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: MORPHOLOGICAL CHARACTERIZATION OF VIRUS-LIKE PARTICLES IN CORAL REEF SPONGES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a really nice piece of work, conducted in a very conscientious way, and which provides novel information about the diversity of sponge-associated viruses.
One could regret the absence of viromic data to accompany the exhaustive morphological description of viruses; however this study is still justified on its own and has sufficient substance for publication in Peer J.
By collecting 15 different sponge species from two constrasted sites (the Great barrier in Australia and the Red Sea in Saudi Arabia), and by using three different protocols for TEM observation, authors have accomplished remarkable sampling and methodological efforts to characterize the viral morphotypes.  Micrographs are also of very good quality which renders the conclusions very convincing. I also compliment the methodological precautions to get ridd off the surrounding viruses of the water column, present in the filtering system of the sponge, and which could be a potential source of bias.  I also appreciated the conclusion about the accuracy of the three tested protocols which will be very helpfull for further morphological studies of viruses in reef invertebrates.
A interesting paper related to this topic (ie, phages in sponge microbial diversity) is missing : Lohr et al 2005, Genomic analysis of bacteriophage JL001: Insights into its interaction with a sponge-associated Alpha-Proteobacterium, Appl. Environ Microbiol 71 : 1598–1609.
Finally I don’t have much to criticize, this was a very pleasant paper to read.
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: MORPHOLOGICAL CHARACTERIZATION OF VIRUS-LIKE PARTICLES IN CORAL REEF SPONGES
Review round: 1
Reviewer: 3

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: Overview. This manuscript describes the results of a microscopy based study that evaluated the viral like particles associated with several species of sponge. Descriptive in nature, this work uses the images to hypothesize what kinds of phage and eukaryotic viruses are associated with these basal metazoans and their symbionts. Overall the work is conducted admirably and the work is novel. Despite this the work requires some editing. While I am enthusiastic about its publication, I feel that the manner in which the authors present the data is quite difficult to read, even for an expert. The authors present the collection of images (figures 1-5) in terms of which sponge species they are evaluting. However, the text refers to the images in terms of hypotheses about what kind of viruses they believe these images represent, for example myophage. I think the authors should instead present first an overall figure showing images from many of the sponges and then the subsequent figures should focus the various kinds of vlps in all of the different sponges. This would align with the presentation of the data in the results text more completely. It would also make it much easier for the readers to follow. An example of this confusing format of the paper comes from lines 165-166 where the authors want the reviews to be able to find 60-200nm vlps in all the figures “Fig. 1A-C, E, G, J; 2A-L; 3B-F, I-L; 4A-C, F, J-166 L; 5D, I, K” by flipping between them. This is very frustrating and time consuming when the authors could have just put those together to make their point more clearly. Any reference to figures that contains more than 1 figure or more than one subsection of multiple figures is simply too hard to follow. Thus, my primary recommendation is that the authors remake all their figures to more conclusively support their hypotheses about to which categories/families these vlps belong.
Specifics:
Line 114 and 133. These are strange references to density gradient ultracentrifugation which has been around for decades. Are there differences in the gradients that warrant these references? What densities were used? What density where the vlps isolated from?
Lines 122-123. Was there no post fixation process conducted to remove the spicules? This seems surprising.
Line 31, 162 and 209. Dumbo-eared? Is this an official term? Many people that are not from western cultures many not get this reference. Elephant eared?
Line 260. Replace Zooxanthellae with Symbiodinium.
Figures. Again, I think these all need to be reorganized. Also, these roman numeral indicators are really not easy to follow. There must be a less complicated way of doing this to make everything easier for the reader to follow.


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: COORDINATIVE CONTROL OF G2/M PHASE OF THE CELL CYCLE BY NON-CODING RNAS IN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript reports some interesting data obtained by microarray analysis of human liver cancer tissues and matched adjacent normal tissues. Further bioinformatics analysis was also performed to identify some signaling pathway or important genes regulated by differentially expressed non-coding RNAs.
Experimental design: The experimental design in the microarray parts was good.
Validity of the findings: The authors claimed that "MiR-125b-2-3p and lncRNAP26302 may affect the G2/M phase of the cell cycle through the regulation of their respective target genes", however, no cell cycle analysis was carried out. The authors need to either revise their conclusion or perform experiments to demonstrate the role of MiR-125b-2-3p and lncRNAP26302 target gene in cell cycle G2/M phase.
Additional comments: The manuscript needs further English editing.


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: COORDINATIVE CONTROL OF G2/M PHASE OF THE CELL CYCLE BY NON-CODING RNAS IN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article uses clear English to describe the effect of MiR-125b-2-3p and IncRNAP26302 on the G2/M phase of the cell cycle through the regulation of CHEK1 and CCNA2 and PLK1.

Tables and figures are appropriate. Results support their hypotheses. There are sufficient references to support their introduction and discussion.
Introduction need more detailed information (e.g. why non-coding RNA research is so important; review more current research in HCC (e.g. HOTTIP and Mir-125b); indicate how your research fills an identified knowledge gap; state the value of your research).

Simply repeating the results (like line 196-209) is not necessary in Discussion section. In addition, it would be better if you can compare and contrast your findings with those of other published results in your discussion. You also need to indicate how your research work is important in the early diagnosis, targeted therapy and prognosis of HCC in the discussion.

