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

Input:
Title: INVESTIGATION OF MICROBIAL COMMUNITY INTERACTIONS BETWEEN LAKE WASHINGTON METHANOTROPHS USING GENOME-SCALE METABOLIC MODELING
Review round: 2
Reviewer: 2

Output:
Basic reporting: The modeling results make more sense after correction of several major issues. One minor issue of Fig 1.: the pictures of Methylomonas and Methylobacter could be misleading. They should not look like E. coli.
Experimental design: Materials and Methods part has provided detailed information of how the model was developed and manually curated. Most major issues, such as no flux through the RuMP cycle and a strong serine cycle, have been corrected. I have no other major concerns on the multi-objective optimization or how the simulation was carried out.
Validity of the findings: No comment.
Additional comments: One interesting finding from this study is that the ED pathway is the predominant glycolytic pathway. The EMP pathway shows little or no flux from G3P to PYR. However, many studies have shown the otherwise for the type I methanotrophs. Of course, no one has yet measured glycolytic fluxes experimentally in Methylobacter and Methylomonas species. The authors may want to mention this or give some explanation in the manuscript.

1) Kalyuzhnaya MG, et al. Highly efficient methane biocatalysis revealed in a methanotrophic bacterium. Nat Commun 4:1–7.
2) He L, et al. Quantifying methane and methanol metabolism of “Methylotuvimicrobium buryatense” 5GB1C under substrate limitation. mSystems 4:1–14
3) Torre A, et al. Genome-scale metabolic reconstructions and theoretical investigation of methane conversion in Methylomicrobium buryatense strain 5G(B1). Microb Cell Fact 14:1–15


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

Input:
Title: BITE FORCE DATA SUGGESTS RELATIONSHIP BETWEEN ACRODONT TOOTH IMPLANTATION AND STRONG BITE FORCE
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I enjoyed reading this paper which proposes an interesting hypothesis backed by convincing data, and absolutely is deserving of publication after what I perceive to be minor but important alterations. I do not wish to remain anonymous, I am Yara Haridy the authors are free to contact me with further questions (yara.haridy@mfn.berlin)

First, the things I thoroughly appreciated:
•	Clear concise writing and getting to the point
•	We need more ‘why did so and so evolve?’ papers that actually go out and test the idea!
•	I really enjoyed the section about “can ‘AVSA’ be reversed?” I’ve considered exactly this, and I too think that Acrodontians basically pigeonholed themselves into this adaptation early on and were unable to adapt or diversify further. I agree with the authors that perhaps this is due to an adaption by their ancestor that is no longer as starkly present in current surviving taxa, great points.

Issues from most to least importance:
•	The acronym of AVSA is not my favorite, however if the authors wish to keep it, I encourage further explanation. I sympathise with the need to easily refer to this specific type of acrodonty however saying ‘Severe ankylosis accompanies acrodonty’ and then saying ‘acrodonty via severe ankylosis’ sounds contradictory and the latter makes it sound like the severe ankylosis causes the acrodonty. I partially agree that the mode of attachment actually changes the ‘look’ of implantation (Haridy, 2018)
•	It is worth mentioning why you chose the term ‘sever ankylosis’ and actually explain how much remodeling and bone invasion there is of the tooth space and why this is not normal ankylosis (Haridy, 2018). This actually lends itself to your argument.
•	One issue is the delineation of implantation vs mode of attachment. Many animals can be considered acrodont or sub-acrodont in implantation (i.e. snakes) but are not traditionally thought of having the same specialized convergent dentition as Acrodontians and Rhynchocephalians. The two aforementioned groups have an amazing convergent trifecta in common, position of teeth, lack of replacement, and extreme ankylosis. I feel like this can be emphasized throughout the manuscript. The authors do a fine job of explaining the modes of implantation but not that of attachment, i.e gomphosis vs ankylosis. Teeth fused to the jawbone are likely ‘sturdier’ than those attached via soft tissue. It should also be noted that most animals which have ankylosed teeth go from a gomphosis to an ankylosis at varying rates (see LeBlanc et al. 2016).
•	Overall my biggest criticism is that I want ‘more’ I found this paper lacking in depth in many of the exceptional points made by the authors. This is not a major issue but adding more to certain points like (line 163-164) “acrodont tooth with a wide base and firm attachment is better suited to withstand higher bite forces” this should go into how and why the fused attachment is better than ligamentous attachment. In the PDF I outline where I’d like areas expanded on and I think it would greatly help in convincing your reader of your hypothesis.
•	I also feel like this paper did not propose alternate hypothesis for the evolution of acrodonty. the proposal of alternate hypothesis then shutting them down systematically would have greatly aided the authors’ biteforce point. An example of an alternate hypothesis could include ‘dietary adaptation’ which is easily refuted by the fact that acrodontians-at least modern ones are varied in their diets as much as non acrodontians so it is unlikely for acrodonty to have evolved for such a reason.
•	While bite-force data is one way to show that ‘acrodonty with ankylosis’ evolved to withstand (or because of) increased force another possibility for testing this idea is via FEA to actually see if the acrodont tooth shape/attachment and uniformity of material interface actually increases the force that can be taken by the tooth structures. Of course I am not suggesting that the authors undergo this lengthy study I am only suggesting that perhaps in can be part of a future directions if the authors wish to include it.


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

Input:
Title: BITE FORCE DATA SUGGESTS RELATIONSHIP BETWEEN ACRODONT TOOTH IMPLANTATION AND STRONG BITE FORCE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting is clear, given the available data from the literature. I would suggest that the authors call this study a Meta-Analysis somewhere in the title and Materials and Methods, though. There are no experiments or direct observations in this study.

I would add that for clarity, the authors should also add a figure of what they think constitutes an acrodont tooth without AVSA. At this point, I do not see why we need to introduce the term AVSA for acrodont reptiles (see the attached PDF for more specific comments on this).
Experimental design: One experimental design issue that I can see is that the data aren't segregated by diet. How is the data set skewed in terms of herbivorous/omnivorous/carnivorous/durophagous lizards for either group? Are the acrodont and pleurodont study groups comparable in the breadth of diets? This should be explored further. Also, show the reader where some of the taxa of interest plot in your figures.

I would also recommend that someone who is more adept than I go through your statistical analyses.
Validity of the findings: My major comment would be that this is a very preliminary correlative study. As a result, the conclusions are quite superficial. Given the proposed correlation between higher bite force and acrodonty, I would like to see far more discussion of why all of the durophagous and more robust-toothed lizards probably do not come from acrodont ancestors. This is particularly true for Varanus niloticus, where I don’t see any evidence of a major shift in tooth implantation between it and any of its relatives. They all look pretty pleurodont to me. So why are these hard-biting or shell-crushing lizards not acrodont? Pleurodonty seems to work just fine for these diets.

I am also unclear as to how AVSA affects the integrity of a tooth. The term seems redundant to me, given that the wide base of the tooth and its position on top of the jaw are principle factors the authors cite as potential adaptations to (or consequences of) high bite forces. These are features of tooth implantation (acrodonty), not degree of attachment (ankylosis). To me, AVSA is synonymous with "acrodont ankylosis" here. Teeth fused to apex of jaw.
Additional comments: Please see my attached PDF for all of the specific comments and suggested edits.


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

Input:
Title: BITE FORCE DATA SUGGESTS RELATIONSHIP BETWEEN ACRODONT TOOTH IMPLANTATION AND STRONG BITE FORCE
Review round: 2
Reviewer: 1

Output:
Basic reporting: Clear, fairly unambiguous, professional writing. I have made some suggestions for clearer reporting in the Materials and Methods. These are minor changes.
Experimental design: Again, clear, easy to follow. I like the scrutiny of the data in the discussion. Much more thorough treatment of the data on the whole this time.
Validity of the findings: Funny thing: I feel as though this revised version carries more weight, not just because of the "positive" results, but the falsified hypotheses as well. It now serves as a reference for just how complex the relationships between tooth characters, body size, and diet are. I have no major issues here.

Please see the attached PDF for all of my minor comments.
Additional comments: Much improved in my opinion. Thanks for trying to appeal to the "Meta-Analysis" concept. This is a very common type of study in (e.g.) ecology (not sure where PeerJ is getting the idea that it is strictly a biomedical article type), but that is a moot point at this stage. Everything is fairly clear in the manuscript.

All of my comments are minor, but I hope you take a look through the attached PDF and find them useful in clarifying a few key sections.


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

Input:
Title: BITE FORCE DATA SUGGESTS RELATIONSHIP BETWEEN ACRODONT TOOTH IMPLANTATION AND STRONG BITE FORCE
Review round: 2
Reviewer: 2

Output:
Basic reporting: This paper is clear and well-written. There are a few references that could be added, see below.
Experimental design: Methods are appropriate for this study.
Validity of the findings: All data are provided, conclusions are well-stated.
Additional comments: This paper is a well-written discussion of the possible functional origins of acrodonty. All previous reviewer comments have been addressed. I had a few ideas and comments while reading the manuscript, listed by PDF line numbers.

Line 40: add reference to Salomies et al., 2019 for development of acrodont dentitions.

Line 45: I think it is known that anchoring the teeth does not inhibit tooth replacement. This is discussed in Haridy (2018). The teeth become anchored after tooth replacement stops. This is also evident in the developmental evidence, where the tissues that make teeth stop making teeth, then the bone develops (Buchtova et al., 2013, Dosedelova et al., 2016, Handrigan and Richman 2010, Salomies et al., 2019).

Line 85: Preuschoft 1991 is a good reference here, since they actually test the function of plicidentine. Also see discussion in Brink et al., 2014.

Line 204: The evolution of the periodontal ligament is also associated with a decrease in tooth replacement rates in the mammalian lineage (see discussion in LeBlanc et al., 2018).

