 was 0 for all participants. Therefore, data from set 5 were excluded from the ANOVA.” Based on these two quotes, the authors should revise the stated limitations of the data by Hackett and colleagues as it doesn’t appear that sets were continued if failure was reached at or before repetition 10, and if failure was reached at repetition 10, the estimation of 0 repetitions remaining was not included in the analysis. Finally, I think the authors do a fantastic job pointing out the issue of an a-priori assumption of accuracy and of the short comings of RIR as a resistance training prescriptive tool. However, on balance, I think the authors should also point out some of the benefits of RIR based load prescription. One, that the authors already point out, is that it is perhaps more appropriate for strength athletes and bodybuilders vs untrained individuals. Indeed, the Hackett 2012 paper showing ~1 repetition away from accuracy was on bodybuilders, and almost the entirety of work by Zourdos and Helms is on competitive powerlifters. I think that should be specifically pointed as both a limitation for general population users, and potentially a benefit for strength and physique athletes who are much less likely to self-select loads that will not be challenging or lead to adaptation.

Also on balance, please point out what the RIR approach improves on. As the authors pointed out in lines 210-213, studies using traditional Borg RPE sometimes report RPE values in the 6-9 out of 10 range (depending on the study) even when training to muscular failure. Thus, as was shown in Hackett 2012, RIR is likely an improvement Borg RPE for resistance training. Additionally, the authors also point out in lines 87-88, there is large variation in the number of repetitions that can be performed at the same relative load (% 1RM) between individuals. In fact, these differences can be quite large; in endurance athletes compared to strength athletes, on average, a difference of 22 reps at 70%, 8 reps at 80% and ~4 reps at 90% of 1RM https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4042664/. Thus, it would seem that even with the inaccuracy found in the present study, RIR is likely to provide a load closer to the intended difficulty than percentage 1RM based load prescription as well. To conclude, excellent work, this data is an important contribution collected in a novel manner, and I would recommend the following amendments in order of importance as: 1. Changing the subjective wording of poor to “not perfectly accurate” or similar, or just stating distance from failure when referring to the present and previous studies (by Hackett) 2. Amending the limitations stated about the Hackett paper. 3. Including the fact that despite its limitations, RIR is likely a better alternative than %1RM or Borg RPE for load prescription in resistance training. 4. Adding additional detail to the greater utility for trained individuals, specifically strength and physique athletes as the subjects in papers by Helms and Zourdos and Hackett 2012, were powerlifters and bodybuilders respectively.

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

Title: ABILITY TO PREDICT REPETITIONS TO MOMENTARY FAILURE IS NOT PERFECTLY ACCURATE, THOUGH IMPROVES WITH RESISTANCE TRAINING EXPERIENCE Review round: 2 Reviewer: 1

Basic reporting: The authors have substantially improved the manuscript…I have only a few more points 1) you use a comma instead of a “.” In the Table for chest press…please be consistent 2) I believe some of the references you deleted in text still show up in your reference list 3) you say repeatedly that people are “not perfectly accurate”…but you never really mention what would be acceptable? What would be considered good..1 rep? It seems unlikely that you’d ever predict it perfectly? Experimental design: See above Validity of the findings: see above Additional comments: 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: ABILITY TO PREDICT REPETITIONS TO MOMENTARY FAILURE IS NOT PERFECTLY ACCURATE, THOUGH IMPROVES WITH RESISTANCE TRAINING EXPERIENCE Review round: 2 Reviewer: 2

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

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

Title: ABILITY TO PREDICT REPETITIONS TO MOMENTARY FAILURE IS NOT PERFECTLY ACCURATE, THOUGH IMPROVES WITH RESISTANCE TRAINING EXPERIENCE Review round: 2 Reviewer: 3

Basic reporting: no comment Experimental design: no comment Validity of the findings: no comment Additional comments: I wish to thank the authors for considering all of my proposed revisions. I have no further suggested revisions, and I humbly suggest the paper is now improved. Well done!

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

Title: ABILITY TO PREDICT REPETITIONS TO MOMENTARY FAILURE IS NOT PERFECTLY ACCURATE, THOUGH IMPROVES WITH RESISTANCE TRAINING EXPERIENCE Review round: 3 Reviewer: 1

Basic reporting: The authors have adequately addressed my concerns. Experimental design: The authors have adequately addressed my concerns. Validity of the findings: The authors have adequately addressed my concerns. Additional comments: The authors have adequately addressed my concerns.

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

Title: ORDINARY KRIGING VS INVERSE DISTANCE WEIGHTING: SPATIAL INTERPOLATION OF THE SESSILE COMMUNITY OF MADAGASCAR REEF, GULF OF MEXICO Review round: 1 Reviewer: 1

Basic reporting: In my review of the document, I went through and made quite a few modifications of sentence structure and grammar. The paper is nearly there, but the overall quality of the writing would be well-served by another edit by a native English speaker. The authors should add the complete R code used in their analyses to the supplementary files available for download. The authors did a fine job structuring the paper and creating some useful, interesting figures. I would like to see a more explicit hypothesis (re: IDW vs. OK) retroactively suggested in the introduction of the paper. Experimental design: The results are interesting and useful for spatial ecologists considering IDW vs. OK procedures. As the authors stated, these methods are infrequently utilized in marine studies. As stated above, the authors need to include their code with their other materials so readers can replicate their work. Validity of the findings: The authors did a good job outlining the impacts and ecological rationale of their findings. I would like to see the Discussion fleshed out with a paragraph added regarding why the IDW outperformed the OK methods. The authors successfully and frequently related the findings of this study to those of relevant prior studies. Additional comments: I enjoyed this paper and hope to see it published. I would like to explore your R code out of my own interest in these methods and curiosity about how they perform in another study region.

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

Title: ORDINARY KRIGING VS INVERSE DISTANCE WEIGHTING: SPATIAL INTERPOLATION OF THE SESSILE COMMUNITY OF MADAGASCAR REEF, GULF OF MEXICO Review round: 1 Reviewer: 2

Basic reporting: The paper is very well written and strcutured, has great figures and is generally a joy to read. It makes sense and is coherent. Experimental design: The experimental design works, and I only have one query: there are a lot of sampling points - Madagascar Reef clearly is the best sampled reef in the world! It would be useful to undertake a sensitivity analysis to find out how many points are needed to get this level of predictive capacity - this would be informative for places with fewer survey points, and also highlight how much sampling effort is required with interpolation to get good predictive capacity. Validity of the findings: no comment, the paper is very well rounded and the findings are valid Additional comments: Spatial interpolation methods generally are weak or unsuitable to support spatially explicit analysis in ecology and conservation. This is why i was extremely skeptical about this article, but you succeeded in convincing me that this is a great contribution. My initial reaction also points to an area of improvement: the abstract and early introduction focus on interpolation methods, but do not explain clearly enough under which circumstances they might indeed be useful. Much spatially contiguous environmental data comes from satellite data and has a given resolution, and for projects that want to spatialise survey data at smaller scales, interpolation might be useful because environmental data that could serve as predictor variables to create spatial models do not exist. I would also recommend to discuss a little more under which circumstances interpolation would be preferred over predictive spatial models.

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

Title: ORDINARY KRIGING VS INVERSE DISTANCE WEIGHTING: SPATIAL INTERPOLATION OF THE SESSILE COMMUNITY OF MADAGASCAR REEF, GULF OF MEXICO Review round: 1 Reviewer: 3

