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: OPTIMISED BIOMOLECULAR EXTRACTION FOR METAGENOMIC ANALYSIS OF MICROBIAL BIOFILMS FROM HIGH-MOUNTAIN STREAMS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Manuscript # 48507
Title: Optimised biomolecular extraction for metagenomics analysis of microbial biofilms from high-mountain streams.
Authors: Susheel Bhanu Busi, Paraskevi Pramateftaki, Jade Brandani, Stylianos Fodelianakis, Hannes Peter, Rashi Halder, Paul Wilmes, Tom Battin

	The manuscript is well written - clear, professional and articulate English has been used throughout. In microbial ecology, we still face several challenges in the area of finding/devising efficient DNA extraction methods to study microbial community dynamics in understudied locations, and therefore studies such as this merit the scope of enhancing our opportunities to explore remote and less-represented sites such as the ones represented in this study.

I have the following comments about the manuscript, please see below.

INTRODUCTION:
	Overall, I see a lack of citations throughout the introduction of the manuscript. While authors make several statements to support the goal of their study, they must also cite enough sources to back up their claims. Please see a few suggestions below:

•	Line 42: Please add a few citations for “under-studied environments”
•	Line 46-48: Are there studies/reviews that more descriptively talk about this? Please include a few citations not just to support this statement, but also to help readers refer back to more detail on this should they wish to.
•	Line 73-75: Please add citation(s).
•	Line 75-77: add citations.
•	Lines 82-83: needs a reference, I am aware of this 50 ng cutoff, there must be some literature on this, just cite a few here.

	Lines 86-93: There’s no need to include results in introduction. Authors should rephrase this paragraph so that this reads to include the goals of your study based on the background and rationale they’ve have provided earlier in the introduction. Results should go under the separate section.

 METHODS:
	Lines 98, 99, 100 – What is CBS, FE, FEU, FED? Please elaborate of abbreviations on first use.
	Line 98: What is a.s.l.? Please elaborate on first use.
	What are the dates and times of sampling? Please add this to sample collection methods section.
	Line 103: What kind of sampling equipment was used?
	This is a comment for ALL the four DNA extraction protocols the authors have included under the methods section: it seems like all of these protocols have been adapted from already existing protocols or commercially-available kits. Authors mention that they’ve modified these existing methods. Since this is methods paper, it is very important to specify in detail the steps that have been specifically modified in your own procedure.

	Line 130: Mention the equipment used for agitation.

	This is also a comment for ALL the extraction methods: Did the authors start with the same weight of sample for all extractions? If not, a systematic and scientific comparison of the methods is not possible. Authors mention between lines 222-224 and in Figure 1 that 5 g was used for all. In the methods, maybe a range should be used. In line 119, it is mentioned that 0.5 g of sediment was the starting sample, whereas in line 128, it is mentioned that 5 g of sediment was the starting material. While there’s no mention of the amount for method 3, method 4 in line 144 again states that 5 g of sediment was the starting material.

	What kind of normalization was achieved for this comparison? In other words, what amount of DNA was the starting material for the DNA sequencing from each of the methods? Authors should mention this in their methods. You could add a line or two in line 158 to address this issue. As this is a methods paper, simply saying ‘sufficient” DNA was obtained is not enough. What were the concentrations obtained based on the starting material? This is could also be provided as a short table to enhance the understanding for the readers.

	How did the authors address contamination issues? For a methods paper concerning DNA extraction, this must be included in methods for readers who might be following your protocols later.

	What were the controls used during the extraction procedures? Please add this to methods.

	Figures 2 and 3 are images, they could be combined together to form one single figure. This helps to make the manuscript concise and precise.

	Figure 2 shows the bands from only one of the extraction methods. Could the authors provide a comparative yield image of all four methods that has been started with the same amount of sediment?
Experimental design: please see my entire review under BASIC REPORTING section
Validity of the findings: please see my entire review under BASIC REPORTING section
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: OPTIMISED BIOMOLECULAR EXTRACTION FOR METAGENOMIC ANALYSIS OF MICROBIAL BIOFILMS FROM HIGH-MOUNTAIN STREAMS
Review round: 1
Reviewer: 3

Output:
Basic reporting: line 42- sentence starting "among the latter" is awkward. Perhaps add "Among the latter include..."

otherwise all is OK.
Experimental design: line 119 - please clarify - these were the beads that are already in that tube? Please provide manufacturer or kit name, only SKU number is listed.

line 136 - list manufacturer of power soil kit. (note, this kit has changed components when MoBio sold it so be clear as to which company it came from)

line 138: what is meant by "elaborate"? Are you just following kit protocol?

line 143 - you switch tenses here. All other methods were past tense.

line 154 - use a method "like the Qubit" or did you actually use a Qubit

line 173: when was quality control of the reads performed?

at the end of methods, you mention a "mock community" yet the methods to create this mock community are not described.

In the results, line 257 you describe a magnetic bead clean up, but this was not clear in the methods.

line 300 - how exactly did you utilize this mock community? Did you add it to sediment and then extract? This needs a section in the methods.

line 347 - please show the data. Considering it comes this late in the manuscript, you could even leave this sentence out.

Figure 3 can be moved to supplemental. It is not informative to your findings.

Figure 4- I'm confused on what 100% success means. Please clarify your units. Do you mean micrograms in resulted in the micrograms out of library prep?

Please provide more detailed information on exactly how many times each condition was tested on the same material.
Validity of the findings: All findings seem valid, details just need to be more clearly explained.
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: OPTIMISED BIOMOLECULAR EXTRACTION FOR METAGENOMIC ANALYSIS OF MICROBIAL BIOFILMS FROM HIGH-MOUNTAIN STREAMS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have made all changes based on my previous review suggestions.
Experimental design: NA
Validity of the findings: NA
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: OPTIMISED BIOMOLECULAR EXTRACTION FOR METAGENOMIC ANALYSIS OF MICROBIAL BIOFILMS FROM HIGH-MOUNTAIN STREAMS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: looks good!


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: EFFECTS OF DIFFERENT DRYING METHODS ON SMEARS OF CANINE BLOOD AND EFFUSION FLUID
Review round: 1
Reviewer: 1

Output:
Basic reporting: The writing was relatively clear and supported understanding of the tables provided. In a  few sections the writing was slightly awkward, but not enough to warrant revision.

One major failing is that very few references provided. Only two of the five references were from primary journal articles (and one of these was from 1984). Support is needed for the rationale behind study and discussion of the findings in the report.

The article was structured adequately and orderly, however, another major failing of the article is that a large portion of the discussion was simply restating the results (lines 189 to 204) as opposed to critically discussing them. Proper discussion with possible reasoning behind the positive and negative results (preferably supported by literature) is required. If no literature is available in either human or veterinary medicine to support these results, then this needs to be addressed.

The experiment is self-contained and the results are relevant to the hypothesis.
Experimental design: The article does appear to provide primary research within the aims and scope of this journal.

A failing of the paper is that the rationale behind the research is not well-defined. Lines 45-46 indicate that "anecdotally" there are varying opinions, which is insufficient justification for the research. The outcome of a non-diagnostic or incorrectly interpreted sample should be discussed in the introduction (i.e. cost to the client, possible consequences of incorrect diagnosis) and supported by literature (either human or veterinary articles). Are there any primary articles that support not using heat fixation because of adverse effects on morphology? Are there any standardized procedures in human medicine for drying slides?  Further investigation into the literature is required to support the rationale behind this study. If there are few to no articles supporting the rationale, this needs to be explained more clearly.

The "Materials and Methods" section also raises some concerns. The duration/time period of study enrollment should be provided. Addressing the reason for the low sample size may also be useful, as the samples (at least blood samples) should not have been difficult to obtain from a clinical pathology laboratory. There is no information on how the "healthy" dogs were assessed to be healthy (i.e. just physical exam, CBC, biochemistry?). The exact number of pleural vs peritoneal effusions should be mentioned, even if they do not directly impact the results. Line 69 should provide a reference to the website as opposed to a direct link.

Lines 71-73 says the slides were prepared "using constant standardized materials and methods" - for blood smears this is likely adequate, but fluid preparation may not be standardized between labs (i.e. in some labs a cytocentrifuge preparation is standard for fluid samples and others will use sediment smear preparation or line smears) - the exact method of slide preparation for these samples should be noted. Similarly, there is no "routine" staining method with Romanowsky-type stains (lines 83-84) - the exact time in fixation, solution I, and solution II should be noted - if not standardized between slides, this should also be noted.

The remaining methods were straightforward and well-described. The scoring system was described well and information for statistical review was provided.

The raw data table is difficult to understand. Are the scores the “average” score from all 4 or 5 evaluators? Or did each evaluator only get 14-15 slides to review? If the latter is the case, this should be noted in the materials and methods and the evaluator should be identified for each slide (i.e. evaluator 1-5).

The rationale for why the reviewer who incorrectly scored the slides was not asked to review the slides again or replaced with another reviewer should be provided.
Validity of the findings: The study, as described in the paper, appears to be novel and has practical implications (though these implications are not currently described in the paper). Although the results are relatively minor, they would still be a meaningful addition to the literature, especially considering this topic is rarely investigated.