Several spelling mistakes are noted including line 45 understanidng (understanding) and line 56 blinding (binding)

Some abbreviations need to be followed by the full name in parentheses, e.g. IncRNA, HULC, CHEK, CCNA, and PLK.
Experimental design: Crosstalk between long noncoding RNAs and MicroRNAs in disease is a hot research field in recent years. Only a few studies involving interaction between IncRNA and MicroRNAs in hepatocellular carcinoma have been published (Liver Int. 2015;35:1597–1606 and Nucleic Acids Res. 2010;38:5366–5383).

This experiment used miRNA, IncRNA and mRNA microarray to identify the novel correlation between MiR-125b-2-3p and IncRNAP26302 in HCC and demonstrate the possible G2/M gene targets, which has not been reported and is a totally novel finding.

The methods described provide sufficient details and information to replicate.
Validity of the findings: The data of this article is totally novel, solid and statically sound and the conclusion are well stated, linked to original research question.

Regarding result1, it would be better if authors could summarize some common features of top significantly dysregulated genes (e.g. they also involve the specific pathway)

Regarding result2, how many co-expressed IncRNAs and miRNAs have been identified? Why do you only report miR125b-2-3p and IncRNA P26302? Is it due to highest correlation coefficient and/or lowest P value?

Regarding result3, it would be better if you use one or two sentences to highlight the most important points.
Additional comments: Overall, this paper is well- designed.  The data is novel and solid, and has not been reported.

However, more detailed information is needed in your introduction. Further discussion is suggested in order to support your novel findings and to show the unique value of your research.


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: COORDINATIVE CONTROL OF G2/M PHASE OF THE CELL CYCLE BY NON-CODING RNAS IN HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Shi et al. had examined the hepatocellular carcinoma tissue and surrounding nourmal tissue of 12 patients and generated the profile of lncRNAs, miRNAs and mRNAs. They demonstrated that CHEK1 and CCNA2 are targets of miRNA-125b—2-3p; and PLK is targeted by lncRNp26302.
The authors are trying to conclude that miRNA-125b—2-3p and lncRNp26302 are thus regulating cell cycle at specifically G2/M phase.
Experimental design: This study is based on RNA screenings, not at the protein level. Thus whether the effect of miRNA-125b—2-3p and lncRNp26302 on the mRNA of CHEK1, CCNA2 and PLK can be carried on to translational products or cellular functionality are not clear. It is still too early to conclude any clinical relationship between these two RNAs and the proliferation or progression of hepatocellular carcinoma. Furthermore, the molecular function of miRNA-125b—2-3p and lncRNp26302 in regulating mRNAs of CHEK1, CCNA2 and PLK is also inconclusive without gain of function or loss of function experiments. Overall, more experiments are required to rule out the possibility of pure correlation, and to confirm the causal relationship.
Validity of the findings: 1.Please make sure to check up the formatting of the manuscript. Some of the periods are not in correct form.
2.There is inconsistency in the miRNA number that are studied. In the abstract it is 81 while in the results it is 57.
3.The correlation analysis showed high score between miRNA-125b—2-3p and lncRNp26302, however the author should make it clearer that there is no direct molecular function between these two RNAs. Instead, they each have their own targets. so in other words the correlation, between the target mRNA expressions, is not due to any result of biological functionality between their upstream regulators.
4.The author should refer to the name of protein instead of the gene name, like CCNA2 in the Kegg pathway, which makes readers hard to track it and understand it.
Additional comments: The title use “simultaneous” to describe the regulation on cell cycle by miRNA-125b—2-3p and lncRNp26302, which is ambiguous. May be changed to “Coordinative control of G2/M phase of the cell cycle by non-coding RNAs in hepatocellular carcinoma”.


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: COORDINATIVE CONTROL OF G2/M PHASE OF THE CELL CYCLE BY NON-CODING RNAS IN HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is a revised manuscript and my concerns have been addressed.
Experimental design: This is a revised manuscript and my concerns have been addressed.
Validity of the findings: This is a revised manuscript and my concerns have been addressed.
Additional comments: This is a revised manuscript and my concerns have been addressed.


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: COORDINATIVE CONTROL OF G2/M PHASE OF THE CELL CYCLE BY NON-CODING RNAS IN HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: as the first review
Experimental design: as the first review
Validity of the findings: as the first review
Additional comments: The authors did well responses for most concern and question, and modified the manuscript following all reviewers' comments. As the authors mentioned, it is a a prospective study.More results need be discussed in future. Currently, The authors need better explain and double-check the data from Fig4., It is the only evidence to verify the microarray findings.


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: COORDINATIVE CONTROL OF G2/M PHASE OF THE CELL CYCLE BY NON-CODING RNAS IN HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: This study is based on RNA screenings, not at the protein level. Thus whether the effect of miRNA-125b—2-3p and lncRNp26302 on the mRNA of CHEK1, CCNA2 and PLK can be carried on to translational products or cellular functionality are not clear. It is still too early to conclude any clinical relationship between these two RNAs and the proliferation or progression of hepatocellular carcinoma. Furthermore, the molecular function of miRNA-125b—2-3p and lncRNp26302 in regulating mRNAs of CHEK1, CCNA2 and PLK is also inconclusive without gain of function or loss of function experiments. Overall, more experiments are required to rule out the possibility of pure correlation, and to confirm the causal relationship.
It had been addressed.
Validity of the findings: no comment
Additional comments: 1.	Please make sure to check up the formatting of the manuscript. Some of the periods are not in correct form.
It had been addressed.
2.	There is inconsistency in the miRNA number that are studied. In the abstract it is 81 while in the results it is 57.
It had been addressed.
3.	The correlation analysis showed high score between miRNA-125b—2-3p and lncRNp26302, however the author should make it clearer that there is no direct molecular function between these two RNAs. Instead, they each have their own targets. so in other words the correlation, between the target mRNA expressions, is not due to any result of biological functionality between their upstream regulators.
It had been addressed.
4.	The author should refer to the name of protein instead of the gene name, like CCNA2 in the Kegg pathway, which makes readers hard to track it and understand it.
It had been addressed.
5.The title use “simultaneous” to describe the regulation on cell cycle by miRNA-125b—2-3p and lncRNp26302, which is ambiguous. May be changed to “Coordinative control of G2/M phase of the cell cycle by non-coding RNAs in hepatocellular carcinoma”.
It had been addressed.