Lines 236-255: In animals that have acrodont teeth at the back of the jaws and pleurodont teeth at the front, the teeth are usually different shapes. Bite force can change depending on where it is measured around the mouth (related to gape), and bite force or bite efficiency changes with tooth shape (e.g., Dumont and Herrel 2003, Huber et al. 2009, and I’m sure there are many more). It should be discussed how in some species, like Agama and Pogona, there is a reason why polyphyodonty is maintained in the anterior teeth, which could be related to bite force, tooth function, heterodonty, jaw function, etc. (see discussion on tooth function in Agama in Cooper et al, 1970). Why does a bearded dragon need pointy anterior teeth while a chameleon does not?

Lines 295-300: From a developmental perspective, there are different ways that acrodont reptiles maintain their dentition. The successional lamina (the tissue that makes the replacement teeth) diminishes after eruption of the first teeth. However, it was recently shown in the bearded dragon that tissue is maintained even after the acrodont teeth have erupted, even though no new teeth are made in the acrodont areas (Salomies et al., 2019). This is interesting when considering your discussion on whether or not acrodonty is reversible. It is possible that teeth could be replaced in the acrodont areas with a few genetic tweaks of that tooth-making tissue. The potential for reversing monophyodonty is there. However, there would need to be a functional reason for this reversal to happen in the evolution of an acrodont lineage.

Line 356: there are pleurodont crushing teeth in Amphisbaena (Pregill 1984).

References:
Brink KS, LeBlanc ARH, Reisz RR. 2014. First record of plicidentine in Synapsida and patterns of tooth root shape change in Early Permian sphenacodontians. Naturwissenschaften. 101:883–892.

Buchtová M, Zahradníček O, Balková S, Tucker AS. 2013. Odontogenesis in the veiled chameleon Chamaeleo calyptratus. Archives of Oral Biology. 58(2):118-133.

Cooper J, Poole D, Lawson R. 1970. The dentition of agamid lizards with special reference to tooth replacement. Journal of Zoology. 162(1):85-98.

Dosedělová H, Štěpánková K, Zikmund T, Lesot H, Kaiser J, Novotný K, Štembírek J, Knotek Z, Zahradníček O, Buchtová M. 2016. Age‐related changes in the tooth–bone interface area of acrodont dentition in the chameleon. Journal of Anatomy. 229(3):356-368.

Dumont ER, Herrel A. 2003. The effects of gape angle and bite point on bite force in bats. Journal of Experimental Biology. 206(13):2117-2123.

Handrigan GR, Richman JM. 2010. Autocrine and paracrine shh signaling are necessary for tooth morphogenesis, but not tooth replacement in snakes and lizards (Squamata). Developmental Biology. 337(1):171-186.

LeBlanc ARH, Brink KS, Whitney MR, Abdala F, Reisz RR. 2018. Dental ontogeny in extinct synapsids reveals a complex evolutionary history of the mammalian tooth attachment system. Proceedings of the Royal Society B. 285(20181792):1-9.

Pregill G. 1984. Durophagous feeding adaptations in an amphisbaenid. Journal of Herpetology.186-191.

Preuschoft H, Reif W-E, Loitsch C, Tepe E. 1991. The function of labyrinthodont teeth: Big teeth in small jaws. In: Schmidt-Kittler N, Vogel K, editors. Constructional morphology and evolution. Berlin: Springer. p. 151-171.

Salomies L, Eymann J, Khan I, Di-Poï N. 2019. The alternative regenerative strategy of
bearded dragon unveils the key processes underlying vertebrate tooth renewal. Elife. eLife 2019;8:e47702.


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

Input:
Title: BITE FORCE DATA SUGGESTS RELATIONSHIP BETWEEN ACRODONT TOOTH IMPLANTATION AND STRONG BITE FORCE
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This is a great paper discussing the functional reasons for the evolution of acrodont dentitions. It is multi-faceted, considering many forms of evidence to support the hypothesis that acrodont ankylosis is an adaptation allowing for (or the result of) a stronger bite force. All previous reviewer comments have been addressed appropriately, and I have no further comments.


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

Input:
Title: THE GENOME OF THE BUTTERNUT CANKER PATHOGEN, OPHIOGNOMONIA CLAVIGIGNENTI-JUGLANDACEARUM SHOWS AN ELEVATED NUMBER OF GENES ASSOCIATED WITH SECONDARY METABOLISM AND PROTECTION FROM HOST RESISTANCE RESPONSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The report and the research are well developed and follow the requirements for clarity, background, data sharing and related. Below some minor comments.
- There are several typos in the scientific names of fungal species and in some other few places. In the corrected file I have attached you can find where this needs revision.
- It is a good idea to check that the names of fungal species are the current ones in use. Names of fungi change frequently so I do this exercise pretty frequently. While most of the names reported by the authors are correct, the current name of one of them, Diaporthe longicolla should be changed to Phomopsis longicolla, wherever is needed. You can consult Mycobank.org or Indexfungorum.org for this. This correction can also be found in the attached file.
- Lines 61-63 need some attention regaarding the statements made. Find the comments in the attached file.
Experimental design: - This is the first report for the pathogen in questions, which fits into the scope of original and primary research.
- Regarding the phylogenetic analyses. Needs a little more information, for example how the authors obtained the core eukaryotic proteins. Orthology? I have seen in other studies only the orthologous genes that are in single copy in all individuals are used for the phylogenetic analysis....was this the same in this case? Also, what was the final length of the alignment? did you use any model of protein evolution?
Validity of the findings: No comments. The authors conclusions are supported by the data they obtained.
Additional comments: It was a very nice, simple work to read.


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

Input:
Title: THE GENOME OF THE BUTTERNUT CANKER PATHOGEN, OPHIOGNOMONIA CLAVIGIGNENTI-JUGLANDACEARUM SHOWS AN ELEVATED NUMBER OF GENES ASSOCIATED WITH SECONDARY METABOLISM AND PROTECTION FROM HOST RESISTANCE RESPONSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: As indicated in my Comment #1, I suggest that thef manuscript incude a table summarizing structural and functional genome statistics.  Some data (on CYP450 gene annotations) seem to be missing from the Supplementary Table provided by the authors.
Experimental design: As indicated in my Comment #2, some of the genomic data provided by the authors (for genomes sequenced by other investigators) can be quite different from data available in public databases or in previous publications. I believe this should be clarified.
Validity of the findings: As indicated in my Comment #3, I feel the discussion is a bit weak and could be strengthened by making a better use of the existing fungal genomics literature.
Additional comments: The work reported by Wu and colleagues, based on the sequencing and comparative analysis of the butternut canker pathogen Ophiognomonia clavigignenti-juglandacearum (Oc-j), represents an interesting and potentially very useful contribution to the study of fungal ganets of cankers. The manuscript is generally well written, concise and easy to read. In my opinion, however, the manuscript would be improved by providing additional information and addressing additional issues. In particular :

1. In the opening section of Results and Discussion (Genome assembly and annotation of Oc-j), the authors should provide more data, including a table summarizing structural and functional genome statistics (including total number of genes identified per genome) for Oc-j and other Diaporthales genomes they analyzed. The estimated size of the Oc-j genome (52.6 Mb) seems quite high given the number of predicted genes (11 247) and I would expect the authors to discuss this issue (assembly artefact or biological reality?).

2. I was surprised by some of the genomic data provided by the authors. For example, on L118, they indicate that the genome of Grosmannia clavigera contains 530 CAZyme-encoding genes, which is far superior to the number (n=269) reported by DiGuistini et al (2011) who sequenced and annotated the G. clavigera genome. Some of the data from non-Diaporthales species originate from a previous paper (Schuelke et al. 2017) but, when I checked this reference, it was not clear to me how Schuelke and colleagues had obtained gene numbers that differed so much from those reported elsewhere. Another example :  in Supplementary Table «Antismash », the authors indicate that the Neurospora crassa contains 10 813 genes, which again is much higher than numbers usually reoprted for this species (e.g. 9 758 coding genes according to Ensembl Fungi). The reader is referred to Suppl. Table 1 for data on CYP genes but, unless I missed something, no relevant information is provided here. I would therefore like the authors to provide background information and further precisions on this issue of genomic data.

3. I would strongly encourage the authors to give further thought to the discussion of their results. While I understand they wanted to focus on the Diaporthales, I feel this weakens several of their interpretations. In the case of genes encoding secreted CAZymes (105-121), the authors’ suggestion that «the large number of secreted CAZymes in Oc-j might help facilitate its life-style infecting and colonizing butternut trees » is based on a limited dataset (this work and the previous work by Schuelke et al. [2017 ]). Furthermore, the authors only consider the breadmold Neurospora crassa when discussing saprotrophs. The genomes of several fungal saprotrophs (e.g. Chaetomium globosum,  Podospora anserina) contain high numbers of CAZy-encoding genes, and this would seem relevant in the discussion.
 When discussing  kinases (L122-129), the authors mention a potentially interesting element (gene family expansion in the domain family of fructosamine kinase PF03881 but do not discuss this finding. The number of CYP-encoding genes annotated in Oc-j (n = 223) is indeed very high compared with numbers estimated for other species considered in the study. I think it would be interesting to put this in a wider perspective by comparing with data from other studies and discussing further the families and potential functions represented in the OC-j CYP gene dataset.

Minor points

L35. Typo. Should read "endophytes"

L60-64.  Two different works published 5 years apart are each presented as «a recent study».  I suggest changing the wording.

L122 and L130 : Suggestion : Use the plural form for « Kinase » and « Cytochrome P450 ».

Legend of Figure 1: Suggestion: indicate whether Figure 1D shows asexual or sexual fruiting bodies of Oc-j.