I trust that the statistics were performed correctly, but am not going to investigate them thoroughly (especially given my concerns regarding the raw data). The tables are reasonably straightforward. It is understandable that the WBC table was omitted as it did not show any significant findings.

The conclusions are adequate for the findings of the paper - however, as mentioned above, further elaboration on these results should be addressed in the discussion.
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: EFFECTS OF DIFFERENT DRYING METHODS ON SMEARS OF CANINE BLOOD AND EFFUSION FLUID
Review round: 1
Reviewer: 2

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: EFFECTS OF DIFFERENT DRYING METHODS ON SMEARS OF CANINE BLOOD AND EFFUSION FLUID
Review round: 1
Reviewer: 3

Output:
Basic reporting: I was interested to read this manuscript and have always wondered how the various drying methods and or environmental conditions may interfere with interpretation of blood smears, however I was unaware that a heat block method is still used.
Your style, presentation and English is generally clear, and you hypothesis is clear. Your introduction and literature review is sparse, there is no mention of echinocyte morphology despite scoring of echinocytes, there should be some mention of why these cells are important using journal article references as opposed to textbooks. There may be no published reports on drying methods but there must be reports on the type of artifacts seen in cells and their significance/cause.
The raw data is shared, but needs more explanation. Are the scores an average or mean of the all investigators, are they a cumulative score? it is not specified. Can the raw data be presented in a way that divides the healthy and sick dogs?
The structure of the article and tables is good, but there is no need to present the data from all 5 reviewers in Table 2 when the data was deemed to be unreliable.
Experimental design: The research question is well defined and meaningful, but maybe there should be more explanation in the introduction about why distinguishing artefact from pathology in morphology is so important.
Line 71: were all smears made by the same person (or equally trained and experienced people)  to have consistent quality and not introduce another confounding factor that may affect morphology?
Line 80 was there an average time to drying with the methods, as environmental factors can effect drying time (humidity).
Line 87: were the reviewers blinded as to health and illness?
Line 96: I am interested why the reviewers made comments, as presented in the raw data, but that data was not reflected on or appear to be analyzed. What was the purpose? How was it used?
Line 100: I a curious how you modified Weiss's scoring as the journal article did not score artifacts from what I read, or did you use it only for the scoring of the echinocytes. If so the reference should be with the RBC echinocytes not with the artifact scoring.
What was your definition of an artifact, or was that left up to the reviewer?
Validity of the findings: Underlying data were provided, but not all data was used (see comments above). I am concerned about the use of the alpha value of 0.5 as being acceptable (line 121,183), most papers (see Bland and Altman, Tavokol) indicate that anything <0.7 is of questionable value for internal consistency, the minimum recommended is between 0.65-8 with >0.9 being the best result. Using this value the WBC are of questions significance even after eliminating the one reviewer.
Line 159: the raw data for the effusions does not appear complete, as only a morphology score is presented, not raw scores for RBC's or WBC'S, and most comments are low cellularity and 'difficult to evaluate cells'. These comments would make any assessment of the data suspect, maybe data should maybe presented in a descriptive way as any conclusions bases on statistics may not be reliable or representative based on the sample quality.
In the conclusions it is stated that the difference in health and heat block is due to a type 1 error. Do you have any other speculation? There is much mention in the raw data of 'smudge cells', were these found in the healthy dogs due to lipemia? if so, there may be another reason that does relate to health on dogs indirectly, and that might be if all samples were not consistently fasted. This aspect should be considered.

The limitations were clearly outlined, and the conclusions are likely valid for the red cell smears, but are questionable for the effusions smears.
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: EFFECTS OF DIFFERENT DRYING METHODS ON SMEARS OF CANINE BLOOD AND EFFUSION FLUID
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Most concerns from previous review have been addressed and manuscript appears ok for 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: EFFECTS OF DIFFERENT DRYING METHODS ON SMEARS OF CANINE BLOOD AND EFFUSION FLUID
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment, see below
Experimental design: no comment, see below
Validity of the findings: no comment, see below
Additional comments: Thank you for the replies, corrections and additions to the queries raised. The supplemental data is easier to understand with the lay out and the additional data. Most of the reviewers and editor’s comments were addressed. There are still a few that may require some additional comments.

Line 50 There may not be information directly related to blood smears and heat fixation but there are papers that look at the morphology of bacteria, red cells and cell morphology with heat fixation vs methanol fixation from Gram stains (in 1980’s) and more recently papers that looked at ear cytology without and without heat fixation.

Line 106-108 In the methods you requested the evaluators make subjective observations on the smears and the information is presented in the supplementary data. I realize you mention in the discussion how this was in case you wanted to analyze the data at some later point, but since there are instructions to do this and the data is presented, it should be analyzed in some way, or not included in either the methods, supplement or discussion.

Line 112. It would be helpful for you to define what you think is an morphologic abnormality due to or suspected to be artifact of drying, as in the discussion you mention this again with no indication of what this is but leave it up to the evaluator.  People differ on what they associate with an artifact of drying vs smears making etc (essentially you have that information in the subjective observations). Not everyone reading this manuscript will be clinical pathologists. some may be student/researchers in other fields making smears but not realizing what artifacts are.

Line 244-, the sentence seems out of place and might be better suited to be modified to fit into the prior section on echinocytes Line 225


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 OSTEOLOGY AND PHYLOGENETIC POSITION OF THE LORICATAN (ARCHOSAURIA: PSEUDOSUCHIA) HEPTASUCHUS CLARKI, FROM THE ?MID-UPPER TRIASSIC, SOUTHEASTERN BIG HORN MOUNTAINS, CENTRAL WYOMING (USA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This mauscript  is very important for better understanding of archosaurs evolution and biostratigraphy. My main suugst is to add new figure with reconstruction of the skull in lateral and dorsal views.


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 OSTEOLOGY AND PHYLOGENETIC POSITION OF THE LORICATAN (ARCHOSAURIA: PSEUDOSUCHIA) HEPTASUCHUS CLARKI, FROM THE ?MID-UPPER TRIASSIC, SOUTHEASTERN BIG HORN MOUNTAINS, CENTRAL WYOMING (USA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript covers all four of the basic reporting areas.
This paper is an important contribution to the understanding of paracrocodylomorphs archosaurs, and subsequently, the distant ancestry of crocodylians. It is nice to see a thorough description of this taxon finally! Generally speaking, the paper is well written and clear. I have annotated the word version of the paper.
The paper reflects a current understanding of this group of reptiles with relevant literature cited with very few exceptions (see line 120).
There are some grammatical and punctuation issues (e.g., see lines 70, 286, 540, 574, 586, etc.). And I have made some suggestions here and there (e.g., lines 326, 556, 595). The figures, for the most part, look fine; however, there are a few formatting errors with some of the fig. #’s (e.g., see lines 260-265).
Experimental design: Materials and methods are fine. The description is thorough and the phylogenetic analysis has been built upon previous analyses with the results congruent with current thinking.
There are a few characters that may benefit from a closer examination (e.g., lines 441, 563). One of the characters consistent in your analyses, is the "fused interdental plates" being present in Postosuchus. As I mention in a comment in your paper, can you better differentiate what you mean by this, since you can make out the separate plates in both TTU P 9000 & 9002?
Overall, the methodology and characters used in the analysis are in line with that of other researchers within the field and should be easily replicated.
Validity of the findings: As mentioned above, a few of the characters should be reassessed (e.g., lines 441, 563), but most likely this will not dramatically change the topology of the provided trees.
The conclusions follow the results of other authors and seems to place Heptasuchus in a taxonomic position that makes sense, given understanding of known taxa.
As stated by the authors, this taxon fits into a little understood gap of geologic time in North America, so any new information is welcome, and increases the significance of this paper.
Additional comments: Overall, a nice, thorough description of the known Heptasuchus material. Please see comments throughout the attached word file. I have attempted to fix some minor errors, a few of them stylistic, so take that as you will, and some suggestions to make the paper flow better. Several, just grammar and punctuation. There are some missing words and other minor things, but generally speaking, a welcome addition to the literature on paracrocodylomorph archosaurs. Would of course be nice to better constrain the time of deposition of the fossils, but it's nice to see more careful consideration of the relationship of this material to the Popo Agie Fm.


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 OSTEOLOGY AND PHYLOGENETIC POSITION OF THE LORICATAN (ARCHOSAURIA: PSEUDOSUCHIA) HEPTASUCHUS CLARKI, FROM THE ?MID-UPPER TRIASSIC, SOUTHEASTERN BIG HORN MOUNTAINS, CENTRAL WYOMING (USA)
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall this is an important paper, it’s amazing that this fossil has escaped further serious scrutiny since its first publication 40 years ago. Overall, the paper reads well, but there are numerous minor grammatical points that require repair. I think most should be obvious from the uploaded copy of the manuscript, but feel free to contact me if not.

The single most pressing need is to universally replace the many instances of “kuperferzellensis” with “kupferzellensis,” the correct specific epithet for Batrachotomus. Both are used throughout the text.