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: ANALYSIS OF ADULT DAMSELFLY FECAL MATERIAL AIDS IN THE ESTIMATION OF ANTIBIOTIC-RESISTANT ENTEROBACTERALES CONTAMINATION OF THE LOCAL ENVIRONMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript  can be shortened. There are currently unnecessary repetition and redundancies.
Experimental design: Experimental design is basically appropriate.
However, I cannot understand why bacteria grown on SCD agar with an antibiotic were environmental bacteria.
Validity of the findings: This manuscript contains unique and interesting observation concerning antimicrobial resistant bacteria in environments and their monitoring procedure.
Additional comments: Line 38-41: Gut bacteria and environmental bacteria are in reverse order.
Line 250-251: The authors say that "Although there were no obvious differences in the MIC ranges between the sampling sites". However, MIC90 for KAN and CIP of bacteria derived from Shinoroshinkawa is very high. This seems to be obvious.
Figure 5 and Table S1: I cannot understand "SCD-Antibiotics" and "Mc-antibiotics". Do these mean that bacterial growth was observed on agar plate containing either antibiotic? Furthermore, I cannot understand "SCD-MC-all antibiotics".


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: ANALYSIS OF ADULT DAMSELFLY FECAL MATERIAL AIDS IN THE ESTIMATION OF ANTIBIOTIC-RESISTANT ENTEROBACTERALES CONTAMINATION OF THE LOCAL ENVIRONMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: Need to add more background information especially in introduction section
Experimental design: no comment
Validity of the findings: no comments
Additional comments: Comments:
The paper describes the prevalence of antibiotic-resistant bacteria, including Enterobacterales, in fecal material damselflies (adults and larvae) collected from seven locations around Sapporo City, Japan. This is a well-written manuscript and a thorough and well-designed study. Few typological and grammatical errors are present that need to be corrected. In terms of bacterial identification, a wide range of API identification cut-off value have been considered which usually raise the question of accurate identification of the isolates. It would be therefore highly accepted if it is possible to confirm the bacteria species using 16S rRNA sequencing of the pure isolates.

Introduction
The introduction is comprehensive and well written, provides all the required background.
Line 75-77: “This approach …and urban planners” is not relevant to the context of the study. May delete it.
Line 79-81: Please add more justification why Damselflies could be good indicator of antibiotic resistant bacteria that have potential to infect human and animals.

Materials and methods
Line 95: What was the basis of choosing these antibiotics. Would be great if the can mention it.
Line 99: Delete period (.) after sample collection.
Line 101: what are the distances among all seven locations? I can see it is mentioned in results sections. Please bring it here. Are there any environmental variations among the locations? Please explain it.
Fig 2. “Prevalence of different damselfly species at each sampling site”. Prevalence will not be the right word to use here. What sampling techniques have been used to determine the prevalence? Please explain if do so..
Line 129: Why do incubate at 25o C temperature? Enterobacteriales usually grow at a wide rage of temperature. There might be a possiblily of missing the bacteria that grow above 30o C temperature.
Line 131: Some Enterobacterales are naturally resistant to tetracycline or ampicillin. Therefore, how logical it is to consider these isolates as antibiotic resistant bacteria. Please explain it.
Line 136-137: What cut off value was used to identify the bacterial species using an API 20E kit? Please mention it. As far I can see in table S2, a wide range of cut-off value have been chosen to identify the bacteria which raise the question of accurate identification of the Enterobacteriales species.
Line 149: What alignment technique was used in phylogenetic tree construction? Please mention it.
Line 153-154: “the prevalence of bacteria on SCD-A, the prevalence of bacteria on MacConkey-A”. The authors have not explained how they have determined the prevalence of bacteria on these media plates? Is it prevalence or only presence? Please explain it and correct it throughout the manuscript.

Results:
Line 178-187: I think, this information has already mentioned in materials and methods. Please rewrite the paragraph without repetition of information. Some information might go in materials and methods.
Line 187-193: In my opinion, the author hasn’t determined the prevalence of Damselfly in this study. Therefore, please replace the word “prevalence” with other appropriate words such as “dominant”.

Discussion:
Discussion is clear and well-written. However, the author should focus also on the importance of the Enterobacteriales isolated from the damselfly. The author should add another paragraph describing briefly the importance of these bacteria identified in this study. The author should also describe the potential role of damselfly in spreading human important Enterobateriales as damselflies focally and domestically inhabit areas with minimal migration.

Conclusion:
Conclusion is well-written

Figures and tables:
Table S2: It can be shown in table 2S that many bacterial species have been identified at below 70% API identification cut-off level. I wonder how accurate the identification of these isolates as most of the Enterobacteriales species are closely related. Need more clarification on results.