Figure 2 :  Not a bad idea to include genome size and gene number data in the figure but I think it would help the reader if this oinformation (and additoinal genomic information) was also provided in a Table within the manuscript.

Legend of Figure 2. Typo in last sentence. Should read: "...number of predicted..."


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

Input:
Title: THE GENOME OF THE BUTTERNUT CANKER PATHOGEN, OPHIOGNOMONIA CLAVIGIGNENTI-JUGLANDACEARUM SHOWS AN ELEVATED NUMBER OF GENES ASSOCIATED WITH SECONDARY METABOLISM AND PROTECTION FROM HOST RESISTANCE RESPONSES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I have read the revised manuscript submitted by the authors and I feel thay have addressed concerns expressed by both reviewers of the original manuscript. In particular, I find the inclusion of Table 1 (Assembly statistics) a useful addition and I believe the conclusions are now presented in a more nuanced way. I am also happy with the authors' detailed response to reviewers' comments.

I have attached an annotated copy of the revised manuscript, with only a few very minor corrections (in red) and comments (mostly related to typos). I suggest adding one reference when discussing protein deglycation by fructosamine kinases in eukaryotes other than fungi (L181-182).


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

Input:
Title: A NEW SPECIES OF FRESHWATER CRAB GENUS FREDIUS PRETZMANN, 1967 (CRUSTACEA: BRACHYURA: PSEUDOTHELPHUSIDAE) FROM A NATURALLY ISOLATED OROGRAPHIC FOREST ENCLAVE WITHIN THE SEMIARID CAATINGA IN CEARÁ, NORTHEASTERN BRAZIL
Review round: 1
Reviewer: 1

Output:
Basic reporting: Parts of the manuscript requires careful edits, in particular, the description of the G1, which is quite complicated in the structure.
Background of the study is well explained.
Structure of the article is appropriate in general.
The establishment of the new species taxon seems to be supported by the given data.
Comments and suggestions for clarification are marked directly on the PDF main text file.
Experimental design: This article describes a new species of freshwater crab with morphological and molecular evidence. It match aims and scope of the journal.
The discovery of the new species provides some insights on the phylogeny and diversity of the genus Fredius.
The authors use the mitochondrial 16S rRNA gene to assess species delimitation, but only a single gene analysis may be insufficient for estimating phylogeny. Nevertheless, the primary purpose of this article is to describe a new species, which could be justified.
Information on DNA experiments is sufficient.
Newly obtained sequence data are not yet registered in GenBank database.
I could not open the MEGA 6 file, because I am MEGA 7 user. I do not like to spend much time to check the raw data.
Validity of the findings: The discovery of the new species is important in areas of biodiversity study.
The morphological comparison is restricted to the male gonopod 1. I would like to recommend the authors to examine other characters, in particular, female characters. Only with differentiation based on male gonopod characters, it is impossible to identify female specimens morphologically.
The discussion section needs much elaboration.
Additional comments: I strongly recommend the authors to reconsider the terminology for G1 structure. Please see attached main text remarked for details.


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

Input:
Title: A NEW SPECIES OF FRESHWATER CRAB GENUS FREDIUS PRETZMANN, 1967 (CRUSTACEA: BRACHYURA: PSEUDOTHELPHUSIDAE) FROM A NATURALLY ISOLATED OROGRAPHIC FOREST ENCLAVE WITHIN THE SEMIARID CAATINGA IN CEARÁ, NORTHEASTERN BRAZIL
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript brings the description of a new species of a pseudothelphusid freshwater crab of the genus *Fredius* from a forest enclave in the state of Ceará, Brazil. The work is original and it is a straightforward taxonomic work, representing a potentially relevant contribution to the knowledge of the Neotropical freshwater crab fauna. The manuscript is well written and structured according to good taxonomic articles on the description of new taxa, and conforms to professional standards. The figures are very good and appropriate as well as the tables. As I am not a native English speaker or even sufficiently fluent in English, I cannot make a correct grammatical assessment of the wording, but it seemed to me to be a clear and technically correct text, allowing for fluent reading. I only highlighted a few minor parts where the authors could double-check the wording.

However, the manuscript has some issues that should be improved. In addition to some minor corrections made throughout the text using MS Word Correction Tools, the main issues I noticed are the following:

a) The Introduction is not sufficient to clearly demonstrate the importance of this work (the introduction of this new taxon). It should bring a little more background on the distribution of the family and/or this genus in particular. I believe the paper would be much improved if the authors comment a little bit about the overall distribution of the pseudothelphusid family or at least the genus *Fredius*,  and how this new species is situated regarding this distribution (in addition to its occurrence in between the Amazon and Atlantic Forest Biomes).

b) The Introduction does not give an overview of previous work on the group in the region studied (the state of Ceará, at least). It would be interesting for potential readers to know about studies on the group that has already been done in the region or at least in the state of Ceará. At least the papers of Magalhães et al. (2005) and Pinheiro & Santana (2016) should be mentioned here.

c) Concerning the publication by Magalhães et al. (2005), despite this reviewer is the senior author of that paper, I think it deserves special attention because it dealt with material from the Ibiapaba plateau and, moreover, with material coming from the very same place of the type locality of the new species described in this manuscript. As the material examined in 2005 was determined to be *F. reflexifrons*, a question is raised about the real identity of those specimens: are they co-specific with the new species? (In that case, the determination should appear on the synonymic list of the new species.) This question should necessarily be addressed by the authors, especially considering that they are aware of this paper (it is cited in the manuscript, although in another context) and that the senior author should have examined that material during his acknowledged visit to the INPA’s collection.

d) As the molecular analysis did not include all species currently assigned to the genus *Fredius* and using only the 16S mitochondrial gene, I believe that the analysis is more a genetic distance analysis than a phylogenetic analysis of the genus itself and as such, it should be discussed. In this sense, it would be nice if the authors also compare their results with those presented by Magalhães et al. (2014) for the same genus and briefly comment on it.

e) Magalhães et al. (2005) also did a similar discussion on the zoogeographic occurrence of a *Fredius* species in the Ibiapaba plateau. Although this subject is improved in the present manuscript, I think that some mention of what was discussed by Magalhães et al. (2005) should be made here.

In addition, some other minor corrections and suggestions are also made throughout the text.

Finally, I would like to add that, although it could be considered that I might have some conflict of interest (planned collaboration with any of the authors), I conducted this review with integrity and with the idea of providing constructive criticism, and I believe that I have an appropriate level of expertise to confirm that this submission is of an acceptable scientific standard and suitable to be published in PeerJ as long as the improvements suggested above are implemented
Experimental design: The research question is well defined (description of a new taxon) and the result is relevant, representing an important contribution to improve the knowledge on the diversity and distribution of the Neotropical freshwater crab fauna. The specimens data are well presented in the “Material examined” section, but I made a few suggestions to improve it. The “Discussion” is well conducted, although some mention of what was discussed by Magalhães et al. (2005) should be made. Prior literature is adequately referenced. However, as it is a taxonomy article, it seems to me that references that cite the taxonomic authorities of the taxons mentioned in the text should also be included, although this, of course,  depends on the journal’s rules.
Validity of the findings: The work is scientifically sound, with rigorous and appropriate taxonomic comparisons and references.  The morphological description and the figures are relevant and of excellent quality. The evidence presented, whether molecular, morphological or zoogeographic, clearly shows that the species is new and valid, making it an original and significant research contribution. I would perhaps suggest that the authors stress a bit more that *F. ibiapaba*  sp. n. and *F. reflexifrons* are pseudocriptic species differentiated only by very subtle morphological characters.
Additional comments: I made several suggestions for minor modifications and corrections using MS Word Correction Tools in order to suggest improvements to the text. Please see the attached file. However, I believe that this work lacks an essential aspect: to compare your material with that studied by Magalhães et al. (2005) and define the identity of the former. As it is material of the species considered in this manuscript as a sister species of the new taxon, a question is immediately raised: are the specimens from both studies co-specific? It is imperative that you answer it. That article cannot be ignored in this regard, otherwise, your work will be incomplete and the discussion of zoogeographic aspects would be compromised.


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

Input:
Title: A NEW SPECIES OF FRESHWATER CRAB GENUS FREDIUS PRETZMANN, 1967 (CRUSTACEA: BRACHYURA: PSEUDOTHELPHUSIDAE) FROM A NATURALLY ISOLATED OROGRAPHIC FOREST ENCLAVE WITHIN THE SEMIARID CAATINGA IN CEARÁ, NORTHEASTERN BRAZIL
Review round: 1
Reviewer: 3

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: The manuscript contains the description of a new species of the freshwater crab genus Fredius Pretzmann, 1967, based on morphological and molecular data. The title of the manuscript is precise and reflects the content. The description of the new species is according to the International Code of Zoological Nomenclature. However, the manuscript has many issues that need to be fixed by the authors before edit it in detail.
 I would, therefore, suggest accepting the manuscript with Major Revision if and only if  the authors carefully take into account the following suggestions/comments. – And I would be happy to receive the revise version and help the authors to improve their manuscript.
1.	From the title, please precise in which genus you are describing a new species.
2.	Please remove this statement from the manuscript…  and references therein…
3.	 We need only Fig. 1E that shows the biotope of specimens of the new species. Then please, I suggest to remove Fig. 1A-D, F-G. – This is a taxonomic work.
4.	Under the Material and Methods section, precise that cw: carapace width. –This is missed from the manuscript.
5.	Please remove Figure 2 from the manuscript and under the Material and Methods section, please re-write this sentence … The terminology used in the description of the G1 is referred in the figure 2. Then simply provide the reference document from where you took the terminology used for the description.
6.	From where exactly you extracted DNA ? You said ‘Muscle tissue samples were obtained from the pereopods or abdomen’. Then It looks like you alternatively used pereopods  and abdomen. – Why? Please provide reference.
7.	Why did you decide to choose Prionothelphusa eliasi and Trichodactylus dentatus as outgroups? I also noticed that there are two distant phylogenetic taxa, then I cannot trace why...  In general, the outgroup should be as close possible to the ingroup.  Please select the appropriate outgroup taxon and provide justification to your choice. (Re- run the phylogenetic tree).
8.	I cannot see scale bars from your figures except figure 3 and figure 7. Then, I’m not convinced that all your diagnosis characters warrant for new species recognition. For example, you said G1 robust… without scale bars from, nobody can trace this… I suggested to revise all this accordingly. This holds true for the description section. After the revision, I suggest to link each statement of the diagnostic section to the corresponding figure. - This also holds true for the description section, where you have to link each statement to the corresponding figure.- What you did, seem not enough and cannot help readers to trace your work.