Basic reporting: I suggest the basic reporting can be improved. It is often ambiguous and intended messages can be unclear. The English can be improved. The literature references and background context are good. The introduction was clear and well structured for the study. However, I suggest some of the methods is more appropriate to the discussion or Introduction. I suggest the comparisons made in the discussion between the study site and other regions can be summarised in a table. Attention is needed to correct english in the figure legends. The results are relevant to the aims however the structure of the manuscript can be improved with simplified listing of the objectives. Experimental design: I believe attention is necessary to describing the methods more clearly. Overall I understand from the manuscript that the research is relevant and adresses an overlooked approach however this can be communicated more clearly. Validity of the findings: The conclusions are clear, however can be communicated more clearly. I believe more focus should be given to the comparison of the methods for interpolation and less to the description of the community structure at Madagascar reef. Community descriptions can be detailed in supplementary information, but I suggest this is not appropriate for a research publication and is more fitting of a survey report. It is speculated in the abstract that patchy habitats and community composition "could benefit many mobile species" but this is not supported by findings in the paper. Additional comments: Abstract Text although clear could be simplified to focus on the objectives. For example from the sentence starting with “we compare…” at line 20 in the Abstract , text could be modified as follows and to also follow the order of objectives outlined at the end of the introduction: We compare the accuracy of inverse distance weighting (IDW) and ordinary kriging (OK), to predict the distribution and abundance and identify hotspots of habitat forming organisms on a poorly studied coral reef. Hard corals, octocorals, macroalgae, sponges and zoanthids of Madagascar reef, of the Yucatan continental shelf are important biodiversity and fishery resources. Followed by a description of the habitat: “The deeper sandy environments of the leeward and windward regions are dominated by macroalgae and octocorals. However, the shallow rocky environments of the reef crest have the highest richness of habitat-forming organisms with high abundances of octocorals and macroalgae, and patchy dominance of sponges, Millepora alcicornis and zoanthids, creating high habitat heterogeneity.” Then followed by the summary results from comparing IDW and OK… Introduction: The manuscript provides suitable content, structure and references in the introduction. I suggest attention is given to improve the text in line with the following examples: Line 75: can the authors identify the “complex procedures” and/or explain why they are undesirable here. Lines 76-78: Over use of “it’s” becomes confusing. I suggest: “However, accuracy of remote sensing diminishes as water turbidity and depth increase light absorption by the water column (Lucas & Goodman, 2014). Remote sensing also has limited ability to identify taxa and accurately estimate abundance (Kutser & Jupp, 2006).” Lines 84-86: Use of “having” is awkward English and I suggest modification for example as: However, there are many relatively small coral reefs (e.g. ~1 km2), in deep or turbid environments, which are a conservation priority (Cohen & Foale, 2013). Lines 111-114: I suggest the outline of the objectes is reworded to combine i with ii, and iii with iv. For example: “The present study (i) gathered baseline information of the abundance and community structure of HFO at Madagascar Reef, and (ii) compared the accuracy of IDW and OK to interpolate abundances of HFO and synthesize this information” Methodology Line 118: the Sampling design is a little unclear. I believe the explanation can be improved. I suggest clearly stating how many transects were used and how long they were, then how many photo quadrats were taken on each transect, followed by the size of each photoquadrat. Line 129: I suggest changing the wording to : “Relative abundance of habitat forming organisms was estimated as percent cover in each photograph…” Line 139: I suggest changing “of each photography” to “from each photograph” Line 142-143 “…are of each other,” please change to “…are to each other…” Information at lines 143 to 149 is possible more appropriate in the introduction or discussion. Lines 156-157: the following sentence is unclear: “The parameters of searching window were the same for both methodologies.” Line 163: in reference to “Millepora alcicornis (millepora)” is this a taxonomic subspecies in backrets or is this to indicate further reference in the text or graphs uses “millepora” Lines 163-165: “…the inclusion in the model of scleractinian corals produced overestimations on the predictions given the small colony sizes found in situ (< 25 cm2).” This should be discussed in terms of where the methodology is appropriate, for example are small corals common in deep and turbid environments and therefore is this method appropriate for corals? Results Line 192: I suggest avoinding language such as “so did”. This can be unclear for the intended meaning. A suggested alternative is: “…the percent cover of sandy substrate and macroalgae increased…” Legend of Figure 1 Change “Localization” to “Location” Legend of Figure 6: Correct “fistribution” Figure 7: It is unclear to me the term HFO “richness” is appropriate. The description of how this was calculated at line 169 in the methods indicates it is a presence absence result to show how many types of HFO are present. Discussion Line 303: Start sentene with a capital “however,…” I suggest that the comparison of the community structure of Madagascar reefs is not the key result and therefore should not occupy a large initial section of the discussion. Perhaps this can be summarized in a table in the results? It is currently confusing to follow, partly as a result of the use Endlish language, the multiple locations and organisms and also because the organisms referred to are not always specified. For example Lines 307 to 310 do not specify octocorals. Lines 318-319: I suggest rewording similarly to: “Our study found IDW to be a better methodology than OK for interpolating the abundance of coral reef habitat forming organisms.” However I also suggest the authors discuss the limitations for small corals, as suggested in reference for Lines 163-165 Line 343-344 I suggest changing the wording as underlined: “Our interpolations showed how the space of the reef was occupied by the HFO in a mosaic fashion.” I suggest the discussion can be started with reference to the current statement at lines 396-399, because this is the main conclusion of the study: “The only published past studies using IDW in coral reef sessile organisms found this method as a good interpolator for coral cover (Walker et al., 2012; D’Antonio et al., 2016); our results agree with them and we extend its applicability to other important sessile organisms that are emerging under climate change (Norström et al., 2009).”

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

Title: ORDINARY KRIGING VS INVERSE DISTANCE WEIGHTING: SPATIAL INTERPOLATION OF THE SESSILE COMMUNITY OF MADAGASCAR REEF, GULF OF MEXICO Review round: 2 Reviewer: 1

Basic reporting: Good basic design and implementation of the study. Experimental design: Abstract: This is a good summary of the study Introduction: Scleractinian needs to be capitalised (line 47 and elsewhere) line 63 - just a couple - replace with few good intro Methods make sense Results Figure 3 would be better placed in the methods, where the different habitats are introduced Discussion terminology of Millepora is still confused, the authors use: Millepora alcicornis (millepora) or just the species name or just millepora. at first mention, the authority is missing. I suggest using Millepora alcicornis or M alcicornis for consistency. Validity of the findings: no comment Additional comments: na

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

Title: HERMIT CRAB RESPONSE TO A VISUAL THREAT IS SENSITIVE TO LOOMING CUES Review round: 1 Reviewer: 1

Basic reporting: Figures 1 and 2 don't contain any scale information. Authors should provide scale. Figure 3: Authors suggest that the stimuli expanded and contracted exponentially to mimic a predator approach with constant speed (lines 160, 166). This is inconsistent with this figure where the size in pixels is increasing linearly. Authors should clarify the discrepancy and depict the stimulus accurately in this figure. Figure 5: Authors should indicate on the figure whether these differences are statistically significant. Authors conducted 15 stimulus presentations for each crab. I suggest that they include the data for each individual crab's probability of hiding and the number of times it took for each crab to hide in the data. Line 45, 56: Reference to Yilmaz and Meister (Curr Biol. 2013 Oct 21;23(20):2011-5. doi: 10.1016/j.cub.2013.08.015. Epub 2013 Oct 10.) would be appropriate as it is a more recent study than the ones the authors cited. Experimental design: No comment Validity of the findings: The authors claim in the abstract that the quicker the stimulus expanded the slower the rate of habituation, however in the results they say (line 223) “because large number of subjects failed to habituate, trials to habituation was not analyzed as a measure of reaction.” I suggest taking this out of the abstract as a main finding. Additional comments: The authors presented the results using clear language. I suggest the paper will be ready for publication after the modifications stated above.

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

Title: HERMIT CRAB RESPONSE TO A VISUAL THREAT IS SENSITIVE TO LOOMING CUES Review round: 1 Reviewer: 2

Basic reporting: The paper is well-written and clear, and appropriate literature is cited. However, the organization of the Methods and Results sections are confusing and seem to overlap, and there seem to be multiple Results and Methods headings. I suggest that the authors have a methods section where they describe the experimental setup, animals, etc., and then a Results section where they describe all the results. Experimental design: I'm curious as to why the first experiment used a star stimulus, and the second used naturalistic stimuli? That makes it difficult to ask whether the naturalistic stimulus was more effective. Did all the crabs escape in response to the hawk and crab images? If it's not possible to do additional experiments, I would not reject for this, but the data should be included if available. Validity of the findings: It is not clear from figure 2 whether there was any statistically significant difference in the hide latency for the different expansion rates. 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: BGDMDOCKER: A DOCKER WORKFLOW FOR DATA MINING AND VISUALIZATION OF BACTERIAL PAN-GENOMES AND BIOSYNTHETIC GENE CLUSTERS Review round: 1 Reviewer: 1