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 BACKMAP PYTHON MODULE: HOW A SIMPLER RAMACHANDRAN NUMBER CAN SIMPLIFY THE LIFE OF A PROTEIN SIMULATOR
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article is well written in generally clear language.  The author should be commended for his clarity.  The introduction and background effectively provide context and motivation for the work.

A new piece of software (BackMAP) is a major part of the paper, and it is available and works out of the box in my hands, which is great.

The included references are reasonable.  However, the author references himself quite frequently (which is related to some weakness of the paper – see below), and omits some salient papers on Ramachandran plots.  For example:
•	Lovell et al. Proteins (2003): high-quality Rama plots + smoothed distributions
•	Ting et al. PLoS Comp Biol (2010): more general exploration of neighbor-dependent Rama plots

The figures are generally clear and quite aesthetically pleasing, which is a credit to the new software package, which was used to generate many of the figure panels.  However, there are a few weaknesses:
•	Several figures do not have a caption, which makes them more difficult to interpret.  Please add captions.
•	Other figures have insufficient captions.  For example, in the Figure 2 caption, it is not clear what the dots in panel b are, nor what the different colors mean in b-c (presumably for emphasis, but this is not stated).
•	Some figures are incorrectly referenced in the text and captions.  For example, in the Figure 2 caption, I think “qualitative sense of peptide backbone structure (a, c), it is not a convenient representation for exploring peptide backbone dynamics (c)” should be “qualitative sense of peptide backbone structure (a, b), it is not a convenient representation for exploring peptide backbone dynamics (c)”.
•	Why are Figure 8c-d so “dotty” as compared to all other MAP images?  Describe this in the caption?

Although the messaging and language of the manuscript are generally clear, they could be improved in places:
•	In e.g. lines 116 onward, “chirality” is discussed with respect to protein backbone.  Typically this means L vs. D amino acids, i.e. which direction the sidechain diverges from the backbone.  Here the definition is something different, but is not clearly defined.  It seems to mean a property that would be observed if several sequential residues were to adopt the same conformation.  This idea was apparently explored more in the author’s earlier papers on this topic, but is not made sufficiently clear here for this standalone paper, which is a repeated theme.
•	Similarly, peptoids are discussed in the “Ramachandran codes are stackable” section, but it is not made clear how they differ from peptides.  Peptoids were previously studied by the author.
•	Lines 88-91: In this overly terse section, the residue-level parameter R is related to several protein-level parameters, but it is not made clear in the text how such a mapping makes any sense – this is only described in the middle of the associated figure’s caption.  Please (briefly) clarify this in the main text.
•	Something called “MAPs” are introduced in line 73, but they are not defined until much later.  Please (briefly) define when first introduced.
•	Lines 38-39: “Today, protein structures described in context of the two-dimensional (φ , ψ )-space are called Ramachandran plots.”  This is imprecise: the structures themselves are not called Rama plots; the structures are described by the plots.
•	Figures 7 and 8 are titled “Ramachandran lines are stackable”, yet these figures are not referenced in the text section called “Ramachandran codes are stackable”, which cites a different figure…  This is unnecessarily confusing for the reader.
•	Abstract: “closed form” vs. “closed-form” – be consistent (or maybe “closed-form expression”)
•	Lines 263-264: Explain how these quantities are defined (and see below for suggestions regarding RMSD/RMSF)
•	This paper uses RMSD and RMSF here very differently from the usual definitions.  Here, both RMSD and RMSF are defined as a difference in two R numbers, squared, and square-rooted.  Usually they are sums of many such squared terms that are then square-rooted.  Without a sum, an absolute value operator is sufficient.  Moreover, RMSF typically refers to deviations from an average, but here it simply refers to deviations from the previous entry in the sequence.  Please simplify these equations by switching to absolute value operators and change the nomenclature to avoid overloading these terms.
•	How were the “Stand Alone Example” structures obtained?  Mention the experimental (or computational) method.
•	Line 81: “most” -> “more”

Generally, there are several careless errors scattered through the manuscript.  I list a few of them here:
•	Lines 320 & 323: “Fig X” = placeholder?
•	Figure 12 caption – typo: “evident available by”
•	Line 327: typo
•	Last line: missing period
•	Line 81: missing Section #
•	Line “254: Panel (b)” should be “Panel (a)”
•	Figure 7 caption: “Ramachanran”
•	(maybe more)
Experimental design: The experimental design, so to speak, of the paper is solid.  The paper provides several relevant examples that bridge Rama plots, the notion of R, multiple-model ensembles, different types of ensembles, and several possible bioinformatics applications.  The paper is relatively heavily focused on a new software package, so naturally a good portion of it is dedicated to that rather than “experimental design” per se.
Validity of the findings: The findings and discussion of the paper are generally valid, with a couple of exceptions:

•	I have read the section “Ramachandran numbers are more compact than one might realize” several times, and still think it makes no sense.  The paper states that “for any N-length peptide, any ordering of [φ1,φ2,...,φN,ψ1,ψ2,...,ψN] can not describe the structure”.  I don’t see how this is true (at least for the backbone structure alone, and ignoring bond lengths and angles, as I assume the author is doing)…  A series of φ and ψ angles certainly CAN define a backbone conformation in this sense…  I’m not sure what is meant by “any ordering” – of course a random order would specify the wrong conformation – but I don’t think that’s what the author is going for…  Anyway, please either clarify this or (more likely) just remove this section – the point is already made that the dimensionality reduction of R can be useful!
•	In the section “Case study: picking out subtle differences from high volume of data” and Figure 8, it is stated that pre-Pro has distinct conformational preferences, which makes sense.  But it is also stated that pre-Gly has distinct conformational preferences, which is not well supported by the data shown.  It is, however, clear that Gly residues themselves have distinct conformational preferences.  The references included on line 142 seem to address different issues: Gly and Pro positions within helices, and energetics of Gly and pre-Pro.  (See above, however, for other references to include.)
Additional comments: This manuscript discusses a previously introduced parameter called the Ramachandran number (R), which effectively reduces a 2D Ramachandran plot to a 1D variable.  This seems difficult initially since the 2D Ramachandran plot is rich with features, but the paper does a reasonable job of alleviating those concerns, largely since the major features of protein structures in the Rama plot (perhaps fortuitously) do not directly overlap in R space.  R thus may be generally useful for structural bioinformatics.  The paper surveys some potential uses of this number, and offers a software package which makes it easy to use for the reader to explore structures of interest to them – in particular ensembles and dynamic trajectories, which is a forward-looking perspective.

My main criticism is that, in contrast to the majority of the paper, a few aspects of the work are not well explained.  These aspects seem to correspond to aspects the author has explored in his previous papers already – it seems he does not want to repeat himself.  However, without these inclusions, the paper has readability gaps.  Most notably, how does the Ramachandran number R map onto a Ramachandran plot?  A simple heatmap of R in a 2D Rama plot as a Figure 1 would help build intuition quickly, which would aid the reader in digesting the rest of the paper.  Only much later, lines 280-281 say “The Ramachandran number increases in value from the bottom left of the Ramachandran plot to the top right in sweeps that are parallel to the negative sloping diagonal.”  The reader could of course derive this him- or herself based on the equation, but since R is so central to the entire paper, making sure to establish this intuition up-front is important.  Please move this to the very beginning when R is introduced!  Other similar aspects, where the paper is sparse on explanation for topics the author has previously published on, are described elsewhere in the review (peptoids, chirality).

Also, while the use of the new(ish) R metric may be useful for many applications, the results presented here are generally retrospective.  It is argued that these findings (e.g. that Gly, Pro, and pre-Pro residues have unique conformational preferences) would be difficult to make without R – but in fact they were made without R.  This is not necessarily a knock against the method, but rather more of a validation that sets up the method to be used for other prospective applications by others later.  However, the language in the paper could be toned down a notch to emphasize these observations were made previously.  (As noted above, some references are included, but other salient ones are not.)

Overall, this paper is a useful contribution that may attract many readers interested in structural bioinformatics and other aspects of computational structural biology.


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 BACKMAP PYTHON MODULE: HOW A SIMPLER RAMACHANDRAN NUMBER CAN SIMPLIFY THE LIFE OF A PROTEIN SIMULATOR
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is well written and clear, but there are abundant typos that need to be fixed. (several are highlighted on attached PDF)

The background is sufficient, although the references are on the light side. A glaring example was a single reference (by the author) supporting the existence of conformational changes.

The figures are fine, but several of the captions are inadequate to describe the content: Fig 2 (explain panels), 6, and 8 (panel d not mentioned).

Database files, code, and scripts are provided.
Experimental design: The goals and methods are well defined and competently carried out.
Validity of the findings: The advancement over the previous publication is subtle, but the author makes a strong case for its inclusion in the literature. It is well suited for this journal.
Additional comments: I am unconvinced by the argument (line 92-102) that the pairing of dihedral angles somehow increases the complexity of the 2N numbers required to specify a backbone configuration. Perhaps the author is referring to the limited region of the Ramachandran plot that is available? But, there are always steric constraints on a polymer, and this leads to coupling over much greater distances along the backbone (Flory swelling). I would argue the other way: 2N numbers are required to specify the backbone, but because so much of the region is sterically forbidden, 2N is greatly in excess of what is actually needed.
The “MAP” acronym is used several times before it is defined.
On line 254 I think he means panel a instead of b.
On line 295 I think the reference should be to panel c.
On line 302 he discusses the “handedness” of amino acids. Based on the following discussion, I think he is referring to the handedness of a helix that would occur if the specified amino acid were propagated, but this could use clarification.


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 ANTI-BIOFILM EFFECT OF SILVER-NANOPARTICLE-DECORATED QUERCETIN NANOPARTICLES ON A MULTI-DRUG RESISTANT ESCHERICHIA COLI STRAIN ISOLATED FROM A DAIRY COW WITH MASTITIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The language of the article needs to be revised and improved.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The manuscript "The anti-biofilm effect of silver nanoparticle-decorated quercetin nanoparticles on a multi-drug resistant Escherichia coli strain isolated from a dairy cow with mastitis" is a continuation of a previous work of the authors and presents interesting findings. However, it must be improved namely in terms of language.
Below, you can find my suggestions and comments.