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

Input:
Title: A NEW SPECIES OF FRESHWATER CRAB GENUS FREDIUS PRETZMANN, 1967 (CRUSTACEA: BRACHYURA: PSEUDOTHELPHUSIDAE) FROM A NATURALLY ISOLATED OROGRAPHIC FOREST ENCLAVE WITHIN THE SEMIARID CAATINGA IN CEARÁ, NORTHEASTERN BRAZIL
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is the re-review. Overall, the manuscript has been sufficiently improved.
Experimental design: See previous comments.
Validity of the findings: See previous comments.
Additional comments: The manuscript has been well improved, being acceptable with minor edits, which are remarked on the main text file.


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

Input:
Title: A NEW SPECIES OF FRESHWATER CRAB GENUS FREDIUS PRETZMANN, 1967 (CRUSTACEA: BRACHYURA: PSEUDOTHELPHUSIDAE) FROM A NATURALLY ISOLATED OROGRAPHIC FOREST ENCLAVE WITHIN THE SEMIARID CAATINGA IN CEARÁ, NORTHEASTERN BRAZIL
Review round: 2
Reviewer: 2

Output:
Basic reporting: As I mentioned in the first round of review, this manuscript is a straightforward taxonomic work, well written and structured according to good taxonomic articles on the description of new taxa, and conforms to professional standards. The authors have followed several suggestions and improved the manuscript. However, there are still some minor points that need to be considered and I have marked them with the Review Tool throughout the text.

The reply letter is very detailed and explanatory and convincingly justifies some of the points whose suggestions have not been followed. Although there are a few cases in which I do not agree with the justification but I understand that they refer to a matter of point of view. Even so, I again stressed my arguments about these issues in comments inserted throughout the manuscript.

The figures are very good and appropriate, but I made some suggestions to improve and to correct tables 1 and 3.
Experimental design: Nothing substantially different from that stated in the first review, except for the following issue. As this is a taxonomic work, I think information about synonymy is very important. This species is not being described on entirely new material but instead, they are considering the material of a population that has been previously interpreted by others according to another concept. In this situation, it is important to make clear the current interpretation of what was previously published in the relevant scientific literature. Therefore, I reinforce the recommendation to include a synonymic list and made a suggestion to that effect.
Validity of the findings: No further comment.
Additional comments: The manuscript has been greatly improved, but I still have a few minor suggestions and corrections, which I marked with the Revision Tool in the files of the main text and in tables 1 and 3. Furthermore, In their reply letter, the authors explain they were not sure about the question I asked if the specimens used in the study by Magalhães et al. (2005) and in this manuscript were conspecific, and replied that they “were not sure what is meant by the reviewer” and that they have clearly stated that *F. ibiapaba*  was a new species. Of course, you did; no doubt about it. However, the question was whether, if you considered that the specimens studied by Magalhães et al. (2005) (which at least the senior author had the opportunity to examine) could be considered, according to the new data and the current new interpretation, as belonging to the new species. In this case, the identification given by Magalhães et al. (2005) should be listed as a synonym for the new species in this study. For a taxonomist, this is indeed an essential issue, as it will clearly indicate to the potential reader the taxonomic history of that taxon and the concept about the specific limits applied to it in a given time, thus avoiding future misinterpretations.


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

Input:
Title: A PREOPERATIVE NOMOGRAM FOR SEPSIS IN PERCUTANEOUS NEPHROLITHOTOMY TREATING SOLITARY, UNILATERAL AND PROXIMAL URETERAL STONES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Grammar and language need correction and some explanations:
1-	Tittle; Better to be in this way: A pre-operative nomogram for sepsis in percutaneous nephrolithotomy treating solitary, unilateral, and proximal ureteral stones. Because you are discussing a method in a procedure.
2-	Line 24; the correct is;  ,and
3-	Line 40; the correct is the receiver, ,and
4-	Line 60; the correct is  the local
5-	Line 64; the correct is; a pre-operative model: The phrase there is an absence of a pre-operative, predictive model may be unnecessarily wordy. Consider using an adjective instead of a noun phrase.
6-	Line 73; the correct is: of the ethics: It appears that the preposition from may be incorrect in this context. Consider changing it.
7-	Line 78; the correct is ; ,and: Your sentence contains a series of three or more words, phrases, or clauses. Consider inserting a comma to separate the elements.
8-	Line 85: the correct is;tests , including albumin and globulin,
9-	Line 96:  the correct is; judgment
10-	Line 98: the correct is; Otherwise, we
11-	Line 100: the correct is; hypertension, and coronary
12-	Line 105: the correct is; t-test
13-	Line 105: the correct is; with a  normal
14-	Line 108: the correct is; converting the regression
15-	Line 105: the correct is: The predictive, the model
16-	Line 110; the correct are  ; ,and.  A calibration
17-	Line 112: the correct is;  the discriminative
18-	Line 113: the correct is ;( In addition)  The phrase In addition may be wordy. Consider changing the wording. Also or beside the clinical
19-	Line 115: the correct is; P-value <0.05 was considered significant.
20-	Line 137: the correct is; the calibration
21-	Line 138: the correct is; by the Hosmer-Lemeshow test
22-	Line 139 and 140; the correct is; the curve, the model
23-	Line 141: the correct is; change had the ability to could
24-	Line 151-153: Monotonous passage; We reported a sepsis rate of 4.7% (35/745) after PCNL for the treatment of proximal ureteral stones. This result was consistent with that (0.3%-9.3%) of a previous study conducted by Gutierrez et al. 13. Sepsis can further lead to organ dysfunction and even septic shock, endangering patients' lives Is 3 similar sentences in a row. These sentences seem repetitive because they all follow the same pattern. Consider changing the word order
25-	Line 162,163; the nutritional and the insufficient
26-	Line 173: the correct is; maybe
27-	Line 183: the correct is: potential (of a potential) The indefinite article, a, may be redundant when used with the uncountable noun infection in your sentence. Consider removing it.
28-	Line 192: the correct is; a comparison ( It appears that an article is missing before the word comparison. Consider adding the article.)
29-	Line 204: the correct is; why female sex
30-	Line 208; the correct is; Second,  the only
31-	Line 212 and 215; the correct are;  , and . a prospective
32-	Line 230; the correct is;   Acknowledgments

References:
No. 7: Line 254; Biomed Res Int. 2019 January 20;2019:8078139.
No. 3,4,5: Line 244-250; change as mentioned later on in the next session.
No. 11: Line 262; Wiesenthal JD1, Ghiculete D, D'A Honey RJ, Pace KT. A comparison of treatment modalities for renal calculi between 100 and 300 mm2: are shockwave lithotripsy, ureteroscopy, and percutaneous nephrolithotomy equivalent? J Endourol. 2011;25(3):481-5.

Tables:
Generally are ok, but a suggestion, if possible, to classify the degree of hydronephrosis.
Experimental design: Line 23; Abstract;
The background doesn't explain the scope of the work; it realizes the aim or the objective of the study. It is better to have a background in the first with the author's speech and an objective of the study.
Line 50,51;
Reference 3 is a guideline book, suggest to use this update reference on management of urolithiasis:
Standard percutaneous nephrolithotomy  (sPCNL) remains the optimal treatment for stones between 1 and 2.5 cm ( Heinze, Alexander; Gozen, Ali S.; Rassweiler, Jens. Tract sizes in percutaneous nephrolithotomy does miniaturization improve outcomes? Current Opinion in Urology: March 2019 - Volume 29 - Issue 2 - p 118-123.)