Basic reporting: The manuscript submitted by Cheng et al. describes BGDM Docker - a tool specifically designed to evaluate bacterial pan-genomes. BGDM Docker integrates a software for genome annotation (Prokka), a software for pan-genome analysis and classification (PanX) and a software dedicated to identify clusters of biosynthetic genes, along with the metabolites that can be produced from such clusters (anti-SMASH). These software are implemented in a single docker container, along with all dependencies necessary for deployment and use in different computing environments. Overall, BGDM Docker represents an interesting tool for comparative genomics analyses and its implementation in a docker platform helps to facilitate installation and ensure reproducibility of data analyses across laboratories. The manuscript is sound and adequately written, highlighting several important aspects regarding the currently available tools for pan-genome analysis and the contribution of docker-based systems to the development of bioinformatics. There are a few points, however, that must be checked by the authors: - The software is available for download from the manuscript´s website and also from github. However, it would be interesting if authors also made it available from Dockerhub (https://hub.docker.com/), since, in this case, the functional docker image would be directly available for testing and download, through docker daemon. - Several references, cited along the text, are not present in the manuscript´s reference list. - Some links in the manuscript´s webpage do not seem to be working properly (such as http://42.96.173.25:8000/bamf_gbk44, for example). Experimental design: Authors provide an adequate description of the software capabilities, as well as detailed instructions for its implementation and use. However, these instructions suggest that users implement the three software available in the BGDMdocker container (Prokka, PanX and anti-SMASH) independently. Wouldn´t it be simpler (particularly to unexperienced users) to integrate them though a single script command (pipeline)? Moreover, as a suggestion, it would be interesting to provide in the instructions an option to run BGDMdocker without having to enter the container, which should facilitate integration of this software with external pipeline-building tools like Snakemake and Luigi, among others. One option would be to provide the command/script in the Docker CMD option, so the end user can execute the pipeline through a command similar to the following: $ sudo docker run -it BGDMdocker $pipelinecommand $filein This command could be called in Snakemake, for example, integrating the entire workflow. Validity of the findings: BGDMdocker provides interesting results and has advantages when compared to many of the currently existing tools for pan-genome analysis. However, the data provided through the manuscript´s website is difficult to download, since the files ad up to more than 600 Mb each, probably due to the presence of large fasta files, which are not relevant for a reader that only wishes to evaluate the software´s capabilities. Thus, while this is not a major point in this critique, I would recommend that authors provide the more relevant output files, such as images, tables and html pages separately in the github or figshare repositories. Additional comments: The manuscript describes a worthwhile tool for pan-genome analyses of bacterial genomes. Its implementation in a docker container shall facilitate its distribution, installation and use in different computing environments, contributing to generate reproducible results across laboratories. The manuscript is sound and can be considerably improved with a few modifications, such as the ones described 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: BGDMDOCKER: A DOCKER WORKFLOW FOR DATA MINING AND VISUALIZATION OF BACTERIAL PAN-GENOMES AND BIOSYNTHETIC GENE CLUSTERS Review round: 1 Reviewer: 2

Basic reporting: The English writing is clear. More background of Docker should be given. The data and software of the manuscript is public available. Experimental design: The research idea is novel, which introduces BDGMdockter to be utilized in biomedical research. Detailed instructions to implement BDGMdockter are also given. Validity of the findings: No comment. Please check General comments. Additional comments: This work describes a pipeline “BDGMdockter” for analysis and visualization of bacterial pan-genomes and biosynthetic gene clusters. The “BDGMdockter” could be used in PC and distributed in cloud service, which might be useful in biomedical research. 1. github is also a popular free platform for software repository. What is the advantage of Docker compared to github? 2. BDGMdockter is basically a wrapper for existing software Prokka, panX, and antiSMASH. However, the utilization of BDGMdockter seems complicated for biologists to understand with too many command lines and needs high privileges of computer such as “sudo”. Is there any way to use BDGMdockter without root privilege? It would be nice if more docker background could be introduced and the command lines of BDGMdockter could be simplified. 3. In the RESULT section, the authors claim “Fast and reproducible building of the BGDMdocker workflow across computing platforms using Docker” line 95. However, I did not see how “fast” BGDMdocker is and how “reproducible” BGDMdocker is. Maybe it is a good idea to show the running time and memory need for the software, and produce the results which have been already reported by other literature. 4. Line 126. The author claim “a website was built for the interactive exploration of the B. amyloliquefaciens pan-genome and biosynthetic gene clusters using the BGDMdocker workflow”. However, I did not see how to upload the bacterial sequences, submit the job and produce the Table 1, Figures 2 &3 from the website http://pangenome.zggskj.com/home if the interactive exploration is what it means. A detailed steps should be given for interactive exploration.

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

Title: BGDMDOCKER: A DOCKER WORKFLOW FOR DATA MINING AND VISUALIZATION OF BACTERIAL PAN-GENOMES AND BIOSYNTHETIC GENE CLUSTERS Review round: 2 Reviewer: 1

Basic reporting: No comments Experimental design: No comments Validity of the findings: No comment Additional comments: The authors address my 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: ON THE RELATIONSHIP BETWEEN TUMOUR GROWTH RATE AND SURVIVAL IN NON-SMALL CELL LUNG CANCER Review round: 1 Reviewer: 1

Basic reporting: The text is well written in clear, professional English to a good standard. Experimental design: Lines 133 - 140. This section would be improved if something could be said about these three trials. For example, were these trials in a primary or metastatic setting? Were patients previously treated? While there are references to the trials, it would aid the reader to have at least some basic background without having to look up three different references. The trial referred to as Paclitaxel/Carboplatin (ref 23) does not appear to have used Paclitaxel - the trial was actually comparing pemetrexed-carboplatin with etoposide-carboplatin. The reference to Paclitaxel/Carboplatin is made in several places in the text and should be corrected throughout. Additionally, there is no discussion of how these trials were selected. Were they the only three trials with data availability for the model, or were these three selected from a pool of candidate studies? If selected from a pool what were the selection criteria? Validity of the findings: No comment. Additional comments: An interesting question for the discussion is whether disease setting has an impact on these analyses? For example the pemetrexed/carboplatin trial was in chemo-naive patients, whereas the other two trials were in previously treated patients. Does it make sense to imagine that the disease trajectory would be the same in these two very different populations? Is it correct to assume that the same model would apply? Similarly, the point is made in the discussion that these results pertain to NSCLC - some discussion on whether these results generalise to other solid tumours would be helpful.

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

Title: ON THE RELATIONSHIP BETWEEN TUMOUR GROWTH RATE AND SURVIVAL IN NON-SMALL CELL LUNG CANCER Review round: 1 Reviewer: 2

Basic reporting: I found this to be a very interesting and well-written paper with appropriate references to the relevant literature. The paper was well-structured and cogently argued. Experimental design: The paper addresses a very important question - how to predict phase III outcome for a new anti-cancer treatment using early clinical data. The statistical analyses carried out on the data (tumour re-growth rate, time to tumour re-growth and survival) were entirely appropriate and well-presented. Validity of the findings: I found the findings compelling and therefore the conclusions of the statistical analysis are very important and have important implications for future design of phase III clinical trials for anti-cancer drugs. Additional comments: This is a very sound paper with interesting findings which deserves to be published. Some minor typographical errors which should be corrected are: lines 51, 52: "the evaluation of a drugs’ efficacy..." -> "the evaluation of a drug's efficacy..." line 52: "The drugs effect on target lesions are reported..." -> "The drug's effect on target lesions is reported..." line 199: "Details of the patients imaging and survival characteristics..." -> "Details of the patients' imaging and survival characteristics..." line 221: "it’s clear that..." -> "it is clear that..." line 240, 241: "However, by re-phrasing the question to, do TTG and GR correlate to survival post progression, to..." -> "However, by re-phrasing the question to, do TTG and GR correlate to survival post progression?, 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: ON THE RELATIONSHIP BETWEEN TUMOUR GROWTH RATE AND SURVIVAL IN NON-SMALL CELL LUNG CANCER Review round: 2 Reviewer: 1

Basic reporting: Pass. Experimental design: Pass. Validity of the findings: Pass. Additional comments: Thank you for addressing the various comments raised in the previous round. I am happy for this interesting paper to be published in its revised form.

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

Title: ON THE RELATIONSHIP BETWEEN TUMOUR GROWTH RATE AND SURVIVAL IN NON-SMALL CELL LUNG CANCER Review round: 2 Reviewer: 2

Basic reporting: All good. Experimental design: All good. Validity of the findings: All good. Additional comments: All requested changes were made - the manuscript is ready for publication in my opinion. Overall, a very timely, original and interesting piece of 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: TRANSCRIPTOMIC PROFILING OF MTOR AND RYANODINE RECEPTOR SIGNALING MOLECULES IN DEVELOPING ZEBRAFISH IN THE ABSENCE AND PRESENCE OF PCB 95 Review round: 1 Reviewer: 1

Basic reporting: Well-written paper, but some of the figures are not necessary or could be moved to supplemental information. Raw data were not shared as supplemental information. Discussion is too long given the limited scope of the study. Experimental design: Solid study but very limited in scope, as the authors only evaluated baseline expression profiles of a handful of genes at three stages of embryonic/larval development. Validity of the findings: Findings are valid, but the authors should just present relative expression or absolute transcript numbers (not both). Additional comments: Line 11: Please spell out mTOR upon first use. Lines 60-61: Recommend moving Figures 1 and 2 to supplemental information. Lines 230-343: The Discussion is way too long (~5 pages), especially given the limited scope of this study. Therefore, the Discussion could easily be trimmed down to no more than 2.5-3 pages. Lines 262-262: While this is true, it's unknown whether chemical uptake into the yolk sac interferes with nutrient availability and utilization during embryonic development. Please revise this sentence to address this possibility. Tables 1 and 2: Move to supplemental information. Figures 3 and 4: These two figures could easily be combined into a single heat-map or volcano plot. Figure 5: I'm not convinced that these data add value in addition to Figures 3 and 4. Why not just present as a fold-change or absolute copy number? Both are not necessary, as these figures rely on the same qPCR data. Figure 6: Please include images for sense ryr3 RNA probes (negative controls) in order to account for potential non-specific hybridization in situ.