Some other issues I would suggest:
Figure 1—the map really is an outcrop or bedrock map of the Chugwater, not a map of
“surface deposits.” Perhaps “Distribution of Chugwater Group strata.” Also, I’d like to see the age of the Gypsum Spring Fm indicated.
Figure 2 would benefit greatly from some indication of scale, which should be easy to extrapolate from Figure 4.

Generally speaking, I would like to see more call-outs to the figures in the text, including at the beginning of the Systematic Paleontology and at least once a paragraph when dealing with the individual bones, especially the various minute processes or other details that may not be labelled. Some indication is provided on the attached copy of the manuscript.

The Jelm is a nefarious beast that is remarkably understudied (words to that effect in the following), but it is a more recent and more synthetic treatment that I think is worth citing.

 Blakey, R.C., Peterson, F., and Kocurek, G. 1988. Synthesis of late Paleozoic and Mesozoic eolian deposits of the Western Interior of the United States. Sedimentary Geology, 56(1-4):3-125.

For completeness’ sake the authors may wish to include Lucas (1994) for discussion of the Triassic of Wyoming.

Lucas, S.G. 1994. The beginning of the age of dinosaurs in Wyoming. Wyoming Geological Association Guidebook, 44:105-113.

References:

Somehow Gower (1999) escaped the bibliography

Gower, D.J. 1999. The cranial and mandibular osteology of a new rauisuchian archosaur from the Middle Triassic of southern Germany. Stuttgarter Beiträge zur Naturkunde Serie B (Geologie und Paläontologie), 280:1-49.

References in the text are to Bittencourt et al. (2014) but in the references is Bittencourt et al. (2015).

I don’t believe Brusatte et al. (2011) is cited in the paper, but is in the references. If cited, capitalize and italicize Ctenosauriscus.

I don’t believe Colbert (1952) is cited in the text, but is in the references.

The text refers to both Nesbitt & Desojo (2017) and 2018, but only 2017 is in the text

Line 135—High and Picard (1965) is listed as High and Picard (1969) in the references. Lines 158-159 do refer to H&P 1969, so either H&P 1965 is missing or misidentified.

Johnson (1993) does not appear to be cited and appears to be an incomplete citation of a USGS publication.

Maddison & Maddison (2015) does not appear to be cited.

Line 505—Nesbitt & Desojo 2018 is probably 2017, or else needs to be added to references.

Lines 1681 and 1722—Schoch & Desojo is 2016, not 2015

Line 992—Sill is 1974, not 1971,

Finally, within either the acknowledgments or the materials & methods the authors may wish to acknowledge the preparation methods used (and/or the preparators), as I do think that this has long-term benefits.
Experimental design: This was, of course, well-executed and probably the highlight of the paper. It is great to see this fossil documented so thoroughly and in a modern phylogenetic context. This longer contribution is the kind of thing that PeerJ is well known for and this paper is certainly a valid contribution.

I would like to see a few more of the features discussed in the text identified on the figures and, as noted earlier, some more call-outs so that the reader always knows which sub-figure is best for some of the details that are perhaps difficult to label.
Validity of the findings: Overall, I find all of this readily acceptable, it is a thorough revision that clears up a taxon about which we know too little. The necessary data are present and robust.

Two points require clarification, one minor and one major.

Minor clarification: The abstract and Line 301 indicate that there are four individuals based on ulnae, but Line 948 indicates that the MNI is 3 based on the ulnae, so this needs to be clarified.

Major clarification:

The only part of this manuscript I would like to see again is the text “The phylogenetic position of Heptasuchus clarki among archosaurs,” specifically lines 1150-1170. To me the paragraphs beginning at 1150 and 1159 both seem to be indicating a list of character states supporting a sister-group relationship between H. clarki and B. kupferzellensis. I suspect that the paragraph at 1150 is actually listing states that support inclusion of both taxa within Loricata—and I also find this a little less than convincing as H. clarki is only scored for 2 of the 6 characters listed.

I absolutely do not think that this requires re-review, but I would appreciate it if the authors could generate revised paragraphs for this section that clarify their results.
Additional comments: All told I think this is valuable contribution that really only requires a couple days' work to be publishable with minor revisions.

In addition to the (mainly) minor points listed previously, I think the following will dramatically improve the paper for relatively little effort:

(1)	Please include a larger view of the reconstructed skull as part of Figure 3B. While it may be tentative, that will significantly increase the impact of the paper. I think this is valid even if it’s only slightly redrawn from Gower (1999).
(2)	Figure 8 is entirely too small, I assume it will be two columns in the final.

(3)	Figure 9 is also pretty small—it could easily be larger as a 1-column figure (see attached), but even if it’s landscape format, the bones could be larger.


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 OSTEOLOGY AND PHYLOGENETIC POSITION OF THE LORICATAN (ARCHOSAURIA: PSEUDOSUCHIA) HEPTASUCHUS CLARKI, FROM THE ?MID-UPPER TRIASSIC, SOUTHEASTERN BIG HORN MOUNTAINS, CENTRAL WYOMING (USA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Generally, the paper is fine and most issues have been resolved from the initial review. There are a few mostly minor grammatical and punctuation issues, however and only a couple of questions, e.g., see line 990 regarding the auricular recess.
Experimental design: The experimental design is perfectly fine.
Validity of the findings: All good here.
Additional comments: As previously stated, the paper is a great addition to archosaur literature. It reads much better now. I found a few more grammar/punctuation things mentioned in the attached word file. Otherwise, it's a very nice paper.
I do want to let you know, due to not being able to just upload the word file with track changes, some of the minor punctuation stuff is really tough to catch, I could see a few things you missed after the initial review. Otherwise, great!


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 BACTERIA GENOME PIPELINE (BAGEP): AN AUTOMATED, SCALABLE WORKFLOW FOR BACTERIA GENOMES WITH SNAKEMAKE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The pipeline put forward by authors seems mostly suitable for the task at hand, which is assembly and visualiation of monomorphic bacterial WGS data. My main concern was that it did not take into account recombination for phylogenetics, which is very important for many bacteria. Authors state at the end of the discusison that the pipeline is only suitable for monomorphic bacteria: this should be clearly stated much earlier on, including in the abstract, so readers do not think this approach will work for bacteria that undergo any level of recombination.

Similarly, the abstract and the instriduction state "However, there is the need to analyze multiple genomes within a short time, in order to provide critical information about a pathogen of interest such as drug resistance, mutations and patient-to-patient transmission in an outbreak setting". I don't see how this pipeline addresses this need, as it does not do drug resistance prediction or transmission analyses, only SNP visualisation and phylgenetics. Thus, the need for another wrapper of existing software needs to be more clearly stated so the reader can better decide why to use this pipeline over a rival such as MTBseq or Nullarbor.
Experimental design: I was not able to install the pipeline on either an OSX laptop or a Linux sever. For both it hung at the 'solving environment' stage of the conda command. After 1 hour of wiating I canceled the install.
Both the laptop and the server have regularly used conda installs and others were checked afterwards to ensure it was the package and not the conda or hardware. This should be fixed so the software can be evaluated.

Authors state that the software has only been tested on a linux machine, yet say the target audience is LMIC researchers and those using a mid-range laptop. I would posit that most researchers that fall into this category are not using a Linux machine and thus the software should be tested on other platforms as well, or at least give instructions or pointers on their github to how to install virtual boxes or similar.

Line 123-125 states that dependancies come from bioconda but a bash script is needed for the R libraries. R libraries can be installed through conda so I dont see why this is 2 steps, especially if the authors are putting this forward as a '1 install/pipeline only' solution.

Similarly, I am unsure why the authors have a git install that then requires a conda environment for use, and ask that R already be installed. If using conda, they should use conda throughout the installation as a single command (e.g. conda install BAGEP), maybe from the bioconda channel, which would install the pipeline and the associated dependancies, including R and the R libraries. As it stands, it is not a single command install, which is what snakemake and conda were made to do.

Methods and github page do not indicate if parameters can be changed, such as model of evolution for IQ-TREE or cut-off parameters for snippy. This would be important as different bacteria require different parameters for phylogenetics. Without the ability to change IQ-TREE parameters, I see this as a massive drawback.
Validity of the findings: No comparison to existing software was undertaken. Authors state that MTBseq installationw as tried but failed. However, this is an M. tuberculosis-specific pipeline and not a general bacterial pipeline, which is what authors should be comparing to. The most obvious candidate for comparison would be nullarbor (https://github.com/tseemann/nullarbor) which undertakes most of what the authors are doing here, plush additional tasks and is created by the author of snippy. A comparison to this, to see the need for such a competing pipeline, should be done.