 Line 51 and 53;
This; Compared with the latter modality, PCNL is associated with less ureter injury, more fluent intrarenal irrigation, better visibility, and a superior stone-free rate 4,5. It has no relation with the main topics; it is about two different endourological procedures. It is better to be removed. Suggest to use this reference:
Overall, PCNL provides significantly higher stone-free rates than RIRS, at the expense of higher complication rates, blood loss, and longer length of stay, with no differences in surgical time and secondary procedures. ( Shuba DeRiccardo AutorinoFernando J. KimHomayoun ZargarHumberto LaydnerRaffaele BalsamoFabio C. TorricelliCarmine Di PalmaWilson R. MolinaManoj MongaMarco De SioCorrigendum re: "Percutaneous Nephrolithotomy VersusRetrograde Intrarenal Surgery: A Systematic Review and meta-analysis" [Eur Urol 2015;67:125–37]
Line 57;
Better not to mention the author's name in the introduction, writ in a passive word as long as it has a reference number.
Line 54-56;
It has no reference number.
Patients and methods;
Line 76;
The inclusion criteria should include the size of the stone; according to EAU guideline 2019, page 303, ureteric stones are classified to <10mm and >10mm. <10mm stones are treated by ESWL or URS.
Line 84;
1-Before surgery: As long as this study is a retrospective type, you cant perform a test or a procedure on patients already treated.
2-Patients with fever and treated by antibiotics either they resolve or have a suppression.
3- Tubes (nephrostomy and JJ ) usually removes in the same session of surgery to avoid two anesthesia or recurrence of the infection.
This part of the study need to be written again in this way:
The study was conducted under the approval of the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (2019S1035). We retrospectively enrolled 745 patients who underwent PCNL from January 2012 to December 2018. The inclusion criteria were the following: (1) PCNL was performed to treat unilateral, solitary, and proximal ureteral stones; and (2) age ≥18 years. Stones size bigger than 10mm.The exclusion criteria were anatomical renal abnormalities (horseshoe kidney, solitary kidney, transplant kidney, and kidney duplication).
Patients information were collected from our hospital's database, including age, sex, body mass index (BMI), comorbidities (diabetes, hypertension, and coronary heart disease), stone size and laterality, prior indwelling stent, blood tests (cholesterol, creatinine, albumin, and globulin levels and white blood cell count), fever (defined as body temperature >38°C), urine tests (white blood cell and nitrite), urine culture, and ASA score. Patients imaging, including abdominal computed tomography CT, confirmed the presence and size of proximal ureteral stones (above the fourth lumbar spine or in the ureteropelvic junction). Types of anesthesia, preoperative antibiotics, surgeons years experience, PNL operation details were identified and recorded. Postoperative patients' data were revised, collected, and recorded.
Sepsis was defined according to the 2001 International Sepsis Definitions Conference; occurrence of an infection and a minimum of two of the following within 48 hours of surgery: (1) heart rate >90/minute, (2) body temperature >38°C, (3) leukocyte count <4,000 cells/μL or >12,000 cells/μL, and (4) respiratory rate >20/minute (9).
All statistical analyses were performed using SPSS 24.0 and R software 3.6.2. The student t-test was used to detect differences between continuous variables with a normal distribution. The chi-square test or Fisher's exact test was used to compare categorical variables. The multivariable logistic regression method was used to determine independent risk factors for sepsis. Then predictive nomogram was generated based on converting the regression coefficient to a 0- to 100-point scale proportionally. The predictive performance of the model was measured by validation, discrimination, and decision analysis10. A calibration curve was generated with 1000 bootstrap samples to reduce the overfit bias. Hosmer-Lemeshow (HL) test implied good calibration when the test is insignificant. The discriminative performance was assessed by area under the receiver operating characteristic (ROC) curve. Also, the clinical usefulness of the nomogram is evaluated by decision curve analysis (DCA) by evaluating net benefits at different threshold probabilities. P-value <0.05 was considered significant.
Validity of the findings: Is there any information about the Shape of the stones( extensions), condition of the patients (paraplegia is associated with postoperative sepsis in Campbell- Walsh  Urology Edition 10th), and degree of hydronephrosis (Kuldeep Sharma et al.  Factors predicting infectious complications following percutaneous nephrolithotomy. Urology Annals; Year : 2016  |  Volume : 8  |  Issue : 4  |  Page : 434-43. Post-PCNL infectious complications were found to be more common in patients with renal failure, diabetes mellitus, preoperative PCN placement, staghorn calculi, severe HDN, multiple punctures, and prolonged duration of surgery. ?
Line 121; No need for Figure 1; is extra-explanation.
Line 125: change hight to size ( more informative).

Conclusion:
Is too long, I suggest this:
A new nomogram is developed to predict sepsis in PNL for an upper ureteric single stone if the patient has these characters: female sex, lower AGR, positive urine culture, and leukocytosis.
Additional comments: Discussion:
Line 152 and 153 ; (Sepsis can further lead to organ dysfunction and even septic shock, endangering patients' lives 6,14.) Has no advisory role in the discussion, consider removal. Especially the reference (6) has been used before.

Line 165, reference 17: has a different opinion about low and high-normale serum albumin levels? See this reference talk about stone size as a predictor for sepsis and the albumin.
It was concluded that patients with larger stone size and preoperative urinary tract infection might have a higher risk of developing SIRS and fever after the operation, while a high-normal level of serum albumin might be the protective factor for postoperative fever. ( Tian Yang et al. , The Evaluation of Risk Factors for Postoperative Infectious Complications after Percutaneous Nephrolithotomy. January 11, 2017)

Line 166: Abbreviation written without preceding details; high-sensitive C-reactive protein/albumin (hs-CRP/Alb) ratio.

Line 172; references 14,8 has been repeated twice. Consider new references!

Line 178: It is not clear where and how the sepsis increased by three times in patients with a preoperative positive culture.

Line 192; Ryan et al. decision may be simple, but it is not effective for urologist decisions. The upper urinary tract urine is highly dependable for postoperative suspecting sepsis. See reference;
The results of this study suggest that positive stone C&S and pelvic urine C&S are better predictors of potential urosepsis than bladder urine. Therefore, a routine collection of these specimens is recommended. ( PARAMANANTHAN MARIAPPAN, GORDON SMITH, SIMON V. BARIOL, SAMI A. MOUSSA, and DAVID A. STONE AND PELVIC URINE CULTURE AND SENSITIVITY ARE BETTER THAN BLADDER URINE AS PREDICTORS OF UROSEPSIS FOLLOWING PERCUTANEOUS NEPHROLITHOTOMY: A PROSPECTIVE CLINICAL STUDY. Journal of UrologyAdult Urology: Urolithiasis/Endourology1 May 2005)


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

Input:
Title: A PREOPERATIVE NOMOGRAM FOR SEPSIS IN PERCUTANEOUS NEPHROLITHOTOMY TREATING SOLITARY, UNILATERAL AND PROXIMAL URETERAL STONES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study reported a pre-operative nomogram for sepsis after percutaneous
nephrolithotomy treating solitary, unilateral, and proximal ureteral stones. There were many studies reported on this topic
Experimental design: 1.An internal or external verification is required to confirm the useful of the crearted nomogram.
2.Definition of ”sepsis” according to the 2001 International Sepsis Definitions Conference in the study is the same to systemic inflammatory response syndrome（SIRS）which make difficulty to follow which the author want to study.
Validity of the findings: The authors present a nomogram to predict  sepsis after percutaneous nephrolithotomy treating solitary, unilateral, and proximal ureteral stones. there were many studies reported on this topic，and the stone location specific to proximal ureter is the only difference compared to other  studies. However，the stone location in ureter is not a risk factor to sepsis after PCNL, thus this limited case series is not presenting a novel idea and is unlikely to alter management.
Additional comments: none


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

Input:
Title: A PREOPERATIVE NOMOGRAM FOR SEPSIS IN PERCUTANEOUS NEPHROLITHOTOMY TREATING SOLITARY, UNILATERAL AND PROXIMAL URETERAL STONES
Review round: 1
Reviewer: 3

Output:
Basic reporting: No Comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Title: A pre-operative nomogram for sepsis after percutaneous nephrolithotomy treating solitary, unilateral, and proximal ureteral stones (#46687)

The paper aimed to determine the preoperative predictors of sepsis after percutaneous nephrolithotomy (PCNL) in patients with unilateral, solitary and proximal ureteral stones, and developed the nomogram to predict sepsis with significant and independent predictors.
Female, positive urine culture and leukocytosis are well known risk factors for sepsis following PCNL and lower albumin globulin ratio (AGR) is new finding in this paper.
There are a few comments to address before publication.

1. The author analyzed sepsis in this paper, how many cases presented septic shock in enrolled cases with sepsis?

2. The cut-off point of AGR was set at 1.5 in this paper. I am wondering how the author determine this cut-off point. Need to make it clear in method section.

3. This paper contained a bit complicated statistical analysis, such as developing nomogram and validation of that. Is there any proper statistician in co-author? If so, please make it clear in affiliation section.


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

Input:
Title: A PREOPERATIVE NOMOGRAM FOR SEPSIS IN PERCUTANEOUS NEPHROLITHOTOMY TREATING SOLITARY, UNILATERAL AND PROXIMAL URETERAL STONES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Language and grammar were corrected.
The study is clear for readers.
All requirements were written in order, the references provided, except reference 6 written more than once.
Experimental design: Background added
Abstract is written
The concept of the study is clear
Questions were answered and changes were made.
Validity of the findings: Findings are clear and more organized.
The data provided better.
All my questions were answered and the changes were made
The conclusion now is simple and more informative.
Additional comments: Use one of these; either Pre-operative or preoperative style throughout the manuscript, same for postoperative.

Cancel the writing of reference No. 6 inline 63, as it has been repeated 3 times.
Line 213 Sen V et. al.
Reference 22 inline 215 is 23.


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

Input:
Title: HEMOGRAM DATA AS A TOOL FOR DECISION-MAKING IN COVID-19 MANAGEMENT: APPLICATIONS TO RESOURCE SCARCITY SCENARIOS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The work presenting by the authors are interesting and addressed an important issue in the resource-limited country. However, some improvement should be implemented before it can be accepted.
1. The paper is not well written. for example, the abstract is poorly written, and is not informative. many important information is missing and readers cannot have a general idea of the study design (such as sample size) and main results (such as performance metrics of the ML model). I strongly suggest the authors to report this paper  as per  the TRIPOD checklist (https://med.stanford.edu/content/dam/sm/s-spire/documents/ManuscriptQualityChecklists/Tripod-Checklist-Prediction-Model-Development-and-Validation.pdf)!
2. The baseline characteristics should be reported.
3. Many other important variables such as demographics, past history of pulmonary disease and smoking; these are readily available in resource-limited country; why not include these variables for your ML model?
Experimental design: Due to the imbalanced dataset, the accuracy cannot be used for evaluating the model. since you can predict all patients without COVID-19 and the accuracy can be 90% in your situation. Thus, I suggest to report the AUROC or PR curve for the evaluation. Furthermore, You also need to compare your model to the baseline model. The baseline model refers to a naive model that predict all patients without COVID-19.
Validity of the findings: The model testing is limited by  the small sample size; there are only 10 events.
Additional comments: The work presenting by the authors are interesting and addressed an important issue in the resource-limited country. However, some improvement should be implemented before it can be accepted.