You are one of the reviewers, your task is to write a review for the article. You will be 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 PROFILING OF MTOR AND RYANODINE RECEPTOR SIGNALING MOLECULES IN DEVELOPING ZEBRAFISH IN THE ABSENCE AND PRESENCE OF PCB 95 Review round: 1 Reviewer: 2

Basic reporting: The article is well written and the background for the study is sufficient. The figures and tables are professional. There are no hypotheses stated as this is an observational study of gene expression during ontogeny of zebrafish. Experimental design: In general the experimental design section is well written. There is a question about how the RNA check gel was performed and it would be good to have included a picture for the quality of the RNA. Validity of the findings: The findings are valid but the study is observational. There was no experiment included. Additional comments: In this manuscript, the authors map gene expression of key transcripts in the mTOR and RyR signaling pathways in wild type zebrafish embryos at three times points during development. The genes that they selected are excellent genes for these pathways and they provide primer pairs for the genes of interest. While it is interesting to know the timing of expression of these genes, I found the study to be descriptive and without an experimental hypothesis. Apparently this study is setting the baseline for future studies in which they will examine changes in these genes after exposure to contaminants. In my opinion, the study would be improved if the authors incorporated an actual experiment. Comments: 1. The authors extract the total RNA from a pooled set of whole embryos for a set of genes that might give information for brain development. Since these genes are likely expressed differentially in tissues during organogenesis, they dilute their signal by using whole embryos. This is a major limitation of the study as the authors point out in line 292. They might get better results if they could micro-dissect out the various tissues of interest. 2. How closely were the fish embryos that were pooled synchronized for development? Since development is rapid, a half hour difference for pooled embryos could erase a significant change in gene expression. 3. Line 207 – absolute transcript copy numbers -- I have an issue with this. The authors make cDNA for the amplified segment and purify it and measure the quantity by nanodrop. However, when doing PCR from a transcript, there is the step of reverse transcriptase that is not necessarily 100% efficient. If they want to have absolute copy number, they should start with RNA. They could get RNA for each of their genes by adding a T3 RNA polymerase promoter to one end and transcribing the RNA and quantifying that product before starting the PCR part. Also it is not clear if the absolute transcript copy number is per embryo or per ug total RNA or??? It would probably be most interesting to have the calculation be per embryo. 4. Line 43-45 – statement that the two pathways are critical for the development of the nervous system needs a reference. It seems to me that mTOR would be critical for the whole organism as would the RyR signaling pathways since they both play critical roles in homeostasis. 5. Line 114. Was total RNA integrity measured on a denaturing agarose gel – for example MOPS gel? It might be good to provide an example of the gel since RIN numbers are not provided. 6. Line 186 – in the text the gene is called “rictora” but in the figures it is referred to as “rictor” Please correct and make them the same. 7. Line 228. I don’t think that it is possible to quantitatively compare the staining for in situ across the different embryos shown in Figure 6. The magnification seems to be different for the embryos and there should be a standard against which to measure the intensity of the stain for quantitation. The statement …. “even though expression throughout the rest of the body is still low…” doesn’t seem to match the very dark staining seen in the rest of the body in the other panels at earlier stages without appearance in the brain. 8. Line 249 -- I don’t think the authors can comment on whether the increased expression of rictora and rptor promote mTOR activity since (a) they have not measured mTOR activity and (b) mTOR activity is promoted at the level of phosphorylation 9. Line 253 – Chemical perturbations may change the expression patterns for these genes, but the authors have not performed these experiments. If they have, they should include them in the paper as it would strengthen the paper. 10. Line 263 – not all yolk sacs are nutritionally equivalent and provide the same amount of nutrients. There are some eggs of lower quality that may have inferior yolk composition, especially if mom was exposed to contaminants. 11. Lines 307-310. The authors have not shown in situ for Ryr1 or Ryr2 and thus cannot comment on spatial expression in the brain in zebrafish in their experiment. They quote other authors, suggesting that RyR spatial expression is already known for zebrafish and thus takes away the novelty for this study. Minor comments Line 47 delete “the” should read …throughout lifespan…. Line 91 Add “was” should read….and RNA was extracted….. Line 246 rictora or rictor?

You are one of the reviewers, your task is to write a review for the article. You will be 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 PROFILING OF MTOR AND RYANODINE RECEPTOR SIGNALING MOLECULES IN DEVELOPING ZEBRAFISH IN THE ABSENCE AND PRESENCE OF PCB 95 Review round: 2 Reviewer: 1

Basic reporting: The authors have adequately addressed all previous comments. Experimental design: The authors have adequately addressed all previous comments. Validity of the findings: The authors have adequately addressed all previous comments. Additional comments: Recommend accepting 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: AGE-BASED AND REPRODUCTIVE BIOLOGY OF THE PACIFIC LONGNOSE PARROTFISH HIPPOSCARUS LONGICEPS FROM GUAM Review round: 1 Reviewer: 1

Basic reporting: No comment Experimental design: No comment Validity of the findings: No comment Additional comments: The paper ‘Age-based and reproductive biology of the Pacific longnose parrotfish Hipposcarus longiceps from Guam’ by Taylor and Cruz provides important demographic information about a commercially important species. The manuscript is written very well. The Introduction provides an excellent coverage of the background information and sets up the reasons for this study in the context of information gaps. The Methods and Results were explained well and, despite being unfamiliar with a couple of the analyses performed, literature support was provided to justify their use. I particularly liked the clear structure of the Discussion, which clearly outlined the importance of the findings in the context of relevant literature. Thank you to the authors for providing a manuscript that was enjoyable to read. I have very few suggestions for improving this manuscript. However, the comments listed below should be considered… Abstract: (i) Line 20 – “… and tend to change sex to males…” This is rather ambiguous. I suggest rewording this. Introduction: (i) Lines 43-44 (and elsewhere) – The common name for this species is stated in the title so I do not see a reason for it to be included frequently throughout the manuscript. All references to the common name should be changed to the scientific name. This is the second time the common name for this species is mentioned in this paragraph. (ii) Lines 45-46 – What is meant by “… among the larger species that occurs in Guam…”? Larger species of what? Of parrotfishes? (iii) Lines 55-56 – “… as can be confirmed histologically.” This needs to be referenced. (iv) Lines 60-61 – What is meant by “…whereby age at female maturation had the strongest relationship to…”? Does this mean they mature earlier? (v) Lines 62-63 – Similar to my comment above, what does “… with corresponding life-history traits generally at the far-end of the spectrum.” actually mean? Does this mean at the lower/higher ends of the spectrum or at both ends? Also, what life-history traits are actually getting referred to here? Is it age/size at maturation and sex change? (vi) Lines 66-67 – The way this sentence is constructed it implies that Taylor (2014) suggests that the Guam populations will be larger-bodied and longer-lived. Is this actually the case? If so, it may be more appropriate to place this reference at the end of the sentence. If not, then an additional reference is needed at the end of the paragraph. Methods: (i) Line 133 – There appears to be an issue with the insertion of a symbol here with f() being in a dashed, square box. Results: (i) Line 209 – What is meant by ‘slightly invariable’? Discussion: (i) Lines 272 & 356 –Why is the common name mentioned here and not the scientific name? (see earlier comment) Conclusion: (i) Lines 358-360 – I find this final sentence of the Conclusion a little confusing in what was otherwise a well-written and clear manuscript. I suggest clarifying this sentence to strengthen the ‘take-away’ message from this study. Figures: (i) Figure 2 (caption) – There is repetition of words in this caption that is not needed. I suggest shortening it to read “(a) Sex-specific length- and (b) age-frequency distributions of sampled Hipposcarus longiceps from Guam.”