Abstract: The authors presented two slightly different abstracts. Please rewrite the abstract more succinctly, taking into account the language. Example: Line39/42: repeated information…rephrase.
According to PeerJ journal “Headings in structured abstracts should be bold and followed by a period.” The headings of the abstract are : background, Methods, results and discussion.
Keywords: I suggest adding Quercetin and silver nanoparticles (in substitution of nanoparticles).
Line 109: “3,3′,4′,5,7-Pentahydroxyflavone (quercetin, Qe), a kind of flavonoid compound, can be extracted from the flowers, leaves, and fruits of some plants” give some examples.
Line 128: “…diluted to a final concentration of 0.03 by optical density at 600 nm” change to: diluted to a final optical density (600 nm) of 0.03.
Line 144: “Qe, Ag NPs, or QA NPs were added to the LB medium with 0.5% milk solution and E. coli cells at different concentrations, respectively. Confusing sentence. Rephrase please.
Line 145: Why did you not use the same incubation time?
Line 176: correct the reference (Miller et al. 1993).
Line 209: refer to figure 1C.
Line 230/231: Change to:  “Meanwhile, biofilms of the Ag NPs treatment group were only in slight surface crack (3C). However, biofilms of the QA NPs treatment group were seriously damaged (Fig. 3D).
Line 250/253: sentence too long, please rephrase.
Line 266: Reference?
Line 267: “…has been used to treat some infectious diseases” Give some examples please or mention if it refers among other infectious diseases to bovine mastitis.
Line 270/273: repetition of information.
Please review all figures captions (more succinct and objective). They are too long.
Graphic 1C: start the y-axis at 0.


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 ANTI-BIOFILM EFFECT OF SILVER-NANOPARTICLE-DECORATED QUERCETIN NANOPARTICLES ON A MULTI-DRUG RESISTANT ESCHERICHIA COLI STRAIN ISOLATED FROM A DAIRY COW WITH MASTITIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: pdf attached


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: PUSHING THE LIMITS OF WHOLE GENOME AMPLIFICATION: SUCCESSFUL SEQUENCING OF RADSEQ LIBRARY FROM A SINGLE MICROHYMENOPTERAN (CHALCIDOIDEA, TRICHOGRAMMA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study uses the GenomiPhi kit (V2) to test for potential biases introduced through whole genome amplification (WGA) followed by RADseq. The authors were specifically interested in testing the potential utility for minute wasps that generally yield extracts with DNA quantities too low for standard RADseq library preparation. These wasps are often used as biological control agents, so proper genetic characterization is necessary. A relatively complex crossing experiment is performed in which WGA is performed on both F0 and F1 flies. RADtags are then compared to those produced from pooling F2 males and generating standard RAD libraries without WGA. The authors analyze their data using the standard Stacks pipeline and find no substantial bias in WGA samples. These conclusions are based off of the mode of reproduction of their study species and Mendelian assumptions. These results confirm previous studies and suggest that WGA through MDA may be suitable for next-generation sequencing of specimens with low DNA quantity.

Although I found the study interesting, especially as it is the first to examine the potential utility of WGA + RADseq with very low DNA quantity, I am unable to recommend the manuscript for publication in its present form. One major concern is with some of the writing, which was difficult to follow. For example, I would recommend using the term ‘70% ethanol’ versus ‘ethanol 70%.’  Along similar lines, use ‘F1 females’ instead of ‘females F1.’ Lines ~190-200 are quite difficult to follow. If not done yet, I would recommend editing by a native English speaker to improve clarity. If the authors can address the experimental design issues and quality of the writing I am sure this will make a valuable contribution to the literature.
Experimental design: My primary concern lies with the experimental design, which I found to be quite confusing throughout the paper. As someone who does not work on parasitoid wasps, I would have liked to see a bit more discussion about breeding systems in the species and how this mode of reproduction lends itself to being tested using the techniques the authors use. Figure 1 essentially has no figure legend, thus it is a bit difficult to understand how the breeding experiment was performed. What do the colored bars represent? What does the side arrow branching off of the F0 cross represent? If a reader cannot fully comprehend the experimental setup, all results and potential implications are meaningless. My recommendation is to modify Fig. 1, include an informative legend, and elaborate on the experimental setup in the Methods
Validity of the findings: The authors conclude that WGA through the GenomiPhi V2 kit combined with RADseq may be useful to study genomic patterns and processes in specimens with DNA quantities too low for standard RADseq protocols. These are potentially interesting findings as no study to date has tested for potential bias in WGA and RAD sequencing when starting from very low DNA quantities. Unfortunately, I remain a bit skeptical about the findings for two primary reasons: (1) The methods/experimental design is a bit difficult to follow. In the revision I would highly recommend modifying Figure 1 and including an informative legend. I would also elaborate on the study system in the Methods and how it can be used to test for potential biases in WGA. (2) It appears that the majority of the conclusions are based on low sample sizes. If I understand correctly, only three pools of F2 males were used for the comparisons. Can the authors justify this sample size?
Additional comments: Line 63: There should be a citation here.

Line 87: Make it clear early on what you are comparing. Are you looking at both coverage of RAD loci and individual SNPs?

Line 97: If identification to species is so difficult, how was this done in the study?

Line 139: Five pools consisting of what? How many/which samples per pool? Were RADtags amplified through PCR? I assume so, but this isn’t stated. Again, I feel that a lot of important information was omitted.

Line 164: Only three crosses had enough F2 males for pooled library preparation? So essentially you have a sample size of three? Do you think this is high enough to make a recommendation/conclusion?

Line 204 and throughout: What exactly is ‘a Mendelian inheritance?’

Line 248: So you did perform PCR?

Line 250: This is not a proper sentence.