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

Input:
Title: HEMOGRAM DATA AS A TOOL FOR DECISION-MAKING IN COVID-19 MANAGEMENT: APPLICATIONS TO RESOURCE SCARCITY SCENARIOS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The overall quality of the writing is fine, but there are a number of grammatical and typographical mistakes that should be corrected. Please proofread.
Experimental design: The article meets the criteria, but would have been a lot stronger if a few things had been corrected:

1. The authors argue that one of the main benefits of using naive Bayes is the interpretability of the model, then state that in this case the model should not be interpreted. I agree with neither of these: the interpretability of the model is not an important benefit, and the model can be interpreted, with caution.
2. The authors completely ignore the capacity of the model to deal with missing data, when this is a key aspect of the problem
3. It is not clear whether the test set is actually resulting in fair evaluation. In the description, it is stated that it is used for training, and that the missing values are filled with the mean value _of the class_. If that is true, the test set is not unbiased and the corresponding results are not valid.
3. The graphs are not completely clear and seem to be slightly incorrect: the y-axis is not fully labelled, and results that should be 1 (the maximum possible value in this case) don't seem to be.
Validity of the findings: See above: the validity of some results is in doubt, and some results are not reported unambiguously.

The conclusions and the potential use cases are well developed.
Additional comments: No additional comments


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

Input:
Title: HEMOGRAM DATA AS A TOOL FOR DECISION-MAKING IN COVID-19 MANAGEMENT: APPLICATIONS TO RESOURCE SCARCITY SCENARIOS
Review round: 2
Reviewer: 1

Output:
Basic reporting: My previous comments have been well addressed.
Experimental design: the experimental design is not solid and sound
Validity of the findings: good
Additional comments: I have no further comments.


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

Input:
Title: PHYLOGENETIC COMPARISON BETWEEN TYPE IX SECRETION SYSTEM (T9SS) PROTEIN COMPONENTS SUGGESTS EVIDENCE OF HORIZONTAL GENE TRANSFER
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1 - some parts of the manuscript are hard to understand. I think that one of the difficulties it to understand the area's terms. For instance, all the acronyms should be written out, even if you that they are obvious, such as IM, OM, SeqYeg, Wbp.

2 - no comment

3- Figures 2 - 5 have no sufficient resolution to see the bootstrap values or the names of the organisms. If the names are important, you should ensure that your reader can read them.

4 - no comment
Experimental design: 1 - no comment

2 - The authors claim that the study "aims to investigate the phylogenetic relationship and taxonomic distribution between putative members of T9SS component protein families". Lines 46 - 47. I think that their problem was not limited to the inference of phylogenies. If I understood, they searched for evidence of horizontal transfer of T9SS. However, they already knew that such proteins were acquired horizontally from other bacteria and at the end,  they concluded that only PorR was "acquired by T9SS-acquiring bacteria through horizontal gene transfer" (line 56).

3 - no comment

4 - Methods were described with sufficient detail. However, they built ML trees with only 100 bootstrap replications. Once they found several clades with low support, It would be hard to find the same topologies in a second round of each analysis. I suggest that they re-perform the phylogenies, but using a Bayesian approach with a minimum of 50 millions of Markov Chain steps. They can easily do it using MrBayes at the Cipres Gateway (https://www.phylo.org/), that saves time and provides very good results.
Validity of the findings: 1- no comment

2- I disagree with the conclusion that 19 of the 20 genes provide trees where the OTUs were "clustered based on their classes under Bacteroidetes phylum".  Hence, Figs. 2A - 2F, 3A-3C; 4A, 4C,4D ,4E; and 5A-5C present at least one OTU out of its expected cluster. If it is true that al T9SS were acquired from other bacteria, so the ancestral of each "monophyletic group of sequences" acquired the genes and passed them to their descendants. Therefore, I understand that such "outsiders" inherited the gene from bacteria of other groups, which should also be interpreted as a recent horizontal transfer.

_ Another point to be highlighted is that authors treated as monophyletic groups, clades with very low bootstrap support [e.g. group 3 of Fig. 2A (Bootstrap - BT = 65%); groups 1 and 2 of Figs. 2B and 2C (BT = 55 and 70%, respectively); group 3 of Fig. 3A (BT = 72%); group 4 of Fig. 3B (BT = 67%); group 5 of Fig. 3B (BT = 30%)]

3 - I think the authors focused only on PorR and should mention that most of the other proteins also show evidence of recent horizontal transfers (after the first transfer to Bacteroidetes or even the first transfer for each of its families). However, they showed evidence that PorR and its neighbors are flanked by IS5 transposons, which is very nice if it is a new finding. I think that the proper way to treat this point is that this operon was easily exchanged between the bacteria families (explaining why the figure 6 is so different from the other trees).
- I'm curious about why they did not test one of the neighbors, such as PorS, only to show that it has the same topology than PorR and reinforce that the recent horizontal transfer took place because of the IS5.

4 - no comment
Additional comments: Even though I have pointed out several flaws in the article, I know how hard is to mining genomes to search for protein families and define the homologous sequences to infer the phylogenies. Because of that, I recommended major revisions, once I would like to see a new version of this article.

I also would suggest that authors use a clearer way to explain their point. I'm not a specialist in cell membrane's proteins and found very difficult to follow some parts of the introduction. I think that a broad audience journal such as PeerJ has lots of readers that would be interested in recent horizontal gene transfer between pathogenic bacteria. Please, consider to re-wright the introduction to the sake of clarity.


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

Input:
Title: PHYLOGENETIC COMPARISON BETWEEN TYPE IX SECRETION SYSTEM (T9SS) PROTEIN COMPONENTS SUGGESTS EVIDENCE OF HORIZONTAL GENE TRANSFER
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this paper, phylogenetic analysis revealed that PorR is different to other Por component proteins. This conclusion is not surprising. Because, PorR protein has similarity to the degT gene product of Bacillus stearothermophilus in Gram-positive bacteria and the homologs are present in broad (Shoji et al. 2002 Microbiology. 148(Pt 4):1183-1191.). In other words, PorR protein is not specific in Bacteroidetes phylum. On the other hand, PorT protein and other T9SS component proteins are specific in Bacteroidetes phylum. Therefore, the conclusions shown in this paper are not contrary to expectations.
Experimental design: No comment
Validity of the findings: In the last part of the results, authors found that additional species in Bacteroidetes phylum lack the proteins of PorG, PorL PorN, PorP, PorQ, PorT, PorU, and PorZ. This finding is interesting. However, it needs to be discussed with more experimental findings.
Additional comments: In addition, I present some advises to improve the manuscript.

1.	As I mentioned above, PorR homologs are shown in Shoji et al. (2002) in Microbiology. The paper should be referred.

2.	It is difficult for new readers to understand many PGN_ or PG_ numbers. Recently, T9SS related proteins are designated by Naito et al. (2019). [Naito M, Tominaga T, Shoji M, Nakayama K. PGN_0297 is an essential component of the type IX secretion system (T9SS) in Porphyromonas gingivalis: Tn-seq analysis for exhaustive identification of T9SS-related genes. Microbiol Immunol. 2019 63(1):11-20.] I recommend that gene numbers shown in the results are changed to PorF, SigP, PorE, and PorG as appropriate. [PG_0534, PGN_1437: PorF], [PG_0162, PGN_0274: SigP], [PG_1058, PGN_1296: PorE], [PG_0189, PGN_0297: PorG], and the paper of Naito et al. (2019) should be also referred.

3.	In Figure 1, L3 are drawn by two rectangles. It should be drawn by three rectangles because small 3 in the right of L indicates the number of PorL.

4.	In Figure 1, A-LPS is drawn outside of outer membrane. This seems to be weird. It should be drawn on the outer membrane.

5.	In Figure 1, as the representative of T9SS cargo proteins, Kgp is shown. It should be changed to RgpB because RgpB is known to be clearly modified with A-LPS.

6.	In Figure 1, “Lipid A transport pathway “should be changed to “LPS transport pathway”.

7.	In Figure 1, R indicating PorR is colored in dark yellow. It should be changed to another different color. Because, the color is same to SigP. PorR function is known to be different to SigP one.

8.	In Figure 8, it is drawn that PorR and PGN_0300 belong to regulation colored in red. PorR should be drawn as A-LPS synthesis shown by another color. PGN_0300 also should be drawn as unknown shown by color in grey or another color. PGN_0300 may be written as Omp17 or Skp (Taguchi et al. 2015).

9.	In line 166 of the main text, “PorR might be acquired by T9SS-acquireing bacteria thorough horizontal gene transfer”. It seems overstatement. I recommend you change that “PorR might be acquired by other bacteria thorough horizontal gene transfer”.

10.	In materials and methods section of the main text, ATCC 33277 should be non-italic font. Likewise, Rhodothermus marinus (italic font) DSM 4252 (non-italic font) and Salinibacter ruber (italic font)  DSM 13855 (non-italic font) .

11.	In line 353 of the main text, “porR gene” should be changed to “porR (italic font) gene”.

12.	In line 364 of the main text, “similar to wzx “should be changed to “similar to wzx  (italic font)“.


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

Input:
Title: PHYLOGENETIC COMPARISON BETWEEN TYPE IX SECRETION SYSTEM (T9SS) PROTEIN COMPONENTS SUGGESTS EVIDENCE OF HORIZONTAL GENE TRANSFER
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors had improved the article according to former recommendations. They inferred the phylogenetic trees using Bayesian Inference using appropriated models of amino acid substitution.