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

Title: AGE-BASED AND REPRODUCTIVE BIOLOGY OF THE PACIFIC LONGNOSE PARROTFISH HIPPOSCARUS LONGICEPS FROM GUAM Review round: 1 Reviewer: 2

Basic reporting: The quality of the manuscript is so high, so I gave only a handful suggesting and comments. Experimental design: The research question was well defined, and perfect for the journal. Validity of the findings: Data is enough for this large sized species, and all result are well reliable. 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: FACILITATIVE AND COMPETITIVE INTERACTION COMPONENTS AMONG NEW ENGLAND SALT MARSH PLANTS Review round: 1 Reviewer: 1

Basic reporting: This paper describes a well-designed experiment that tests the degree to which plant-plant interactions are positive and negative across the life history of a perennial forb and determines the redundancy of three matrix species. The analyses are appropriate. The writing is clear and professional. The authors cite the relevant literature both within and outside their study system to support their approach. To prepare the reader for the methods, the authors should be more specific in the introduction regarding the meaning of light environment and salinity. They should also be more detailed about the importance of (reasoning behind) the 'mimic' treatment. Specific comments for the introduction: Line 81 an e.g. or example is needed here Line 89 examples of species specific factors that preclude functional redundancy should be included here. Experimental design: no comment Validity of the findings: The authors are careful in interpreting their results, but so much so that there is little that those working outside the salt marsh system can take away from the paper. The authors should add context to their conclusions: how do these results apply to other ecological systems? Improving the text supporting the light and the salinity measurements will help with this. Additional comments: none

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

Title: FACILITATIVE AND COMPETITIVE INTERACTION COMPONENTS AMONG NEW ENGLAND SALT MARSH PLANTS Review round: 1 Reviewer: 2

Basic reporting: ABSTRACT: Lines 27, 29: “Three matrix-forming grass and rush species” implies three of each. Use instead, “one species of rush and two grass species” for clarity. Line 31: Suggest changing “forbs” to simply the species name Aster tenuifolius since this was the only forb analyzed in the experiment, and “forbs” implies more than one forb species. Line 33-35: “There was no statistically significant variation among matrix species in their net or component effects, however, the competitive effect of J. gerardi was negligible, especially compared to that of D. spicata.” Unclear wording. Line 34: “…net or component effects. However, …” INTRO: Line 50: Although elucidating facilitation within your study system is the main aim of the research, this first line should be inclusive of both general types of interaction (facilitation and competition) as the concept is introduced initially. I.e. “Research on community organization in ecology has long recognized that species interactions are often composed…”. Then, honing that down through this first paragraph to the last lines, Lines 63-65: Try “The prevalence of facilitation in instances of environmental stress or particular life-history stages does not minimize the role played by competition…” Omit “trivialize”, I’m not sure any ecologist would ever consider competition a ‘trivial’ component of a community, but I think I get at your point. Line 99: ”…may not, leading to a de-coupling…” Lines 107-108: Same comment as above in abstract regarding clarity of grasses and a single rush species. Also, comma unnecessary after “rushes” METHODS Line 113-114: It might be good to include the nearest town or other geographical landmark (or Lat/Long?) to specifically identify Nag Creek Marsh. Being unfamiliar with the area, it took a bit of time to locate the marsh online. Line 143: You state that A. tenuifolius both is a perennial and that it germinates in early spring. An individual of A. tenuifolius from a previous season is not ‘germinating’, but rather re-emerging from established below-ground rootstock. Is there a difference in emergence/germination times? DISCUSSION Line 265: “…standing dead biomass, …” Experimental design: Line162-163: “Shade cloth…in the field.” The white painting may have provided elevated photosynthesis (over either darker bare substrate or surrounding vegetative matrix). How was this either accounted for or monitored? Lines 170-171: Were the dug J. gerardii plots replaced in the exact same hole from which they were dug, or were they moved to one of the four plots within the block to simulate hole size or local substrate mismatch likely part of transplanting the other matrix species? Lines 188-189: is this growth timeline (June to August) the full breadth of seasonal growth of these species? There needs to be more life-history information within the Introduction, or here, to show why these times were chosen. The Introduction identifies “early spring” as the germination time (and see my previous comment regarding perennials), and August seems early for the end of growth (thinking more September/October). Validity of the findings: No Comment. Additional comments: It is noted that this research was done in the summer of 1999. The majority of the references for this work are 15 or more years old, with a few exceptions. Before publication, I strongly recommend including more current research on the topics included, with attendant descriptions of how this work remains novel and contributory within that context.

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

Title: DNA BARCODING REVEALS THE MISLABELING OF FISH IN A POPULAR TOURIST DESTINATION IN BRAZIL Review round: 1 Reviewer: 1

Basic reporting: The main issue I had with this paper was its English style. While understandable, it has many errors in grammar and usage. I suggest the authors firstly revise the paper (after considering more specific comments made below) and then get this revised version carefully read and corrected either by a native English speaker or by a professional editing company (there are on-line companies that do this). The study itself I found to be interesting and satisfactorily carried out, and is within the scope of PeerJ. Raw data, in the form of the barcode sequences, are supplied. Experimental design: Analytical methods, both the initial barcoding protocols and subsequent assignment of species, were appropriate and generally satisfactorily described. Validity of the findings: The results and findings were interesting and adequately referenced. Conclusions were well supported by the data. Additional comments: 1. The major recommendation – apart from improving its English style – is to reconsider and improve the layout of the two Tables. In my view these can be considerably improved by: a. A: Re-arranging the numbering, so that the correct assignations form Table 1 and the incorrect assignations Table 2. This will obviously then require some small textual changes too. b. B: Subdividing each Table into two subsections, one being the fishmongers and the other the Japanese restaurants. c. C: Spell out the names of the genera – as there are many of them, it’s not sufficient to identify them here simply by initial letters. d. D: Aren’t the numbers in parentheses the best or highest match comparisons, in percent, not the lowest match comparisons? 2. Lines 29-33. Explanation of the Cat by Hare Project is not readily intelligible as given – please clarify. 3. Line 30. Lahiri and Nurnberger, not K. and Nurnberger. 4. Line 89. Blast and BOLD respectively, not BOLD and Blast respectively. 5. Lines 96-103. Please explain the relevance of giving BOLD BIN numbers for these species (or omit them). 6. Line 133-134. Genus not genre. 7. Please check spellings of all scientific names in text and tables. I noticed misspellings of L. niloticus (Table 2) and line 162 O. kisutch. Also give all genus names in full in the text, as you are dealing with a wide variety of genera, not just one or two. 8. Check references. DNA should be given as DNA not as Dna. Line 274 – delete d. Some journals also have part numbers given – e.g. (1) in Barbuto et al. Is this necessary? I suspect these should all be deleted.

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

Title: DNA BARCODING REVEALS THE MISLABELING OF FISH IN A POPULAR TOURIST DESTINATION IN BRAZIL Review round: 1 Reviewer: 2

Basic reporting: The results are described in a way that is not always easy to follow and the language is confusing at some points. An editing by an English native speaker or an editing service is highly recommended. The introduction should include a brief description of the DNA-based methods used in species identification. It should describe the concept of DNA barcode and its advantages and disadvantages. For example: L. Wilson-Wilde, J. Norman, J. Robertson, S. Sarre, A. Georges, Current issues in species identi?cation for forensic science and the validity of using the cytochrome oxidase I (COI) gene, Forensic Science, Medicine, and Pathology 6 (2010) 233–241. The authors should mention in the Introduction section the previous initiatives for the implementation of forensic standards in the identification of species, particularly in the field of food forensics. For example: F. Teletchea, C. Maudet, C. Hänni, Food and forensic molecular identification: Update and challenges, Trends in Biotechnology 23 (2005) 359–366. M. Woolfe, S. Primrose, Food forensics: using DNA technology to combate misdescription and fraud, TRENDS in Biotechnology 22 (2004) 222–226. Experimental design: The methods should be better described. Which type of material and conditions were implemented for the sample collection? Sterile material? I’m worried about the possible contamination of the samples during the tissue collection. The authors should describe in more detail the sampling process and discuss possible contamination issues. For example, the sample fish piece might have been in contact with pieces from other species, and a cross-contamination is possible. The sequencing reaction should be briefly explained. Which reaction conditions were used? Which primers? Do the authors sequence the PCR products in both directions? The parameters used in the Blast and BOLD searches should be indicated (blast algorithm, database, etc). The experiment has to be described in a way that anyone could replicate it. Lines 92-103. The paragraph is not clear. Why do they used the IUCN Red List of Threatened Species status? Which species are listed here? Lines 105-108: The English should be revised. Line 35: parenthesis are missing in the reference Lines 48-48: the sentence should not be in italics Line 72: PCR reaction is redundant. The R is for reaction. Lines 83-85: The sentence should be revised. The meaning is not clear. Line 172: it should be “species” Validity of the findings: The approach used by the author is valid as it has been used by others before. 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: DNA BARCODING REVEALS THE MISLABELING OF FISH IN A POPULAR TOURIST DESTINATION IN BRAZIL Review round: 2 Reviewer: 1

Basic reporting: I found the results of this study were presented much more clearly than in the original submission, but the English style remains, for me, poor. In fact I find it hard to believe that two ‘professionals’ had separately read the manuscript and all their comments were incorporated – they cannot have been highly proficient in the English language! The title of the paper is now greatly improved, but the wording of the Abstract (for example) remains virtually identical to the original Abstract. It is true that the text can, with a little difficulty, be understood by an English speaker, but in my view it still needs improvement prior to publication. I continue to recommend editing by a professional English speaker or editing service. Experimental design: Acceptable Validity of the findings: Acceptable Additional comments: Some specific points: 1. Line 18. ‘30% of fraud in fisheries’ Isn’t this really 30% mislabelling in fisheries? Later in the Abstract the authors mention that not all cases of mislabelling are necessarily fraudulent. 2. Lines 52-58. I did not find the short explanation of DNA barcoding to be useful, and could even be confusing to newcomers to the topic. What is meant by a ‘global system of different mutational rates’? The implication that nucleotides mutate randomly so that 4 * 15 sites would generate 1bn new codes is a considerable simplification of reality – most sites are conserved! Please clarify this paragraph. 3. Line 79. e to and 4. Line 80-82. I couldn’t understand this sentence 5. There are still some spelling errors in scientific names, e.g. for tilapia in Table 1 (but OK in Table 2) and for coho salmon (line 172) 6. Line 115. What is ‘GL’ – is it degrees of freedom? 7. Line 169 autosomal 8. Line 183 what is ‘abastance’?