It has been shown previously that removal of contamination from read sets before mapping is required to reduce false SNPs for phylogenetics and other tasks (see Goig et al https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-020-0748-z). This pipeline does not do this, and thus may gibve false results to the user. Authors should indicate why this is omitted or add a step that does this., However, contamination removal unfortunately tends to require large databases (this is why UVP is so large, as the authors state), so is incompatible with a laptop setup for such pipeline, which is one reason why such pipelines dont exist. Even still, authors need to justify why their pipeline doesnt do this, which likely will increase the rate of false SNP discovery.
Additional comments: I think the technological gap the authors are trying to fill here is admirable and necessary. Indeed many LMIC are being left behind in the WGS era because much analyses require high performance computing and standalone pipelines that are pathogen specific (see https://pubmed.ncbi.nlm.nih.gov/31209399/ for a discussion of this for M. tuberculosis, for example). I agree that a pipeline that bypasses this is required. However, there is always pitfalls in trying to create cross-pathogen, low resource pipelines, including omission of heavy processing steps (like contamination removal) that tend to still be very necessary, and not undertaking pathogen specific steps (like rtecombination removal or masking of repeat regions, which both have massive impacts on downstream analyses).
Thus, I think the idea for such a pipeline is comendable, it is tough to justify how this one addresses that issue and wont just give incorrect results due to the generalisability and low resource setup. Authors need to address these points to ensure incorrect results from their pipelines are not perpetuated in the resource community.


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 BACTERIA GENOME PIPELINE (BAGEP): AN AUTOMATED, SCALABLE WORKFLOW FOR BACTERIA GENOMES WITH SNAKEMAKE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Introduction
Line 69. “as next generation sequencing gradually replaced Sanger sequencing”. NGS do not replaced Sanger sequencing. WGS is very useful for large scale works as epidemiology, infection control, contact tracing, surveillance etc, however most of research does not require WGS and Sanger sequencing is still an important tool for research. One of the problems of WGS are the ambiguous results due to sequences of low quality that can result from several incorrect procedures that frequently are solved/verified by Sanger sequencing. I suggest rephrase this statement as it can induce the readers in error.
Materials & Methods
In the MM should be included information about the genome sequences used to evaluate the pipeline. Their origin (published sequences or not – it is in the beginning of results but that information could be added here for contextualization), method used for DNA extraction (no need to be detailed), if the DNA was obtained from cultures or direct from clinical samples, were the sequences are deposited (database) and their accession numbers and study accession. Patient information should not be included.

Other remarks
Designation of the species: some inconsistency was observed. The species need to be mentioned in full at the first time in the main text (excluding abstract). The same for Salmonella. In the middle of the results appears MTB; should be maintained the terminology used from the beginning. If want to designate the species name abbreviated, please change at the very beginning.

MTBseq and the remaining pipelines/software’s – without italic please; there is no need. Italic should be reserved for the name of the bacterial species.
Experimental design: No comment.
Validity of the findings: The article meet PeerJ standards, with suggested improvements.
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: THE BACTERIA GENOME PIPELINE (BAGEP): AN AUTOMATED, SCALABLE WORKFLOW FOR BACTERIA GENOMES WITH SNAKEMAKE
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1. Authors should acknowledge, more recent work in the filed. For example when referring to GWAS (lines 92-93), Farhat et al, 2019 (Nature Communications).
2. The figures are in low resolution (for example the text in y axis is overriding itself in figure 2), and I would use better representation for SNPs along a chromosome (see example in Liu et al 2020, Scientific Advance).
3. Results does not demonstrate improvement in performance when compared to existing tools.
Experimental design: - When developing a new computational research methods / tools which aim to out-preform other existing methods, it is essential to compare performance in terms of accuracy, memory usage, running time, etc. This was not done here.
- Bacterial clinical samples tend to contain non-target contaminations, which generates noise and hampers downstream analyses. In order to address such contaminations, there are quality control measures that are used (example can be Centrifuge by Kim et al, 2016 Genome Research). Such measures are not included in the BAGEP pipeline.
- Details and instructions for installation of BAGEP are only referred to a git page, which should also be included in the MS.
Validity of the findings: - The data sets used for demonstration of the applicability of the developed tool is too small to make a good assessment (n=20).
Additional comments: The idea of making a 'light version' tool for WGS data of bacterial genomes is very compelling. There is a clear need for providing user friendly tools with minimum hardware requirements and high performances, and therefore I command the authors for giving it a try. I think the BAGEP tool has great potential and will help scientists in low income settings, however the MS lacks basic features for publishing a new computational pipeline.


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 BACTERIA GENOME PIPELINE (BAGEP): AN AUTOMATED, SCALABLE WORKFLOW FOR BACTERIA GENOMES WITH SNAKEMAKE
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: All my comments have ben addressed sufficiently.


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 BACTERIA GENOME PIPELINE (BAGEP): AN AUTOMATED, SCALABLE WORKFLOW FOR BACTERIA GENOMES WITH SNAKEMAKE
Review round: 2
Reviewer: 2

Output:
Basic reporting: Minor corrections to the text:
L89. Please revise “single nucleotide polymorphisms (SNP)”
L90. Please revise “(indels) and/or translocations.”
L91. Please revise “ways to detect variants”
L92. Please revise “genomics of bacterial isolates”
L93. Please revise “bacterial species”
L94. Please revise “to understand evolution of bacterial genomes”
L98. Please revise “and/or”
L102. Please revise “bacterial genomes”
L110. Please revise “who are becoming users of WGS of pathogens”
L111. Please revise “also to provide”
L115. Please revise “income countries (LMICs)”
L180-181. Reference year are in bold. Please revise
L199. Please revise “The great advantage about Abricate”
L270-271. “This pipeline will be useful for researchers in low-to-middle income countries and people with little or no bioinformatics skills in analyzing raw genomics data.” I think that this software will be useful not only by the reasons pointed by the authors but also for those that have bioinformatics skills but no time available to perform such laborious analysis. As an example, prefer to outsource this kind of services to have the complete genome sequence due to lack of time. If this software can reduce the time spend for assembly and annotation making the process user-friendly will be an add on to this area leaving more time for other tasks. sentence could be improved in this sense.
L278-280. This sentence is repeated in line 270-271. Please revise.
L288. Please revise “Salmonella enterica serovar Typhy”
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: THE BACTERIA GENOME PIPELINE (BAGEP): AN AUTOMATED, SCALABLE WORKFLOW FOR BACTERIA GENOMES WITH SNAKEMAKE
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Authors satisfied my concerns raised in the first round of review.


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 SIZE OF SIGNAL DETECTION AND EMISSION ORGANS IN A SYNCHRONOUS FIREFLY: SEXUAL DIMORPHISM, ALLOMETRY AND ASSORTATIVE MATING
Review round: 1
Reviewer: 1

Output:
Basic reporting: L53-55, please specify if these species have synchronous or asynchronous males

L59, I agree that synchronous species are good subjects due to the density of signaling males. But it would be helpful to discuss the downside which is that males with a smaller lantern might be able to exploit larger neighboring males without paying the severe metabolic costs.   Also at line 62 aren’t the authors assuming that there is no pre-copulatory courtship or cryptic female choice by the females, as discussed by Eberhard and others?

L72, change for to of

L82, at first I agreed that selection should favor males with larger eyes for improved detection ability but then I wondered more about the natural history of how far a male can or will fly to try to encounter female:   larger eyes should capture more photons meaning a male could detect a female at a greater distance.   However under intense competition, by the time he actually arrives, another male closer by may have already gotten there.  In synchronous species presumably females are not responding to just one particular male, but to the collective source of light (followed by scramble competition among males to get there first).  If so, males be selected to respond via controlled flight to point sources only within a certain range, which may be a constraint on eye size enlargement.

I wonder if the situation might be similar to some deep-sea fishes that, if I recall correctly, have relatively smaller eyes, which is hypothesized to be associated with being relatively weak swimmers (see Warrant and colleagues, Biol Rev 2004).  It may be unfair to claim that fireflies are relatively weak flyers but I think the argument is at least worth considering, as it would flip the prediction the authors make.

L121, it was not clear to me how female body size was estimated given size was based on an estimate of elytra length IF the elytra were not reduced, but how can this be known?

L125ff,  it would be useful to show that there are, or are not, allometric relationships between body size and eye size, and lantern size.

L154,  I am not persuaded these data showing female elytra are shorter than those of males are worth presenting since we already know they are brachyopterous.

L190, in discussing support for their hypothesis, the authors seemingly jump from intra-specific comparisons to inter-specific comparisons.  That is, they set up to study by predicting that males with larger eyes and or larger lanterns should be found in copula than males with smaller structures.  They found no evidence for this. But then these inferences really are inter-specific, showing that males are larger bodied, and have larger lanterns, than females.  To fully substantiate the claim that it is due to sexual selection requires out-group comparisons, showing that the derived species have larger lanterns, for example.  This should be clarified (similarly, for female eye size line 194).

L197,  here the authors need to again clarify which species are synchronous and which are asynchronous, as  I think the predictions differ.

L204, as above is this species synchronous or asynchronous?

L211, the observation that most meetings involve solitary males contradicts the notion of intense competitiveness described in line 205.  Please reconcile.

L228, correct spelling of species name

L 233, fix typo in Laughlin

L239, change webbing to web (also L 252)
Experimental design: No comments in addition to the above
Validity of the findings: No comments apart from the above
Additional comments: I enjoyed reading this manuscript very much, and found the study well done, interesting and compelling.