However, their findings were not completely convincing. They infer phylogenetic hypotheses of 20 T9SS proteins using Bayesian Inference (BI) and found different topologies (as expected). They argued that 19 of the 20 topologies presented "well-supported clusters" for the three major classes under Bacteroidetes. I have concerns about the concept of a cluster: "The definition of a cluster in this work is the cluster formed by majority of the terminal nodes of the tree (> 50%) that belong to the same class under Bacteroidetes" (lines 285 and 286). They used this particular concept instead of monophyletic clades containing the Bacteroidetes classes. The authors cited the phylogenetic inference of Karlsson et al. (2011) based on the 16S sequences as a reference for the phylogenetic relationships among Bacteroidetes. I consulted the referred article and could not find the three Bacteroidetes classes as monophyletic clades (it is not my research field, so I had difficulties to find the clusters). I think the authors could reproduce the 16S tree coloring the OTUs according to the cited classes.

Because of their particular concept of clusters, they found such "clusters" in 19 of the 20 trees but had to construct a table (Table S1), including all OTUs that grouped in unexpected clusters. Also, they compared the PorS and PorR trees and argued that they have the same "clusters" of Cytophagia (blue) and Bacterioidia (green). The problem with this state is that the "conserved clades" of PorS tree contain only one genus of Bacterioidia (Porphyromonas) and only one genus of Cytophagia (Hymenobacter).
Experimental design: The authors treated as "well suppported", clusters with posterior probability (PP) values > 0.8 (line 288), which is unusual since we consider as well supported clusters with PP > 0.95 when using BI.

In Abstract and Discussion, the authors refer to clusters in terms of their position at the printed figure, which is unusual in phylogenetic studies.  At lines 48-49 they stated that: "General topology of PorR BI tree is different from the other 19 BI trees that are multifurcating at its centre into three big clusters that are approximately 60o apart from each other except for PorN BI tree that has two big clusters that are almost 180o apart. However, PorR BI tree has one big cluster at bottom half of the tree and branches sequentially diverging out from its centre at top half of the tree". At lines 397-399, they repeated the same idea: "This is because the 19 BI trees are multifurcating at its centre into three big clusters that are approximately 60o apart from each other (or two big clusters that are almost 180o apart from each other for BI tree of PorN) (Figs. 2-6). However, the BI tree of PorR has one big cluster at bottom half of the tree and branches sequentially diverging out from its centre at top half of the tree (Fig. 3)".
Validity of the findings: I could not understand the point of the horizontal transfer of genes in this work. It would be useful to inform (or infer) the consequences of the transfers for the organisms that received the genes. I think that authors had a great job in working with the Bayesian Inference. Still, I feel that the work would gain with the aid of a phylogenist to help them to describe the trees and interpret their results.
Additional comments: When I evaluated the first version of the manuscript, there were problems with the figure resolution as well as concerns about the methods used to infer the phylogenies and interpret the results. The authors resolved the first part of such problems, and now it is possible to see the results. However, I still have concerns about the interpretation of the results. My main suggestion is that the authors invite a phylogenist to help them interpret their findings. Also, it will be necessary to investigate the possible consequences of horizontal transfers of some of the T9SS family genes among Bacteroidetes.


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

Input:
Title: PHYLOGENETIC COMPARISON BETWEEN TYPE IX SECRETION SYSTEM (T9SS) PROTEIN COMPONENTS SUGGESTS EVIDENCE OF HORIZONTAL GENE TRANSFER
Review round: 2
Reviewer: 2

Output:
Basic reporting: I believe that the revised paper entitled as “Phylogenetic comparison between  Type IX Secretion System (T9SS) protein components suggest evidence of horizontal gene transfer (#38397)’’ is very improved.
However, I recommend  the authors to change one thing to improve as shown in general comments for the author.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I recommend  the authors to change one thing to improve.
1. In Figure 1, A-LPS attachment property should be RgpB, not Kgp.  This must be a mistake.


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

Input:
Title: WHOLE GENOME SEQUENCING ANALYSIS IDENTIFIES RECURRENT STRUCTURAL ALTERATIONS IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study uses clear language to introduce the topic, the approach and the findings.

There were a few issues:
1. References are missing for some statements. Examples are lines 52-55 and 111 (R language).
2. The results section includes description and references to the importance/interpretations of the findings which may be appropriately placed in the discussion section (not sure what is the journal policy on this item or how strict they are on enforcing this).
3. Figure 1. Top and right annotation (bar graphs) are missing y and x-axis labels (number of mutations and number of patients). In C, binary variables can be represented by 0/1 or white/black instead of unique colors.
4. Figure S1. doesn’t show place the calling of INDELs in the pipeline. In addition, the figure can be more informative by including the tools that were used at each step.
5. Figures 3A and S3 are uninformative. They don’t clearly (obviously) show the trends described in the text referencing them.
6. Figure 4. Axes labels are not provided.
7. Figure legends: The figure legends are brief and are not self-contained. The reader would have to refer to the methods and the results to be able to read the figures. The legends for the supplementary figures are missing.
8. There is no reference to the raw data. The raw data should be deposited in the appropriate repository and the repository identifiers are listed in the manuscript (check the journal policy).

The authors provided ethical approval statement for the use of data from human subjects and no identifiable info of the subjects are in the text.
Experimental design: The authors defined their research question and documented their approach. .
Here are a few issues and suggestions:

1. The methods used to assess druggable genes are not described.
2. Line 68. The incidence rate and the risk factor association of a disease are presumably independent. This sentence presents an unclear contrast between the two facts.
3. Line 69. The authors can present direct statistics of the prevalence of the ESCC in men and women instead of relying on the mortality rate ration, which depending on how it was calculated it may or may not reflect the prevalence of the disease.
4. There is no statement on the reproducibility of the study. It is on the authors to assess the reproducibility of their study and to guide the reader to the appropriate resources (data/code/documentation) to achieve that level of reproducibility.

More issues with the methods are raised in the context of making certain claims (Validity of the findings).
Validity of the findings: The study provides useful exploration of the mutational landscape of ESCC in a specific population of a japanese city. They identified certain sequence and structural variations in the samples. However, some of the claims in the text went beyond exploration to establish correlation between the disease and the patient characteristics.

Here are two examples and why they might be problematic:

1. Specific claim such as “Enrichment of signature W4 in smokers confirms the carcinogenic role of tobacco smoking in ESCC” are harder to establish in this study for several reasons.
a. The small sample size.
b. The use of alternating logic to associate signatures with patient metadata and similarity with the COSMIC signatures. If any correlation to be established, the signatures and patient metadata from this study and the COSMIC study should be shown. But alternating between the similarity to another study and the enrichment in one study is not enough reason to establish correlations.
c. Other variables (confounders) such as gender, age or drinking were not ruled out (or not shown) as an explanation of the correlations. For example, if most smokers were males or over 60 yo, it is hard to see why maleness or old age is the reason for the apparent correlation.
d. The use of t-test and statistical significance (p-value) is unfortunate. First, the two groups are of unequal sizes (n = 14 and 5). Second, the within group variances of the two groups (although are not explicitly mentioned) look very different from the boxplot (Figure 2C).
e. The claim is contradicted in the discussion (line 312). It says that the signature W4 is only common in male patients, and female patients who had it were non-smokers and responded to chemo unlike their male counterparts.

2. Similarly, the authors claim that certain structural variants are mostly common in male “patients with smoking and drinking (line 234, table S5)”, while others are common in patients “irrespective of the same characteristics (line 236, table S5).” The latter being a no claim is hard to justify. The problem with the former is that most of the patients in the study are males and with history of drinking and smoking. If possible, the authors should support the claim with proper visualization and testing.
Additional comments: No additional comments


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

Input:
Title: WHOLE GENOME SEQUENCING ANALYSIS IDENTIFIES RECURRENT STRUCTURAL ALTERATIONS IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this study Munmee Dutta  et al provide a genomic map of Structural variations, CNV and somatic point mutations in ESCC using 20 Whole Genome Sequencing samples. This is a unique dataset in the Japanese population and the authors have done a commendable job in identifying the key driver Structural Variants and Copy Number events. However, the following caveats need to be addressed and this would significantly improve the findings in the manuscript.
Experimental design: The 20 Whole genome cases will be a good resource for the ESCC genomic community.
Validity of the findings: Major Comments
1)	A new signature associated with smoking has been reported. Alexandrov et al have shown a smoking signature in Lung Cancer. They have further validated this signature in-vitro by exposing cell-lines to Benzopyrene, which has also resulted in the same signature. Given the vast amount of literature available on the smoking signature, Munmee at el should clarify the cosine similarity of the smoking signature. Secondly, alcohol as stated is significantly associated with ESCC. While an alcohol related signature is not reported in COSMIC it was reported by Li et al1 in their recent study of 549 exomes of ESCC. Munmee et al should clearly state how faithfully the alcohol signatures identified in both studies concur in their cosine similarity and 96-trinucletide context profile. Alternative signature extractions such as SigProfiler methods if necessary could aid in this.

2)	Chang et el2 have previously sequenced 94 ESCC cases using Whole genome sequencing and in the Chinese population and identified a total of ~610,000 SNVs in their cohort with a mutational burden of 2.1 mutations/Mb. This is in clear contrast to the ~104,000 mutations identified in the 10 samples. Munmee et al should clarify if these differences are due to difference in disease aetiology or technical differences such as depth of coverage or purity.

3)	While the sample size for driver detection using mutations is small for methods like MutsigCV, methods such as dNdsCV should be able to delineate driver genes from those mutated by chance alone.