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: PUSHING THE LIMITS OF WHOLE GENOME AMPLIFICATION: SUCCESSFUL SEQUENCING OF RADSEQ LIBRARY FROM A SINGLE MICROHYMENOPTERAN (CHALCIDOIDEA, TRICHOGRAMMA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: I recommend publication once a more coherent draft is submitted.  There are multiple instances in the article that would benefit from a thorough review.  For instance

Overall Comments/examples of awkwardly worded sentences/ and statements (not complete, just representative):
Please re-think use of “ca.” and possibly remove/reword.  Very Distracting
Second Paragraph (lines 41-56) very important but awkwardly worded
Line 101-105 confusing sequence explain from F0 to F2
Line 122 Consistency Whole Genome Amplification/WGA
Line 164: leaded?
Line 167: that stand?
Line 171: In average?
Line 171: (226 - 2393)? Is this ng?
Line 182: In average?

Tables:
The tables should be self-explanatory… please expand
Experimental design: Well developed assessment that should be published once it is made more coherent.  The experimental design and technical aspects of the work are quite in depth and interesting, but the writing distracts from this.
Validity of the findings: The assessment is novel and the analysis robust.  The Conclusions drawn are relavent to the analysis.
Additional comments: This is an interesting assessment, and the use of the haplodiploid genetics of this organism for the work is rather clever.  I recommend publication once a more coherent draft is submitted.  The assessment needs a thorough review for writing and clarity.  The Discussion does a relatively good job of explaining the significance of the paper, whereas the methods and results are unclear especially when addressing the different crosses.  The Introduction, although relatively clear, is awkwardly worded at points and should be reviewed thoroughly.  The Summary is fine.  I cannot necessarily comment on the whole genome amplification method as I have not used it, but from what I can tell it seems like a very interesting method that was used appropriately.


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: PUSHING THE LIMITS OF WHOLE GENOME AMPLIFICATION: SUCCESSFUL SEQUENCING OF RADSEQ LIBRARY FROM A SINGLE MICROHYMENOPTERAN (CHALCIDOIDEA, TRICHOGRAMMA)
Review round: 1
Reviewer: 3

Output:
Basic reporting: An excellent, unambiguously written article. I have made minor corrections to some of the English early in the article.

I suggest one major, and one minor issue:

1. Major issue: The identity of the origibal culture as T. brassicae needs to be unambiguously established. This does not of course affect the findings of the paper, but it would make it much more useful if the species could be unequivocally named.

This can be done by using the Polaszek et al and the Pino-Perez et al papers cited in the attachment (using ITS 2 sequencing which the Rasplus lab can easily provide). A morphological confirmation using dissected male genitalia would also be useful. I would be happy to provide these details.

2. Minor issue: A recent paper dealing with the use of Trichogramma to control the notorious tomato pest Tuta absoluta (and the banana pest Chrysodeixis chalcites) could be included as a useful reference.
Experimental design: While not an expert on the experimental design area of this paper, I am very familiar with the track records of the 1st and last authors, and am certain of the accuracy and reliability of their protocols. The work is well within the journal's scope.
Validity of the findings: Clearly robust, and most importantly highly innovative findings. The results provide protocols for future work on minute organisms.
Additional comments: Please see general comments above and in the attachment.


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: DESCRIPTIONS OF FOUR NEW SPECIES OF MINYOMERUS HORN, 1876 SEC. JANSEN & FRANZ, 2018 (COLEOPTERA: CURCULIONIDAE), WITH NOTES ON THEIR DISTRIBUTION AND PHYLOGENY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is written in clear, unambiguous and professional English language. The main objective of this work is to describe four new species of the weevil genus Minyomerus (Curculionidae, Entiminae, Tanymecini), previously revised by the authors of the article, and to integrate the new findings in an updated key and a phylogenetic analysis based on morphological characters.
The article is well organized and the structure conforms to PeerJ Standards. The introduction includes the necessary information and background on the taxon under study. The literature is well referenced and relevant.
The taxonomic study was based on a sufficient number of specimens collected by the authors and borrowed from different entomological collections. The descriptions are very detailed and the structures of diagnostic value are illustrated with very good photographs (dorsal and lateral habitus, mouthparts, sternites VIII of female, spermathecae). All figures are relevant, high quality and well labeled.  The literature is well referenced and relevant.
Experimental design: The new species described have been registered in ZooBank, and the information on LSID’s in provided.
A cladistic analysis was performed on a data matrix of 52 morphological characters and 26 terminal taxa (5 outgroups and 21 ingroups), to see the position of the new species in the context of Minyomerus and related genera. The selection of characters and character states was correct. The most parsimonious tree was found using TNT, and NONA was used for the optimization of the characters.  The tree with the characters optimized was conveniently illustrated.
The cladistics methods are sufficiently detailed and the information given can be replicated.
The potential distribution of each new species was assessed applying an ecological modeling approach, using MAXENT, and was shown in maps.
The authors adopted the taxonomic concept approach, including the use of taxonomic concept labels and Region Connection Calculus (RCC–5) articulations and alignments. I am not very familiar with this approach and I find it a bit confusing for reading the results, but I understand that the authors are leaders in this matter, not yet commonly used in the taxonomic papers. The details of the RCC–5 alignment are given in free text form in the Supplemental Information, which also describes the content of the data input and output files.
Validity of the findings: The work provides new taxonomic information. The descriptions of the new species are well justified, same as the results of the cladistics analysis. In the discussion, the authors compared the results of the new phylogeny with those previously obtained. They discussed on the intrageneric relationships and the phylogenetic position of each species in a historical-biogeographic context. In find it interesting and not excessively speculative, however, I have some difficulties in following the indication relative to the taxonomic concept approach.
Conclusions are well stated, linked to original research question and limited to
supporting results
Additional comments: The paper provides new basic taxonomic information, consisting on a description of four new weevil species, rigorously described and illustrated.  The phylogenetic placement of these species is analyzed and their geographic ranges are discussed in relation to those of their putative sister taxa, based on the results of a niche modeling analysis. The methods applied are classical and correct.  All the figures are appropriate and necessary.
In my opinion, the taxonomic concept approach applied in this paper is somewhat confusing, as well as the supplementary information relative to this subject, but I am not an expert on this matter.