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 SIZE OF SIGNAL DETECTION AND EMISSION ORGANS IN A SYNCHRONOUS FIREFLY: SEXUAL DIMORPHISM, ALLOMETRY AND ASSORTATIVE MATING
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors use clear and unambiguous, professional English throughout their work and provide literature that is mostly sufficient to support their statements. However, I have noticed gaps concerning their cited literature works in support of setting up their study and formulating their hypotheses which may potentially question their  justification of the study. Please see my comments below, “Experimental Design.” The structure of the article is professional, and raw data are shared. However, R scripts to replicate their analysis are not supplied which is a shortcoming that must be addressed.
Experimental design: The paper represents original research within the aims and scopes of the journal. The authors identify a gap of knowledge about firefly mating systems and underlying mechanism, but the main research question is not well defined and perhaps may not even be meaningful.
The authors must explain how eye size is linked to detection ability of bioluminescent light flashes. Following Land and Warrant’s extensive body of work on physiological modeling of visual light sensitivity, it is primarily the absolute size of the optical aperture which determines the ability of the eye to detect point light sources. Of course, the size of the aperture will be constrained by the size of the eye and animal, hence one would need to look at absolute aperture size and relative aperture size. Eye size, as used by the authors, may not be meaningful in the detection ability of point light sources such as bioluminescent light flashes. In addition, the authors must justify the use of lantern size as a proxy of emitted light intensity of the signal and provide evidence that the size of the lantern organ is indeed positively correlated with signal intensity.
As for the methods, I notice that R scripts were not supplied, which is not conducive to reproducing the statistical analyses. The authors should share their scripts for full transparency. In addition, the chosen methods are far from ideal. Methods should explicitly address body size as a covariate, hence the analyses should focus on ANCOVAS. ANCOVAS are much more “direct” than PCA and functionally more relevant, because the loadings of the PC represent a mix of the traits. It is therefore difficult to explicitly ascribe a function to the loadings. ANCOVAS also allow for using scaling information as an assessment of selection, such as isometry vs allometry.
Validity of the findings: The validity of the findings largely hinges on the appropriateness of eye size as the proxy for ability to detect point light sources. This is where the authors need to deliver more information and justification.
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: THE SIZE OF SIGNAL DETECTION AND EMISSION ORGANS IN A SYNCHRONOUS FIREFLY: SEXUAL DIMORPHISM, ALLOMETRY AND ASSORTATIVE MATING
Review round: 1
Reviewer: 3

Output:
Basic reporting: Basic reporting is sound and clear and paper is well written. Publishing negative results is important if they are based on reliable data and scientifically sound hypotheses. In the present state, the paper does not reach scientific standards. This paper could become scientifically sound after rewriting and reanalyzing the data.
A) Authors should be aware of the hypotheses proposed to explain sexual size dimorphism in Lampyrid species (relevant literature listed below).
B) Correlational data are based on samples captured from different field populations each collected in different times. The data are relevant to describe sexual dimorphism of the species but not relevant to test hypothesis of mate choice. The data also have a limited value as correlation is not causation.
C) The description of mating system of Photinus palaciosi is lacking.

Detailed comments:
Sexual dimorphism and mating system
1.There are several published hypotheses explaining sexual dimorphism in Lampyridae. Male biased sex ratio is not the only explanation to a large male (and lanterns) size. In many species (flightless) females are larger than males and yet male-male competition is intense (see below).
Sexual size dimorphism in fireflies has been connected to female flightlessness, evolution of female sexual signals and male nuptial gifts (South et al., 2020, see below).
Is P. palaciosi a nuptial a gift giving species?
Most firefly species are capital breeders and adults do not feed. Thus both females and males have a limited energy for reproduction and maintenance. For example, some Photinus species females may loose seven eggs/day if remain unmated (Wing, 1989).
During mating males of many Photinus species deliver a large spermatophore which increases female fecundity and longevity (Lewis et al., 2011). Male nuptial gift size correlates positively with body size. A large nuptial gift delays female remating and increases his fertilization success. A large nuptial gift also increases female fecundity and longevity. Thus large male size may be selected not because of lantern size but because of spermatophore size.
 Is this the case in P. palaciosi as well?
Thus testing correlation between lantern size (signal emission organ) with mating success may be irrelevant.
Large male size may be selected because large spermatophores delay female rematings and increase her fecundity and thus his fertilization success. This should be tested in controlled experiment and cannot be detected in field collected data.

References not found in the ms:
Lewis, S. M., Cratsley, C.K., Rooney. J. A., 2004. Nuptial gifts and
sexual selection in Photinus fireflies. Integr. Comp. Biol. 44:234–237.

South, A., Stanger-Hall, K., Jeng, M.-L., Lewis, S.M., 2011. Correlated evolution of
female neoteny and flightlessness with male spermatophore production in
fireflies (Coleoptera: Lampyridae). Evolution 65, 1099–1113.

Wing, S.R., 1989. Energetic costs of mating in a flightless female firefly, Photinus
collustrans (Coleoptera: Lampyridae). J. Insect Behav. 2:841–847.

2. Male-biased sex ratio
Authors claim “a strong male-biased sex ratio” (line 23).  In their own data male biased sex ratio was found in all eight early samples/localities but in nine late samples sex ratio was males biased only in two samples (22% of samples): it was female biased in three samples and equal in four sampels, see ms: supplementary material).
 In other Photinus species sex ratio is known to vary temporally. Local sex ratios become female-biased later in mating season (Lewis & Wang,1991).   Could that be the case also in P. palaciosi?
References not cited in ms:
Lewis, S. M., and Wang, O. T. 1991. Reproductive ecology of two species of Photinus fireflies (Coleoptera: Lampyridae). Psyche 98:293–307.
Experimental design: The work is based on correlative data from field captures. The data are collected from different localities and different dates. The distance between sampling localities are not presented in supplementary material. There is seventeen days between the first and the last sampling. To pool the data from different samples is valid when testing sexual dimorphism in morphological measurements.
 When testing possible mate choice hypotheses pooled data across sites and localities are not valid. For example, some males (altogether 34) were captured in localities (and on a period) when no females were found. Thus these males could not make any choice or could not be a target for female choice. The data should be reanalysed based only on samples when both solitary and mating individuals are found simultaneously and date (= locality) should be one explanatory factor in the analysis. Figure 2 and Table 4 should be rewritten respectively.
The relationship between signal detection organ (eyes) and lantern size with mating success should be tested experimentally not sampling field data. The effect of male nuptial gift on male mating success should also be tested.
Validity of the findings: When the data are reanalysed, irrespective of the results, the findings may be interesting if they are discussed on the light of mating systems of firefly species (South et al. 2011). Even when reanalysis is done, the present data are correlative and not evidence of causality.
Additional comments: The paper gives correlative data based on field samples. The paper is well written and clear but currently lack strong scientific merits. The authors should include more information on mating system of the species. Sexual size dimorphism should be discussed and tested on the light of published hypotheses. The data on mate choice should be reanalyzed omitting localities where only individuals of one sex are found.


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 SIZE OF SIGNAL DETECTION AND EMISSION ORGANS IN A SYNCHRONOUS FIREFLY: SEXUAL DIMORPHISM, ALLOMETRY AND ASSORTATIVE MATING
Review round: 2
Reviewer: 1

Output:
Basic reporting: I feel there is no need to re-review. The ms has been rewritten well according to suggestions.
Experimental design: .
Validity of the findings: .
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: INTEGRATED TRANSCRIPTOME META-ANALYSIS OF PANCREATIC DUCTAL ADENOCARCINOMA AND MATCHED ADJACENT PANCREATIC TISSUES
Review round: 1
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: The current manuscript by Sevcan Atay presents a transcriptome meta-analysis of pancreatic cancer tissues. GEO, TCGA combined with GTEx data sets and others were utilized to identify pancreatic cancer specific genes. Pathway enrichment analysis was carried out as well as survival analysis using TCGA Pan-Cancer data. Genes specific for pancreatic cancer, correlating with survival of pancreatic cancer patients were identified.
This is an interesting analysis that is solely based on publicly available data and data analysis tools. The novelty of the findings is rather limited. The utilized methods are sound and valid. However, there are further concerns that should be addressed:
It is difficult to follow, which datasets were used as training and validation cohorts. Obviously, these datasets are all different, therefore validation is not trivial. Could the author please comment?
Using an inclusion criterion such as “high quality gene expression microarray data” does not necessarily mean that these data are of higher quality than other published microarray data.
Sample pairs can make sense but need better explanation. In pancreatic cancer, most areas next to caner contain chronic pancreatitis like changes and not ‘normal’ pancreatic tissue. This should be discussed.
The work provides “a list of multifunctional biomarkers that have the potential to distinguish PDAC from both adjacent-to-tumor tissues and healthy pancreatic tissues”. This statement is true, and the methodology is up-to-date. However, there are numerous studies with similar aims and results. Validation and functional characterization remain most important.