4)	Chang and Li et al in their previous publications showed NOTCH1, KMT2D, PIK3CA as the most commonly mutated genes apart from TP53 and FAT1 with frequencies of each gene being mutated being > 10% frequency. However, these mutations are not present in any of the samples in this cohort. Can these differences in mutated genes be attributed to differences in the Chinese and Japanese population at disease presentation? IF mutations are present, showing these driver mutations in Figure 1 is more relevant that showing genes mutated by chance and not conferring the tumors cells any growth advantage.

5)	Cheng et al3 in another WGS study of ESCC have reported recurrent SVs like in this cohort have reported recurrent inactivation of CDKN2A cohort and activations of FGFR1 and CCND1 in their cohort. They report the mechanism of activation of CCND1 is through Breakage-Fusion-Bridge (BFB) and this is clearly seen the WGS data through the stepwise increment in the coverage. In their study, they show chromothripsis as common method of activation of FGFR1. Does a similar pattern of activation found in this cohort?

6)	Chromothripsis, Katetgis, BFB and double-minutes are identified in more than half of the samples in Cheng et al. Have these mechanisms of activation and inactivation’s been identified in your cohort also?

Minor revisions
1)	Gene such as TTN are large and frequently mutated without any known biological relevance due to their size and should be removed from Figure 1A. Driver detection algorithms account for size and such genes are usually excluded.
2)	Samples are referred to as OK007, OK008, etc..(line 208). While Figure 3A has labels like ESCC_007. The label names should be kept consistent.

References
1	Li, X. et al. A mutational signature associated with alcohol consumption and prognostically significantly mutated driver genes in esophageal squamous cell carcinoma. Annals of Oncology 29, 938-944 (2018).
2	Chang, J. et al. Genomic analysis of oesophageal squamous-cell carcinoma identifies alcohol drinking-related mutation signature and genomic alterations. Nature communications 8, 15290 (2017).
3	Cheng, C. et al. Whole-genome sequencing reveals diverse models of structural variations in esophageal squamous cell carcinoma. The American Journal of Human Genetics 98, 256-274 (2016).
Additional comments: No additional comments


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

Input:
Title: WHOLE GENOME SEQUENCING ANALYSIS IDENTIFIES RECURRENT STRUCTURAL ALTERATIONS IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The revised manuscript corrected for most of the issues raised in the review. In particular, some methodological details and missing references were added. In addition, the authors revised several statements in the text that were either vague or unfounded based on their analysis. The issue of reproducibility remains however. While not strictly required by the journal, the authors can still reaffirm their commitment to reproducible analysis by providing 1) links or accession numbers to the deposited data 2) links to the code that was used to perform the analysis and generate the figures/tables.


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

Input:
Title: TIP DATING WITH FOSSIL SITES AND STRATIGRAPHIC SEQUENCES
Review round: 1
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: King and Rücklin present a new module for BEAST2 introducing new operators to update tip ages while accounting for the fact that relative ages of fossils might be known. For instance, two fossils from the same site can have a range of temporal uncertainty associated with the dating of the site but can be assumed to be of identical ages. The new operator constrains the proposal for ages of the two extinct tips to be identical, within their stratigraphic range.

I think the paper is very well written and presents an interesting and useful tool. There are however a couple of things that I would like the authors to clarify about their method. While sampling identical ages for two or more tips from the same site is relatively straightforward, It is unclear how tip ages are sampled in the case of ordered stratigraphic ranges (lines 79-84). In particular, because the update of one tip age is not independent of the other tip ages, it is not clear whether the move is reversible and symmetrical and whether there should be a correction in the form of a Hastings’ ratio. One simple solution would be to

1) draw n ages (for n tips) from a uniform distribution bounded at the stratigraphic boundaries of the section
2) sort them
3) assign them to the tips, sorted by relative age

This move should ensure that the proposal is symmetric and reversible. Maybe this is what the authors have already implemented, in any case I think this point should be clarified in the manuscript.

While I agree with the authors that the possibility of constraining fossil ages based on their sampling site is generally a good idea (in fact my co-authors and I have recently introduced a similar feature in a non-phylogenetic context: doi.org/10.1017/pab.2019.23), I think the authors should briefly discuss the fact that two fossils from same site are not necessarily of identical ages. There are cases in which sedimentation rates are very low resulting in millions of years of evolutionary history being compressed in a few centimeters of rock. Worse even, sediments might be reworked resulting in unclear relative ages of occurrences within a stratigraphic column. I think briefly mentioning these issues a would be a useful cautionary note.

Minor point: Change “I” for “we” in the Abstract.

I hope you will find these comments useful.
Best regards,
Daniele Silvestro


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

Input:
Title: TIP DATING WITH FOSSIL SITES AND STRATIGRAPHIC SEQUENCES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Although reference is made to a github site, it is not clear what is actually available there.

Also, on line 93 the authors refer the reader to methods as used in King et al. 2017.  It is important that the authors report the details of the analysis
as they performed it in this paper (or in Support Mater) and not just refer the reader to another paper.
Experimental design: Methods in King et al. 2017 are not sufficiently described. See above under 1.
Validity of the findings: Conclusions with respect to the test data are perhaps overstated. The authors claim that linking tip-date priors is a good idea, and they provide a way to do this. So far, I agree.

However, their empirical example does not convince me that this linking of priors actually has a significant effect, or is even an improvement.

Brnach support values (posterior probabilities) for Fig. 2B and 2C must be provided.
Additional comments: Line 93. It is important that the authors report the details of the analysis
as they performed it, and not simply refer to King et al. 2017.

Line 104. Mention the Github repository specifically.

The names of the formations from the early Devonian of Spitzbergen in Fig. 1C are not mentioned in the text. Are these the same as MOTH and Turni Hill? It doesn't seem so; there seems to be a mistake here.

Given that Fig. 1C is a histogram and not a probability density function, I don't think it's appropriate to refer to the y-axis as "density."

Figs. 2B and 2C need to have scale bars. Also, B and C are not "cladograms" despite what the
figure legend says. They are chronograms, and support values should also be supplied.

Line 142: "The 50% majority-rule consensus tree for the analysis...". The authors do not state how the trees in Fig. 2B and 2C were produced. Typically, BEAST users use the TreeAnnotator program to produce a maximum clade credibility tree (MCCT). This is not the same as at 50% maj rule, which cannot be produced by Tree Annotator. Please state how
the maj-rule tree was produced, and why should it be preferred over the MCCT.

Line 148: "The results show that linking tip-ages from fossil sites can lead to an increase in the precision of age estimates when compared with independent tip dates"

Line 170: "Linking tip-dates can affect the phylogenetic position of fossils. The affected taxon, Cassidiceps, from the MOTH site, has the oldest age estimate...". The authors argue that the change in the position of Cassidiceps between Fig. 2B and 2C is a significant effect of linking tip-dates. In truth, the difference is minimal, and without branch support values (posterior probabilities), one cannot really assess the importance of the difference.

Fig. 2A. The increased precision that is claimed to result from linking the tip dates is not that striking. Although I think the method in general is important, the actual results in this analysis aren't very convincing that linked tip priors have an important effect, in this case. Nonetheless, I think what the authors propose is an important advance.


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

Input:
Title: TIP DATING WITH FOSSIL SITES AND STRATIGRAPHIC SEQUENCES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well-written and easy to follow along. I only have very minor comments on my reviewer PDF. One thing I think would help out is an additional context figure that compares the stratigraphy/rock formations/ages of the two sites, just to help visualize the issue at hand with the example case. Otherwise, the manuscript is concise and self-contained as they explore their new protocols.
Experimental design: I feel the design was straightforward in the implementation of their new protocols. One thing to consider, is maybe include a second example data set, perhaps one with a wider temporal range on the order of tens of millions of years as the current example is narrower, <10 millions of years.
Validity of the findings: I think the linking of sites and taking stratigraphic ordering into account is a good idea that would be applicable for many researchers that utilize tip-dating methods.
Additional comments: This was a concise read and proportional to what was being explored. I only have minor comments and I look forward to the final publication.


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

Input:
Title: TIP DATING WITH FOSSIL SITES AND STRATIGRAPHIC SEQUENCES
Review round: 2
Reviewer: 1

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


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

Input:
Title: CONFORMATIONAL HETEROGENEITY OF SAVINASE FROM NMR, HDX-MS AND X-RAY DIFFRACTION ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is clear and well written. Need to change line around 35 HDX does not exclusively report on H-bonding. I think there are too many citations, 56 I think for a short paper is too many in my opinion and needs to be focused more on those that are absolutely necessary. I think figure one needs more annotation as it stands it's not possible to see what the authors are referring to in the main text. In particular figure 1b needs a ribbon above it showing the secondary structure (they have provided similar in 2b) and they should highlight residues in the active site. Figure 1c also needs annotation with some arrows pointing to salient structure referred to in the main text. I also think there is a mistake in the gradient bar should go from -8 to 0.
Experimental design: I think the experiments are rigorous and well explained but I find it a shame that the authors have only used HDX-MS to underpin the NMR rather than use the HDX to look for rare states. In the conclusions the authors point to alternative methods that are more sensitive to rare states. However HDX-MS is sensitive to rare conformations but they haven't employed this technique to look for them. The longest labelling time is quite short and I appreciate they have concerns about auto-lysis in the enzyme. But with these short labelling times much of the protein doesn't exchange could there not be some slow EX1 exchange at longer times? Can the authors comment as to why they haven't incubated this protein for long enough to explore EX1 dynamics and potentially unearth a rare state?
Validity of the findings: The findings are not particularly interesting but I still think these types of data have value and have been conducted rigorously. I think the data that has been provided is sufficient to support the conclusions except the lack of long labelling times in the HDX data.
Additional comments: No additional comments