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

Title: DNA BARCODING REVEALS THE MISLABELING OF FISH IN A POPULAR TOURIST DESTINATION IN BRAZIL Review round: 2 Reviewer: 2

Basic reporting: No further comments to the manuscript. Experimental design: No further comments to the manuscript. Validity of the findings: No further comments to the manuscript. Additional comments: No further comments to 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: DNA BARCODING REVEALS THE MISLABELING OF FISH IN A POPULAR TOURIST DESTINATION IN BRAZIL Review round: 3 Reviewer: 1

Basic reporting: English, and the discussion of the barcode approach at the end of the Introduction, is improved and adequate for publication. Experimental design: Satisfactory Validity of the findings: Good Additional comments: None

You are one of the reviewers, your task is to write a review for the article. You will be 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 OF POTENTIAL REFERENCE GENES FOR REAL-TIME QPCR ANALYSIS IN A BIPARENTAL BEETLE, LETHRUS APTERUS (COLEOPTERA: GEOTRUPIDAE) Review round: 1 Reviewer: 1

Basic reporting: The ms is clear, well written in professional English and conforms to professional standards of expression. Literature references basically cover sufficiently the field context. I would suggest to add the following paper and to accomplish the checklist provided in it. The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, by Stephen A. Bustin et al., Clinical Chemistry Apr 2009, 55 (4) 611-622; DOI: 10.1373/clinchem.2008.112797. The ms includes sufficient introduction and background and demonstrates how the work fits into the broader field of knowledge. Figures are relevant to the content of the article and appropriately described and labeled. Resolution of Figure 1 is probably too poor. It is suitable for review, but it would be necessary improve the quality in final version. A comment to figure 1 is missing in result sections. Figure 1 indeed shows that generally thorax samples showed higher Cts than head, especially in males and that generally there were no strong differences between male and female samples. A statistical analysis of these differences (ANOVA on different samples for each genes) could improve this statement and Figure 1 as well. A different table organization is needed to improve ms readability. Tables 1 and 2 could be merged, and Authors should add qPCR parameters for different primer pairs that are now missing (Efficiency, R2, melting temperature). Table 5 and 6 should be provided as supplementary data, since are not crucial. On the other hand, data about different ranking obtained by reffinder on different tissue and different sex are missing. Authors should include new table with reffinder results for different subset of samples analysed, especially to facilitate reading of lines 251-258 in results and 296-300 in discussion. These paragraphs describe key findings of ms, but they need to be rephrased. The ms is ‘self-contained’ and represents an appropriate ‘unit of publication’. Experimental design: Research question is well defined, relevant and meaningful, as author stated in discussion lines 284-287. The investigation was rigorous and performed to a high technical standard. Methods are generally well described. Nevertheless, the different subset of samples analysed should be clearly stated. In other words, if I understood well, authors run different gene stability analysis to establish reference genes on different sample subsets: i) all samples, irrespective of tissue and sex; ii) all females, irrespective of tissue; iii) all males, irrespective of tissue; iv) all thorax samples, irrespective of sex; v) all head samples, irrespective of sex; vi) female thorax samples; vii) male thorax samples; viii) female head samples; ix) male head samples. Authors should better explain this experimental plan of analyses and including a new table at least including reffinder results for all these analyses. Validity of the findings: Presented results are statistically sound, but some details on different subset of analysis are missing, as previously indicated. qPCR Efficiency must be calculated for all primer pairs. Indeed in relative quantity calculation it should be included to obtain more accurate data. Conclusion are well stated, linked to original research question and limited to supporting results. Additional comments: Detailed comments not included in above suggestions are: -In the abstract is missing the fact that L7A and RP18 are also the most stable genes considering all samples irrespective of tissues and sex. - “Lethrus apterus”, “reference gene” and “RT-qPCR” keywords are redundant since already present in the title. -Lines 58-42: please rephrase. - Line 84: Change it, since, reference genes must be evaluated in different specific sample types and treatments. The concept of “universally applicable reference genes” is not reasonable. - Line 89: change in “the expression level of hormone regulating genes involved in parental care” - Line 141-142: please add details about negative controls of no-retrotranscribed samples to exclude genomic DNA contamination. -Line 148 149. Change with the “lowest Ct value” instead of the “highest”. (the lowest Ct value corresponds to the highest cDNA starting quantity) -Caption to Table 5: rephrase the subset of samples analysed.

You are one of the reviewers, your task is to write a review for the article. You will be 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 OF POTENTIAL REFERENCE GENES FOR REAL-TIME QPCR ANALYSIS IN A BIPARENTAL BEETLE, LETHRUS APTERUS (COLEOPTERA: GEOTRUPIDAE) Review round: 1 Reviewer: 2

Basic reporting: No comment Experimental design: 1. In line 116, the author quoted “Using a draft genome of Lethrus apterus (Rácz et al., 2015)”. Looking at the paper, its not a genome paper, it’s a paper identifying microsatellite markers. Can the author clarify this? Is there a genome of Lethrus apterus? 2. The amplicon length of some of the genes are quite big, up to 426bp for a qPCR experiment. Its usually recommended to keep amplicon size small and if possible about the same length across all test genes. Is there any particular reason why some of these genes have big amplicons? 3. The authors mention about sample collection in line 94-96. Was the sample pooled? If so, which samples were pooled? 4. The number of variables are quite limited, only head and thorax of male and female collection. Although it is understandable that the sample is an environmental sample, but in this type of experiment, it is important to test the candidate genes in different variability (e.g. different stages of development) to measure its stability. Validity of the findings: 5. For the gene finder and BestKeeper analysis in Table 4 and 6. The results shown are for which samples? As there are 4 samples chosen by the authors (head and thorax of male and female), why is there only 1 set of data? Can the authors comment to make this clearer. 6. Same goes to NormFinder data in Table 5, why is there only Head and Thorax but there is not male and female data? 7. Did the authors sequence the PCR products to see if the correct genes were amplified? 8. Could the author explain why the housekeeping gene was not tested/validated using another gene? Additional comments: The work presented in this manuscript describe the use of potential reference genes for the use of qPCR based studies in Lethrus apterus. The authors choose 11 candidate genes to test its suitability to become the housekeeping genes. The data presented appeared to be technically sound for the most part. Most papers are using geNorm, NormFinder, BestKeeper and RefFinder to evaluate the suitability of these candidate genes.