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: INTEGRATED TRANSCRIPTOME META-ANALYSIS OF PANCREATIC DUCTAL ADENOCARCINOMA AND MATCHED ADJACENT PANCREATIC TISSUES
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This is a very nice bioinformatics work and a well written manuscript. Using many public databases and analysis tool that all of them are freely available Dr Atay comes to some interesting findings on biomarkers for PDAC. I only wonder if a more detailed analysis that could link biomarkers to staging of PDAC would be able. This could help to identify biomarkers for the early detection of this deadly cancer which is may be the biggest clinical problem.
Also it would be very helpful I believe for the reader Dr Atay to include a paragraph where all abbreviations will be explained (there are a lot!) and a table with all the databases and tools used for the meta-analysis.


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: INTEGRATED TRANSCRIPTOME META-ANALYSIS OF PANCREATIC DUCTAL ADENOCARCINOMA AND MATCHED ADJACENT PANCREATIC TISSUES
Review round: 2
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: The authors have satisfactorily answered most of my and the other reviewers’ questions. I believe that this is an interesting and valid manuscript that has been strengthened by the additions and changes. 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: COMPARING SEDIMENT DNA EXTRACTION METHODS FOR ASSESSING ORGANIC ENRICHMENT ASSOCIATED WITH MARINE AQUACULTURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: With this manuscript, Pearman et al. are presenting their very interesting results on studying biodiversity in marine sediment, comparing a relevant selection of different DNA isolation kits along a sampling gradient. The manuscript is well written with a clear structure and sufficient context and references provided. The scripts and data analysis procedures are clearly described and/or made available. I was unfortunately unable to access the PRJNA657189 dataset at NCBI SRA, I assume this has not yet been made publicly available?
Experimental design: The research question is well defined, relevant & meaningful. The methods followed are mostly described with sufficient detail. Some unclarities are still present. According to the manufacturer, the Favorgen mini kit that is said to have been used is only suitable for up to 0,5g sediment. As the authors have tested this kit with 2g and 5g, I assume they instead have used the midi version of this kit? Please correct throughout the manuscript and supplementary data.
The other quibble here is that, although the volume of the used PCR-primers is mentioned, the concentration is not stated. Please add the used primer concentration.
I have several points regarding the experimental design. These might be hard to correct now, but would be something to take in to consideration in future experiments. If the authors agree with the points below, it would also be beneficial to add these suggestions to the discussion as to guide future research from anybody reading this manuscript.
The DNA concentration and quality directly after the isolation was measured with a nanophotometer. For accurate DNA concentration using a fluorometric approach such as a Qubit (used elsewhere in the experiments) would be much more accurate. Although initial differences in DNA concentration are unlikely to have a major effect on the observed diversity after metabarcode amplification, measurement of these metrics should still be performed using the most appropriate method available. It will also provide additional information on the DNA quality and contamination.
Another improvement could be the use of a isolation controls. This will provide valuable insights in the DNA contamination present in the sampling equipment and the so called kit-contamination. Especially when comparing different kits this would have been interesting. Especially in experiments using eDNA (with usually very low concentrations of target DNA) this would be very valuable.
Finally, for the mentioned difference/bias in extraction efficiency for different species (l.418-420), an inclusion of extraction controls using e.g. zymo microbial standards would have provided valuable information about this bias for the DNA isolation kits used.
Validity of the findings: The findings described in this manuscript are well described and all statistical methods used described in clear detail. Some processed data is provided in the supplemental materials. A table with the sequences of all identified ASV’s is not given, and would be an interesting addition to the supplemental data. Also, as mentioned above, the raw short read data could not be retrieved from NCBI.
The conclusions are clear and based on the data obtained in this study. I would say that even more emphasis could be placed on the fact could that all kits are able to show similar core communities and as such, also the gradient present is at the research site is resolved regardless of the kit used.
It is mentioned that less pure DNA could lead to effects such as PCR inhibition. As the Favorgen kit was clearly producing less pure DNA (line 372-378, Table S2), was such an effect also observed in the PCR results? E.g. in less efficient amplification/lower overall yield/smearing or less pronounced bands after PCR? It would be interesting to report on this if such effects were observed.
Additional comments: From the abundance plot (fig 4) for the eukaryotes, it is apparent that the classes listed in the top 10 most abundant reads only represent very small organisms. To me, it seems highly likely these small organisms are present on and inside the sampled sediment and mostly were alive and intact during sampling, just like the bacteria that were identified. As such, I would not consider this an eDNA study, and would describe this as a study of microbiome and bulk benthos. For an eDNA sample, especially taken at a fully stocked fish pen, I would at least expect high numbers of reads for the animalia, more specifically the exact fish species present in the pen. I am curious is fish DNA was found at all in this study.

Although I understand this paper is mainly about the difference between the DNA isolation kits, the obtained results related to the Enrichment State index and the findings in a ecological context are also of relevance. It is clear from e.g. fig. 2 that the CTL site has a much higher biodiversity compared to the site directly near the fish pen. I would suggest to either shortly discuss these results in the manuscript, or, alternatively, mention more clearly why these are deliberately not discussed.
Also, the use of bacteria at class level in my opinion is unlikely to give a real meaningful picture of the ecology, as there is a very high functional diversity of bacterial strains inside the observed classes. Clearly using bacterial diversity is able to show a gradient, and this might be sufficient in the context of the ES index. It would be worthwhile to shortly discuss this issue in the manuscript to clarify the intention of measuring bacterial diversity.

In the abstract, line 45-46, “to” should be removed.


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: COMPARING SEDIMENT DNA EXTRACTION METHODS FOR ASSESSING ORGANIC ENRICHMENT ASSOCIATED WITH MARINE AQUACULTURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this paper, the authors present the comparison of different sediment DNA extraction methods evaluating their efficiency in the context of using sediment DNA metabarcoding to assess the organic enrichment associated with salmon farms activities. The topic is timely and of great importance for future developments of DNA-based biomonitoring. The reported value of 3% of shared ASVs between the kits shows to which extend this aspect is important for any kind of comparative studies.
The paper is well written and, in my opinion, does not contain any technical or analytical flaws. However, there are few important points that should be taken in consideration while preparing the revised version:

1) The title does not really reflect the content of the paper. The authors do not optimize any DNA extraction method but rather compare the existing protocols. Moreover, the wording “marine farm sediments” sounds very unclear. I would suggest something like.
“Comparing sediment DNA extraction methods for assessing organic enrichment associated with marine aquaculture”

2) The authors should carefully revise the statements concerning the composition of eukaryotic assemblage obtained from sediment DNA. In fact, the figure 4 shows that among the 10 most abundant classes within eukaryotes, there are several metazoan groups (nematodes, copepods, polychaetes). There are certainly not the micro-eukaryotes and their presence in eDNA dataset can be affected by the volume of extracted sediment.
By the way, the apicomplexans that also very abundant in the sediment DNA data are mainly parasites of invertebrates. So, the eukaryotic DNA in marine sediments is dominated by metazoans and their parasites. This aspect might be worth discussing in the paper.

3) The impact of the weight of the sediment on DNA diversity is based on comparison of different type and brand of kits. These results need to be confirmed by the analysis of different sediment weights using the same kit or at least the same brand of kit. Those that have done these analyses with Qiagen PowerMax Soil kit will probably disagree with your conclusions.

4) As far as I understood, all kits provided DNA that could be amplified during this study. So, the discussion about the low quality of DNA generated by some kits (FAV 2 and FAV5) and potential inhibition should be moderated as long as there is no strong evidence for such inhibition. Otherwise, the producers of these kits could feel unfairly treated.

5) The authors omit at least two important papers about DNA extraction from marine sediment. These papers have to be referenced and their results discussed in the paper:
•	Lekang et al. Aquatic Microbial Ecology 2015
•	Hermans et al. Mol Ecol Res 2018
The authors could also mention the papers that focus on ancient sediment DNA, which present several extraction protocols that can be used for recent and fossil sediments:
•	Epp et al. Meth Mol Biol 2019
•	Haile, Meth Mol Biol 2012
•	Armbrecht et al. Mol Ecol Res 2020

Minor points:
Line 26 – “selection of sediment extraction weight and the DNA extraction method” sounds somehow weird – could be replaced by “selection of sediment DNA extraction method” (which anyway contains sediment weight)
Line 45 – “with the to distance” – something is lacking here
Line 359 – benthic health?
Line 420 – taxon-specific
Line 435 – the factor kit ?


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: REINSTATEMENT OF PHASCOLOSOMA (PHASCOLOSOMA) VARIANS KEFERSTEIN, 1865 (SIPUNCULA: PHASCOLOSOMATIDAE) BASED ON MORPHOLOGICAL AND MOLECULAR DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Reinstatement of Phascolosoma (Phascolosoma) varians Keferstein, 1865 (Sipuncula: Phascolosomatidae) based on morphological and molecular data

Itzahí Silva-Morales

Decision: Review of Article ID 51760 is highly favorable, and the Reviewer recommends this manuscript for publication in PeerJ. This manuscript is important to the specific field of sipunculan taxonomy, and a welcome contribution to the field of taxonomy in general.

1. Basic Reporting:

There are issues with use of the English language throughout the manuscript, which includes elements of grammar, word usage, word agreement, sentence structure, insertion of terms, and the clarity of particular statements. These are expected from an Author with English as a second language. Therefore, because of the language issues, the Reviewer has taken a focused interest in improving the manuscript in a non-conventional manner. There are many instances where a line-by-line review of grammatical errors and statement clarity issues would require considerable time, and many pages of review notes to be submitted. Therefore this Reviewer has edited the Word.docx version of the original manuscript with Tracked Changes throughout most of the text, and has placed a number of comments and suggestions along the margin.