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

Title: DIAGNOSTIC VALUE OF SERUM PROCALCITONIN, LACTATE, AND HIGH-SENSITIVITY C-REACTIVE PROTEIN FOR PREDICTING BACTEREMIA IN ADULT PATIENTS IN THE EMERGENCY DEPARTMENT Review round: 1 Reviewer: 1

Basic reporting: 1. Title: the title is misleading. The authors have assessed the diagnostic value of several biomarkers commonly used in predicting positive blood cultures, not diagnosing sepsis. The title should reflect this. 2. Abstract: the authors state the “lactate added to procalcitonin helps to improve the performance to differentiate whether blood culture will be positive with either Gram-positive or Gram-negative pathogen”. However, the results presented do not support this conclusion as no biomarker levels are presented. For example, if the patient has elevated lactate and elevated PCT, this information will not help the clinician to differentiate between Gram-positive or Gram-negative bacteraemia. It is rather just an indicator that the patient is more likely have a positive blood culture. 3. Discussion: too few references are used in the Discussion (only three). The results presented in this paper must be considered in context of other people’s work. 4. Abstract: abbreviations should not be used in the abstract. 5. Introduction: the introduction need more detail and relevant references. For example, the previously published results for PCT as a biomarker have been contradictory, which is not mentioned in the introduction. 6. Introduction: references are missing for several claims. For example, line 79-80; line 86-87. Besides, many of the references are relatively old – there have been a lot of publications in this area the latest years. 7. Material and method: no information whether one-sided or two-sided test were used. 8. Material and method: Line 152: the sentence “The p-value in our analysis was set at 0.05” should be reformulated as one cannot set a p-value. Before you perform a test, you decide the significance level (alpha) which is usually set to 0.05.I suggest that you write: “The significance level was set at 5% (p < 0.05).” or “A two-sided p value of <0.05 was considered statistically significant.” 9. Regarding the number of decimals used for reporting p-values both in text and tables: too many decimals used, I suggest that you use two decimals for p-values >0.01; three decimals for p-values between 0.01-0.001; p-values below 0.001 should be reported as p<0.001. 10. Regarding the number of decimals used for reporting the AUROC values: too many decimals used, I suggest that you use two decimals. 11. The 95% CI for AUROC should also be reported, in the text as well as in the figure legends. 12. Results: Interpretations of results and conclusions are presented in the Result section; this belongs to the Discussion section. For example, line 206: “With regards to the single test item, PCT is still a better choice.” and line 209: “Therefore, a combination of PCT or lactate increases is better against blood culture.”, etc. 13. Results: all paragraphs starts with “Table X shows…”. I suggest that the authors report the key results in the text and then referring to the figures and tables in the text. 14. Sometimes the authors use the term odds ratio (OR) and sometimes diagnostic odds ratios (DOR). This is confusing. The authors should stay put to use one of the terms. 15. Table 3: cut-off for lactate is written as 19.8 mg/dl? Confusing as the authors have stated 1.98 mg/dl as cut-off elsewhere in the paper. 16. Table 7: all abbreviations used in the table is not explained in full (e.g., Y, ED, PCT). 17. Table 7: complete references are missing; only the name of the first author is provided. Experimental design: 1. Statistical comparisons between the ROC curves are missing. In order to determine whether there is a significant difference or not you must test for that, not only rank them in order. I suggest that you use DeLong’s test for comparison of two correlated ROC curves (DeLong et al. Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach. Biometrics. 1988;44(3):837-45). 2. The reasons for selection of the cut-off values for lactate, PCT and CRP should be described. For example, why cut-off values for lactate >1.98 mg/dL and CRP>0.8 mg/dl? These cut-off values have not been used in previous sepsis studies. 3. Material and method: Line 127: information about the blood volume drawn from each patients is missing as well as the number of blood culture bottles incubated for each patient. 4. Material and method: neither information nor reference are given for which bacteria species were interpreted as pathogenic or contaminant. Validity of the findings: There are many caveats in this paper which limits it validity. 1. Definition of sepsis: this is my major concern about this paper. For several reasons, the results presented in this paper cannot be used in diagnosis of sepsis as claimed, only to predict positive blood cultures. Firstly, I would like to stress that positive blood culture is not the same as sepsis. Many patients suffer from sepsis without having a positive blood culture (only 30-40% of the septic patients have a positive blood culture) and vice versa, many patients can have a positive blood culture without having sepsis. Besides, sepsis may be caused by other pathogens than bacteria. The previous definition (Sepsis-2) of sepsis included proven or suspected infection accompanied by systemic inflammatory response syndrome (SIRS), whereas according to the new sepsis definition (Sepsis-3), sepsis should be defined as life-threatening organ dysfunction caused by a dysregulated host response to infection (Singer et al., 2016. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA 801-810:(8)315). Secondly, the patients enrolled in the study were not selected based on a clinical suspicion of sepsis. Patients were enrolled if all four tests (blood culture, PCT, hs-CRP, lactate) were performed within 24 hours after admission to the hospital – that´s a huge different. No information is provided about the clinical diagnosis nor the reason for taking blood cultures. 2. The paper studied the predictability of positive blood cultures in adult patients, not sepsis. This must be made clear throughout the whole paper. 3. Cut-off values: the selected cut-off values have not been used previous sepsis studies and are in disagreement with the ones generally recommended for clinical practices. 4. The levels for the different biomarkers should be presented as well in the Result section. 5. Sometimes the authors write that they have studied blood cultures positive with Gram-negative bacilli or bacteria/Gram-positive cocci, and sometimes blood cultures positive with Gram-negative /Gram-positive pathogens. This inconsistency is confusing – what have actually been studied? How did the authors evaluate findings like Gram-positive bacilli? Where they considered as negative or contaminations or excluded from the study? 6. Raw data file is supplied, that´s excellent. However, no information is given about the numbers of positive blood culture bottles, no either is any information about the findings classified as contaminants. As the number of positive blood cultures are of importance for deciding whether it’s a pathogen and contaminant it should be provided. What about polymicrobial findings? What about fungal findings? The raw data file also contains some Chinese characters not possible for me to understand. Moreover, units for the biomarkers measurement are missing. 7. Accuracy is another useful performance measure, not included in the paper. It would have been a nice addition to the paper. Additional comments: 1. Discussion: the studies you compare with have used other cut-off values which makes it difficult to compare. It is more correct to only compare results between studies using same cut-off values. 2. The use of abbreviations: abbreviations should generally be avoided. For the first reference to a term in the text, the term should be used in full with the abbreviation included in brackets. For the remainder of the text the abbreviation should be used. Moreover, I recommend that the authors only used standard abbreviations commonly used (e.g., DOR, CI, PCT, hs-CRP, ROC). 3. Line 238: sepsis is NOT defined as a bloodstream infection. 4. Line 242: “In this study, PCT was compared with conventional competing tests, such as hs-CRP or lactate,…”. I guess that “or” should be changed to “and”? 5. Line 298: the authors write that PCT has a better diagnostic ability for GNB, but in comparison to 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: DIAGNOSTIC VALUE OF SERUM PROCALCITONIN, LACTATE, AND HIGH-SENSITIVITY C-REACTIVE PROTEIN FOR PREDICTING BACTEREMIA IN ADULT PATIENTS IN THE EMERGENCY DEPARTMENT Review round: 1 Reviewer: 2

Basic reporting: See general comments Experimental design: See general comments Validity of the findings: See general comments Additional comments: Thank you for asking to review this paper. This retrospective analysis assessed the utility of lactate, PCT and hsCRP in prediction of blood stream bacteraemia in patients presenting to ED. General comments: 1. Patients included in this study were treated in 2010 onward, specify the end date. 2. The language used across the manuscript, particularly in the abstract, need significant English editing. 3. Sentences such as “hsCRP should be abandoned ……” should be avoided and deleted. 4. The short title should reflect the biomarker interest. Suggest changing to Biomarkers of sepsis in adult emergency. 5. Why there is no ED physicians included as authors? 6. A sensitivity analysis should have been performed on patients who had one or 2 tests done not just those who have had ALL the 3 tests done. Specific comments: 1. The mini VIDAS biological range is much lower than 0.5 ng/ml. I thought it goes down to 0.06. Methods: It is not helpful having too many categories of culture negative. Stick with a definition and just use the total number of what was categorised as negative culture in the analysis. In the results, graphs and tables. 1. It is very adequate to ONLY separate g-ve and g+ve. No need for ROC for bacilli and cocci. This should apply to all results including the tables. There is little pint dividing cultures into 4 groups in addition to negative, contaminant and others. 2. PCT cut off was chosen at 0.5 ng/ml. Do you have data for PCT cut off at 0.25 or 0.1 ng/ml? In addition is there an upper cut off value for best performance of PCT? 3. Suggest merging tables 1, 2 an 3 into one table. 4. Table 7 is not necessary. This is not a MA. I suggest comparing the results with your data in the discussion section. Discussion should be restructured to address the following: 1. Start with key results 2. Explain why DOR of combinations is better than single DOR. 3. Compare with previous trials 4. Implication of the above finding for ED practice and sepsis management in general 5. Limitations of the study 6. Next step. 7. Shorter conclusion (should not have any results in the conclusion).