Note: the Author may, or may not, accept or use the recommended Tracked Changes, but it is the Reviewer’s opinion that such recommendations will significantly improve the manuscript.

Literature sources are sufficient for this study and the extensive background required to make the case for reinstatement of a species that was previously synonymized with another species. Author should recheck the References section for accuracy and formatting, and see comments in the margin of the Tracked Changes document (e.g. Quatrefages vs. de Quatrefages; Broeke vs. ten Broeke, etc.). Regarding the Material Examined section, Reviewer did not review this.

The structure of the manuscript meets professional standards and general PeerJ formatting. However, the figure captions have also been extensively edited with Tracked Changes by the Reviewer to comply with PeerJ figure caption formats, and they were edited for a more specific, detailed and thorough description of the primary data presented in each of the four figures.
Experimental design: 2. Experimental design:

This manuscript is original research, and it meets the Aims & Scope of PeerJ, particularly in the areas of Biological Sciences, as a data-driven Research Article, and on its methodological soundness. This manuscript goes far in its effort to examine previous research, provide a comprehensive set of specimen observations, with corresponding genetic support for those observations, and aims to correct notable errors by previous workers on this particular topic.

The question is clear, the methods utilized to answer the question are appropriate, and the work therein builds upon previous efforts to answer similar concerns by other investigators who have pursued this same question. There certainly is a gap in our knowledge of species distinctions and the connectivity of assumed con-specific populations. More of this work will be required going forward. And, the Author has made a strong case for such efforts here.

Note: The Reviewer would like to see the Author develop a more comprehensive Introduction section by adding pertinent background on the relevance of using COI sequences (e.g. a Barcode gene) for identifying and distinguishing species within the target group, and other metazoan groups outside Sipuncula, with examples. And, address whether COI alone is sufficient, or should it be complemented with other genetic markers to strengthen the results.  Additionally, there should be more introductory information on sipunculan life histories, in particular the types of larvae within the clade, and the limitations of different larval modes (e.g. lecithotrophic, planktotrophic, teleplanic) to disperse and connect species populations, as suggested by Cutler & Cutler (1990), who have made this manuscript necessary. These two issues, larvae as vectors of connectivity, and genetic markers of species identification, should then be revisited in the Discussion section with more detail than first provided by the Author.

The Reviewer finds no outstanding issues with technical standards as presented, and the methods described are both appropriate and replicable, as required for similar studies on sipunculan taxonomy that must be performed in the future. As noted above, the Reviewer has performed edits, added comments and posed questions that will clarify particular methods and data presentation materials – to improve access to specific details by the Author’s readership.
Validity of the findings: 3. Validity of the Findings:

There are no obvious concerns regarding acceptability of the results, completeness of the data provided or the conclusions made. Although, the Reviewer thinks that there are more conclusions from this investigation than those which are stated in the Conclusions section.

The Reviewer suggests that the Author add support values (X.X%) to the COI tree (Figure 4), and provide more discussion as to how and why those values are acceptable measures, confirming the proposed status of Phascolosoma (Phascolosoma) varians as “reinstated”.  For instance, what does the literature state as ‘acceptable’ for separate species?  This is important because no molecular data was used to support the original synonymies by Cutler and Cutler (1990).

Note: In the Remarks section, Keferstein’s descriptions are interesting and relevant, but they are only general. The Author should highlight the differences between species and then use a separate paragraph to ‘remark’ on the specific differences as stated by Stephen & Edmonds (1972), to perhaps complement the Systematics section (lines 134–157) within the Results.

The Author should also mention the condition and dimensions of the fixed material if known.

Final note: use of the term ‘reinstated’ does not need to be written with every listing of the species, as the purpose of this manuscript is very clear. Please see the edits to figure captions by the Reviewer. The terms, ‘reestablished’ and ‘restored’ are not helpful and were removed.

Final comment/question to Author: will this investigation have any anticipated impact on the current relocation of Sipuncula to the basal branches within the annelid radiation? At some time in the future, sipunculan taxonomic revisions, including genetic comparisons with several polychaete annelid sister taxa, will require similar efforts to distinguish sipunculan species. Does your work anticipate any role in this larger effort?  If so, how?  If not, why not?

And, although this manuscript focuses on a species-specific topic of clarification, does the Author have any future directions for the next species targets within Sipuncula?
Additional comments: Reinstatement of Phascolosoma (Phascolosoma) varians Keferstein, 1865 (Sipuncula: Phascolosomatidae) based on morphological and molecular data

Itzahí Silva-Morales

Decision: Review of Article ID 51760 is highly favorable, and the Reviewer recommends this manuscript for publication in PeerJ. This manuscript is important to the specific field of sipunculan taxonomy, and a welcome contribution to the field of taxonomy in general.

1. Basic Reporting:

There are issues with use of the English language throughout the manuscript, which includes elements of grammar, word usage, word agreement, sentence structure, insertion of terms, and the clarity of particular statements. These are expected from an Author with English as a second language. Therefore, because of the language issues, the Reviewer has taken a focused interest in improving the manuscript in a non-conventional manner. There are many instances where a line-by-line review of grammatical errors and statement clarity issues would require considerable time, and many pages of review notes to be submitted. Therefore this Reviewer has edited the Word.docx version of the original manuscript with Tracked Changes throughout most of the text, and has placed a number of comments and suggestions along the margin.

Note: the Author may, or may not, accept or use the recommended Tracked Changes, but it is the Reviewer’s opinion that such recommendations will significantly improve the manuscript.

Literature sources are sufficient for this study and the extensive background required to make the case for reinstatement of a species that was previously synonymized with another species. Author should recheck the References section for accuracy and formatting, and see comments in the margin of the Tracked Changes document (e.g. Quatrefages vs. de Quatrefages; Broeke vs. ten Broeke, etc.). Regarding the Material Examined section, Reviewer did not review this.

The structure of the manuscript meets professional standards and general PeerJ formatting. However, the figure captions have also been extensively edited with Tracked Changes by the Reviewer to comply with PeerJ figure caption formats, and they were edited for a more specific, detailed and thorough description of the primary data presented in each of the four figures.

2. Experimental design:

This manuscript is original research, and it meets the Aims & Scope of PeerJ, particularly in the areas of Biological Sciences, as a data-driven Research Article, and on its methodological soundness. This manuscript goes far in its effort to examine previous research, provide a comprehensive set of specimen observations, with corresponding genetic support for those observations, and aims to correct notable errors by previous workers on this particular topic.

The question is clear, the methods utilized to answer the question are appropriate, and the work therein builds upon previous efforts to answer similar concerns by other investigators who have pursued this same question. There certainly is a gap in our knowledge of species distinctions and the connectivity of assumed con-specific populations. More of this work will be required going forward. And, the Author has made a strong case for such efforts here.

Note: The Reviewer would like to see the Author develop a more comprehensive Introduction section by adding pertinent background on the relevance of using COI sequences (e.g. a Barcode gene) for identifying and distinguishing species within the target group, and other metazoan groups outside Sipuncula, with examples. And, address whether COI alone is sufficient, or should it be complemented with other genetic markers to strengthen the results.  Additionally, there should be more introductory information on sipunculan life histories, in particular the types of larvae within the clade, and the limitations of different larval modes (e.g. lecithotrophic, planktotrophic, teleplanic) to disperse and connect species populations, as suggested by Cutler & Cutler (1990), who have made this manuscript necessary. These two issues, larvae as vectors of connectivity, and genetic markers of species identification, should then be revisited in the Discussion section with more detail than first provided by the Author.

The Reviewer finds no outstanding issues with technical standards as presented, and the methods described are both appropriate and replicable, as required for similar studies on sipunculan taxonomy that must be performed in the future. As noted above, the Reviewer has performed edits, added comments and posed questions that will clarify particular methods and data presentation materials – to improve access to specific details by the Author’s readership.

3. Validity of the Findings:

There are no obvious concerns regarding acceptability of the results, completeness of the data provided or the conclusions made. Although, the Reviewer thinks that there are more conclusions from this investigation than those which are stated in the Conclusions section.

The Reviewer suggests that the Author add support values (X.X%) to the COI tree (Figure 4), and provide more discussion as to how and why those values are acceptable measures, confirming the proposed status of Phascolosoma (Phascolosoma) varians as “reinstated”.  For instance, what does the literature state as ‘acceptable’ for separate species?  This is important because no molecular data was used to support the original synonymies by Cutler and Cutler (1990).

Note: In the Remarks section, Keferstein’s descriptions are interesting and relevant, but they are only general. The Author should highlight the differences between species and then use a separate paragraph to ‘remark’ on the specific differences as stated by Stephen & Edmonds (1972), to perhaps complement the Systematics section (lines 134–157) within the Results.

The Author should also mention the condition and dimensions of the fixed material if known.

Final note: use of the term ‘reinstated’ does not need to be written with every listing of the species, as the purpose of this manuscript is very clear. Please see the edits to figure captions by the Reviewer. The terms, ‘reestablished’ and ‘restored’ are not helpful and were removed.

Final comment/question to Author: will this investigation have any anticipated impact on the current relocation of Sipuncula to the basal branches within the annelid radiation? At some time in the future, sipunculan taxonomic revisions, including genetic comparisons with several polychaete annelid sister taxa, will require similar efforts to distinguish sipunculan species. Does your work anticipate any role in this larger effort?  If so, how?  If not, why not?

And, although this manuscript focuses on a species-specific topic of clarification, does the Author have any future directions for the next species targets within Sipuncula?

4. General Comments:

This manuscript is likely one of many more to come from the collective efforts of investigators to reexamine and to correct the extensive synonymization of sipunculan taxa, as most recently published by Cutler in 1994. Cutler changed the number of recognized species of sipunculans by ~ 171 species, from an already low-species-number taxon. The Author has referenced previous work, efficiently recognizing the momentum of that work, in order to support the addition (reinstatement) of a species back toward a more realistic number. The Author has also clearly reviewed and referenced several previous workers, since Cutler’s (1994) reduction, who have found not only new species, but also that various groups that were lumped into a single species are most likely represented by a species complex. Yet, none of those workers pursued reinstatement as the Author has done here. The Reviewer has recognized this new study as significant, as it may stimulate others to follow using modern molecular genetic analyses to resolve sipunculan taxonomy. Therefore, this manuscript is timely, well-presented, validated and provocative . . . and it is clearly appropriate for meeting the Aims and Scope of PeerJ.


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: REINSTATEMENT OF PHASCOLOSOMA (PHASCOLOSOMA) VARIANS KEFERSTEIN, 1865 (SIPUNCULA: PHASCOLOSOMATIDAE) BASED ON MORPHOLOGICAL AND MOLECULAR DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: - English must be improve. Maybe, to be read for an English spoken person. Taxonomy has its own style and way to say things. So, sometimes ideas are not well written or lack of correct grammar.
- Literature is enough. Just one reference cited is lacking in the section.
- The document attached has many recommendations, changes, suggestions, questions, etc. that could improve the paper. Nevertheless, the document needs more than that. It needs more argumentation and to clarify many ideas.

The document is accepted with major changes and revisions. I will need to revise a new version.
Experimental design: See comments done in the document.
Validity of the findings: Document needs strong argumentation for being clearer in both morphological and molecular findings and analysis.
In the document, I have put several changes, suggestions, etc. in these sections.
As it is used in taxonomy, the discussion presented here could be divided in two. The first part must me fused to remarks (morphological analysis), and the second part must be fused to molecular analysis.
Thus, all the information will be more compacted and inclusive.
In molecular findings, the discussion and analysis are far to be clear. I recommend, if the molecular section does not improve, could be deleted since in the present state, does not give relevant information to the reinstatement of the species.
On the other hand, morphological argumentation is still weak. Author has to include information about the characters in other close species, as well as the discussion about that the characters chosen are very stable and informative.
Additional comments: The document needs a new version and new revision.
Please, check the English, but particularly, check the telegraphic style in the redescription.
If author does not attend some comments, they have to be explained why they were not included in the new version.
All comments are done in the word 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: GYNOMONOECY IN A MYCOHETEROTROPHIC ORCHID EULOPHIA ZOLLINGERI WITH AUTONOMOUS SELFING HERMAPHRODITIC FLOWERS AND PUTATIVELY OUTCROSSING FEMALE FLOWERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Language needs minor revision
Experimental design: Very small study, design is OK
Validity of the findings: Interesting findings; I have not seen any raw data.
Additional comments: This is an interesting article documenting the presence of single sex flowers in a orchid. As most orchids are hermaphroditic, this is a worthwhile contribution to our knowledge of mating systems in orchids. I have seen a few problems that need fixing before the paper can be published.
Table 1 needs to indicate statistical values
Figure 2 needs labelling of the different organs
L122, Table 2: pollinations between different flowers of the same plant are called geitonogamy, not autogamy (i.e., an only-female flower is not capable of autogamy)
Abstract: it is mentioned that female flowers increase chance of autcrossing. If this was the case, they should be more attractive than hermaphroditic flowers, to increase the income of pollen. I guess what is really meant here is that female flowers avoid autogamy, which is semantically not the same.
There are some spacing problems in the abstract: the word possesses has a space in the middle (2 times)
Abstract: “This mixed mating” not “These”
L63: Better say “These examples” or similar, than “these studies”
L65: Better: “Here I describe”
L97: sentence starting with “however” makes no sense; reformulate.


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: GYNOMONOECY IN A MYCOHETEROTROPHIC ORCHID EULOPHIA ZOLLINGERI WITH AUTONOMOUS SELFING HERMAPHRODITIC FLOWERS AND PUTATIVELY OUTCROSSING FEMALE FLOWERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript is well written with only a few minor grammatical issues.  The literature review is not exhaustive but is intelligently selective to get the breadth of issues to the forefront.  The figures and tables are well-done and the raw data are available in the supplemental files.

L118 “After confirming gynomonoecy in E. zollingeri using the cross-pollination experiment”.   Better: “After confirming with the cross-pollination experiment that both hermaphroditic and females flowers produce fruits …”  It is the seed work that will confirm gynomonoecy.

L151 replace “rostella” [plural] with “rostellum” [singular]

L152 An orchid can have a functional rostellum and still be capable of self-pollination (there are various mechanisms).  Replace with “… and were therefore unlikely autogamous” or words to that effect.  Better yet:  If all those that had a functional rostellum failed to set fruit in bagged experiments, then that is your proof that such flowers are not autogamous.

L162 Sentence should read: “… female flowers face severe pollinator …”

L162-163: The way the sentence reads, severe pollinator limitation improves chances for outcrossing.  Pollinator limitation is not directly related to outcrossing probabilities.  Being a female flower without agamospermy is what improves the probability of outcrossing (selfing may still occur via geitonogamous pollinations).
Experimental design: L119-127
a)	It is not clear how the averages were calculated.  As flowers on an inflorescence are not independent of one another (they receive resources from the same plant; pollinator attraction may be at the inflorescence level, not the flower level; etc.), the sample size for each treatment is 5, not 10.  Furthermore, from an evolutionary perspective, fitness is at the plant level, not the flower.
b)	ANOVAs were calculated to compare treatments for fruit and seed set. The author should state whether the data meet the assumptions of the test, and also mention which test(s) they used to determine that.  Furthermore, at a minimum, F statistics and degrees of freedom must be shown, not just the P-value. This would also help the reader understand the statistical analysis.

L167: It is difficult to imagine that resource constraints would be the reason for the production of female flowers at the top (distal end) of an inflorescence because fruit production is far more expensive than male flowers. Hermaphroditic flowers may be more expensive because of pollen production, but one may argue that the female flowers would generally have genetically superior seeds and that there would be selection (if genetically based variation existed) for a favored place on the inflorescence. Unfortunately, it is not clear whether there is any fitness difference in progeny between self and outcrossed pollinations. A re-analysis of the data as suggested above is necessary.

Under resource constraints, one might expect andromonoecy to be advantageous, with male flowers produced in the upper parts of the inflorescence because they are cheap to make.  Using the raw data on flower position, I divided each inflorescence into two equal halves, distal and proximal halves.  Female flowers averaged 1.17 flowers in the distal half, and 3.33 flowers in the proximal half.  I did not test for differences (non-parametric method would likely be appropriate) but it looks like it would be significant.  So, if that is true, then there is a tendency for female flowers to be on the lower part of the inflorescence.  I suggest you do this analysis or something like it.
Validity of the findings: The basic observation that Eulophia zollingerii is gynomonecious is clear.  Even with the ambiguity of the current data analysis, the figures, coupled with raw and summary data, show that some hermaphroditic flowers are capable of self-pollination and this is a case of reproductive assurance.  At the study site, both hermaphroditic and female flowers are rarely visited by pollinators.  It is not clear whether seed set is different between self and cross pollinations.  The averages are very close.  Usually, if there is a difference, it is much more obvious (see table 6 in Tremblay et al. 2005, Biol J Linn Soc 84: 1-54).
Additional comments: Invoking the Red Queen hypothesis is an interesting angle and worthy of further exploration.  Nonetheless, I have doubts that it could explain gynomonoecy with the parasitic orchid-fungus interaction being the underlying driver. Most evidence indicates that all orchids are parasitic on their mycorrhizal fungi, at least in early stages of development.  And since orchids often have mycorrhizal fungi throughout their life, both terrestrial and epiphytic species, one might think that the Red Queen hypothesis could apply to all orchids.  On the other hand, the amount of nutrients captured by orchids from their fungi may be too insignificant to register under the selection landscapes in which fungi find themselves.  So, much is unknown at this point.  If the Red Queen is dealing cards with orchids via their host-parasite interactions, then it should be most obvious with mycoheterotrophic species. However, it is this reviewer’s perception that most of them are autogamous – more so than the rate in which we see in autotrophic species, which if true would run contrary to expectations.  You might dig into this a little more.