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

Title: DIAGNOSTIC VALUE OF SERUM PROCALCITONIN, LACTATE, AND HIGH-SENSITIVITY C-REACTIVE PROTEIN FOR PREDICTING BACTEREMIA IN ADULT PATIENTS IN THE EMERGENCY DEPARTMENT Review round: 2 Reviewer: 1

Basic reporting: It is still a lack of literature references and context in the Discussion. Only a very few of your results are compared with other peoples work (and here only two other works are cited). The Disucssion must be extended and the obtained results put in a context, i.e., compared with previous studies reporting the performance of the single biomarkers and/or combined biomarkers for predicting positive blood cultures. Reasons for differences/similiarities in findings between studies? Line 249 and 257: No information about the frequency of positive blood cultures for the expanded cohorts. This is important as the group sizes are very important for the performance characteristics. This information must be added in Method and Material section. Line 184-186: this sentence is very hard to understand and must be rewritten. Line 197- : all the data presented in the text here should be presented by tables only. Line 246: threshold for PCT is not defined here. Inconsistent use of hs-CRP vs. high-sensitive CRP. This should be looked over throughout the manuscript. Experimental design: No comments. Validity of the findings: In the Abstract: You write "Among patients with elevated PCT and/or LAC, the odds for predicting positive blood culture was nearly five-fold higher compared to that in patients with normal levels (diagnostic odds ratio, 4.83; 95% CI, 42 2.84–8.69). The diagnostic odds ratio was nearly eight-fold higher for the prediction of positive gram-negative bacteria (GNB) culture (diagnostic odds ratio, 7.75; 95% CI, 3.56–20.03) and it was nearly three-fold higher for the prediction of positive gram-positive bacteria (GPB) culture (diagnostic odds ratio, 2.87; 95% CI, 1.43–6.41)." I wonder where these figures come from as I don´t recognize the from the results reported in the manuscript? The abstract should only report exactly the same results as in the Results section. A diagnostic accuracy of only 65% is not acceptable from a clinical point of view (as stated for example in line 275 and line 347). This is considered to be rather poor actually. It is wrong to say that CRP should be abandoned from the ED; it is still useful as a marker for bacterial infections but is limited by it´s slow kinetics and should be viewed from the timpe point of disease onset. 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: DIAGNOSTIC VALUE OF SERUM PROCALCITONIN, LACTATE, AND HIGH-SENSITIVITY C-REACTIVE PROTEIN FOR PREDICTING BACTEREMIA IN ADULT PATIENTS IN THE EMERGENCY DEPARTMENT Review round: 2 Reviewer: 2

Basic reporting: Nil Experimental design: Nil Validity of the findings: Nil Additional comments: Thank you for a very improved manuscript. I have one suggestion to remove the number of patients from the title.

You are one of the reviewers, your task is to write a review for the article. You will be 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 ZEBRAFISH AS A MODEL SYSTEM FOR ANALYZING MAMMALIAN AND NATIVE Α-CRYSTALLIN PROMOTER FUNCTION Review round: 1 Reviewer: 1

Basic reporting: This paper reports expression patterns of mouse and zebra fish a-crystallin promoter constructs in zebra fish embryos and examines native gene and protein expression using PCR and mass spec. There is some nice data here. The GFP reporter results are clear. They confirm that crystallin promoters can work across species, as has long been known, and also define functional fragments of the zebra fish promoters. Mass spec detected aA and aBa peptides and provided evidence for eye/non-eye regulation of aBa in development. aBb was not detected. Unfortunately the PCR results are not so convincing. Fig 2, shows aA expression throughout development quite well. Fig 5, however, has poor image quality and, unlike tubulin, the aBa,b primers seem to be generating smaller non-specific bands. These might be solvent front, but the aA and tubulin gels are clear in this region. Were these products confirmed by sequencing? Expression in adult is obvious, but the inconsistent results between panels A and B for embryonic expression are hard to explain. Both show strong bands on single developmental days, followed by almost nothing, which by itself seems biologically unlikely. The difference in timing in different samples is also strange and looks artefactual. I think it would help if this figure was repeated using different primers. The lack of detectable peptides for aBb also raises questions about the PCR. As for the control genes, is tubulin really a good control? Might tubulin be developmentally regulated? Although the authors cite the Hou et al 2006 paper on expression of the human bB1promoter in zebra fish in the introduction, they don’t compare their results in the Discussion. Some mention seems appropriate. Minor points: Line 32: Specify chaperone activity refers to a-crystallin. Line 102: aB? Seems to have symbol character. Line 398: Specify a-crystallin. Figs 1,2,3: In legends – state ages of embryos for GFP expression. Experimental design: Experimental design is fine in general, but the paper is let down by the execution of the PCR. Validity of the findings: No further 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: THE ZEBRAFISH AS A MODEL SYSTEM FOR ANALYZING MAMMALIAN AND NATIVE Α-CRYSTALLIN PROMOTER FUNCTION Review round: 1 Reviewer: 2

Basic reporting: In this study, M. Posner and coworkers conducted a series of gene reporter, expression, and proteomic studies to determine expression of zebrafish and mouse -crystallin genes in zebrafish. The A- and B-crystallins are key structural proteins of vertebrate lenses. Although some insights into transcriptional control of these genes are known for mammalian crystallins, understanding of their temporal and spatial control both in and outside of the lens requires additional studies. Zebrafish is an excellent cost effective experimental model to accelerate these studies. The findings are novel and address a number of questions in the field. The present studies thus fill a critical gap in our understanding of lens differentiation and crystalline gene expression. The manuscript is well-written and within the scope of PeerJ. The data are of excellent technical quality. There are several minor issues to be addressed in the revision: 1) Introduction: Lens development in zebrafish and mouse differs as the mouse lens placode invaginates into the lens vesicle, prior the formation of lens fibers. Lens fiber cells in zebrafish are generated through the delamination from lens placode. This should be mentioned as both zebrafish and mouse models are discussed. 2) Methods: the coordinates of DNAs used to generate eGFP reporters should be included. 3) The paper by Zou et al. 2015, Exp. Eye Rees. 138:104-13 should be quoted here. Experimental design: Some technical details regarding the DNAs have to be addressed (see above). Validity of the findings: Addition of supplemental files including three video is highly valuable. 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 ZEBRAFISH AS A MODEL SYSTEM FOR ANALYZING MAMMALIAN AND NATIVE Α-CRYSTALLIN PROMOTER FUNCTION Review round: 1 Reviewer: 3

Basic reporting: The authors present an excellent review of the literature. Clear and well written manuscript. Experimental design: 1. The overall purpose is unclear. Why functionally assess mouse lens promoters in zebrafish? And how would the authors determine whether negative expression in fish generated from a potentially new mouse lens enhancer would be due to species differences or merely that the enhancer was not a true enhancer in mice either? See Intro: “This conservation suggests that mammalian a-crystallin promoters could be functionally assessed in the zebrafish, providing a faster and less expensive system than traditional mouse transgenic approaches.” Furthermore, the authors spend a lot of effort to simply confirm that EXISTING, KNOWN regions of the mouse gene promoter function in the fish. What new is gained here, since these regions were already known to control expression in the mouse as such? See Results: “Our results indicate that the mouse aB-crystallin promoter drives GFP expression in zebrafish embryos, and that the resulting spatial patterns reflect the functional regions first identified in mouse.” 2. The authors attempt to use promoter driven GFP to assess endogenous onset of gene expression. If an enhancer or modifier is missing in their cloning, then their entire conclusions are in error. The characterization of the developmental patterns produced by the promoters should be perform by analyzing the expression of the endogenous products. See Results: “However, no study has characterized developmental patterns produced by the two respective promoter regions. We produced a GFP-linked 3 kb fragment of the zebrafish aBa-crystallin promoter and a series of GFP-linked fragments… We found no difference between the timing of onset for any of these zebrafish promoters… Overall these data suggest that the divergent expression of the two zebrafish aB-crystallin promoters…” They then follow this up with RT-PCR and show that it is essentially present at all timepoints analyzed (no timepoints prior to 1 dpf were analyzed). Since this wasn’t qPCR, assessment of abundance should not be given. I am not sure that the following statement in the Discussion is valid. See Discussion: “Lastly, by examining promoter activity, mRNA expression and protein abundance for zebrafish aBa-crystallin we resolve remaining questions about the timing of expression of this gene.” Validity of the findings: Data are robust and controlled. See point 2 above regarding experimental design issues leading to over interpretation of findings. Additional comments: Annotation of Figure 5 would be appreciated. Legend for colors. A-B: aA, C-D: aB.

You are one of the reviewers, your task is to write a review for the article. You will be 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 ZEBRAFISH AS A MODEL SYSTEM FOR ANALYZING MAMMALIAN AND NATIVE Α-CRYSTALLIN PROMOTER FUNCTION Review round: 2 Reviewer: 1

Basic reporting: I think the manuscript is much improved. Indeed the authors have made several important corrections. I only noticed two minor points: I would drop the last sentence of the Abstract. It is speculative and doesn’t relate to these results. Line 144 in the PDF has a square symbol character that probably should be a roman B. Experimental design: No further comment Validity of the findings: No further 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: THE ZEBRAFISH AS A MODEL SYSTEM FOR ANALYZING MAMMALIAN AND NATIVE Α-CRYSTALLIN PROMOTER FUNCTION Review round: 2 Reviewer: 2

Basic reporting: Satisfactory. Experimental design: Meet standards. Validity of the findings: No comment. Additional comments: The authors have address all main minor points raised in the earlier review.