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: DESCRIPTIONS OF FOUR NEW SPECIES OF MINYOMERUS HORN, 1876 SEC. JANSEN & FRANZ, 2018 (COLEOPTERA: CURCULIONIDAE), WITH NOTES ON THEIR DISTRIBUTION AND PHYLOGENY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a comprehensive, well-written followup to the authors previous paper revising the genus Minyomerus.  I have made a few minor comments in the text of the manuscript PDF.  I am quite sure that the single specimen of M. ampullaceus is from the stomach/gut contents of a roadrunner.  The USNM was quite active in the mid 1900's in supporting identification of gut contents from various species of birds. I have a new Phyxelis from Louisiana known only from USNM specimens from quail stomach contents and the weevil Glaphyrometopus ornithodorus was described from a series of USNM specimens from meadowlark stomach contents.  As such, the paragraph related to the natural history of this species should be rewritten.
The phylogenetic work appears to be well supported and discussed with all characters and states well-explained.  As the authors know, I am not a fan of the taxonomic concept approach but I can understand the authors rationale for its use. The authors have done a nice job of integrating the 4 new species into the key and phylogeny and presenting these results.
Experimental design: See above.
Validity of the findings: See above.
Additional comments: See above.


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: DESCRIPTIONS OF FOUR NEW SPECIES OF MINYOMERUS HORN, 1876 SEC. JANSEN & FRANZ, 2018 (COLEOPTERA: CURCULIONIDAE), WITH NOTES ON THEIR DISTRIBUTION AND PHYLOGENY
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: I suggested in the pdf itself that some information may need to be added in the methods section regarding additive/non-additive coding of multistate characters.
Validity of the findings: No comment.
Additional comments: This is a thorough study that aims to describe and phylogenetically place 4 new species of Minyomerus.  The use of occurrence data to predict distribution ranges for each species is a much welcome approach to this type of study.


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: AGRICHEMICALS AND ANTIBIOTICS IN COMBINATION INCREASE ANTIBIOTIC RESISTANCE EVOLUTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: It is to some extent necessary to read Kurenbacher 2015 and 2017 to get informed on the choice of the study organisms, herbicides and antibiotics and several MIC values. It would ease the reader experience if some more information from these studies were provided to make the rationale for the study and the experimental design more evident. The authors might consider to include MIC values with and without herbicides where relevant in table 1 or in a new table.
Experimental design: With reference to the research question Lines 100-103 “both increases and decreases in MIC caused…”. Why is it not performed experiments on acquired resistance frequencies in drug herbicide combinations that reduced the MIC. This is only explored exclusively in MSC assays with defined pre-existing genetic variation. From figure 1 in Kurenbach et al 2015 it looks like there is several candidate combinations to explore this. Can we assume that this will cause the reverse result of the experiment performed with antibiotic/herbicide combinations resulting in an increased MIC? Also see point 1 in “Validity of results”
Validity of the findings: The experiments can broadly be divided into two different types of assays that is used to test various hypotheses, short term evolution in the form of fitness assays and mutation frequency assays. I have two larger concerns regarding the mutation frequency assays.

1.	Line 193 – 229 and Table 2: It is not clear to me how the authors can claim that acquired resistance increase due to herbicide-induced increases in MIC without designing this assay in a manner allowing for frequency data on LB + CIP (below MIC) as a control in this assay. Was the MSC for LB+CIP+Roundup/Kamba higher than the MIC of the parental strain? In its current form the results obtained from this assay looks somewhat forced since selective pressure on resistance development in essence only was allowed in the CIP+Herb combinations.

2.	The innate mutagenic effect of ciprofloxacin is not tested nor discussed even though that can be considered an alternative explanation for -or bias the results seen in the mutation frequency assays. The higher mutation frequencies reported on LB+CIP in Table 3 compared to LB+CIP+Herbicide in Table 2 might indicate this. See for instance Cirz et al 2005 plos biol,  https://doi.org/10.1371/journal.pbio.0030176 were this issue is addressed.
Additional comments: I struggle to keep the flow through the results section. For instance, Fig 2 and Fig 3B is clearly linked sharing a significant data point applying the exact same environment. However, there is a whole chapter with different methods between the two assays. It would be nice if the authors could try and bridge the different sections better so it is easier to follow.


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: AGRICHEMICALS AND ANTIBIOTICS IN COMBINATION INCREASE ANTIBIOTIC RESISTANCE EVOLUTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript needs additional information to better relay the experimental design and results to the public. There are questions regarding methods that need to be addressed. See comments to authors section.
Experimental design: The experimental design was not clearly articulated. More detail is in the comments to authors section.
Validity of the findings: The reviewer isn't convinced that comparison of bacterial growth curves after exposure to a mixed chemical formulation warrants statements that attribute endpoints to the active ingredient in those formulations.
Additional comments: Review of ms # 27947 – Agrichemicals boost the effects of antibiotics on antibiotic resistant evolution

First thoughts…  This is a very broad title that seems to encompass myriad chemical formulations used by the agricultural complex and then goes further to promise how this knowledge applies to (I’m assuming at this point) bacterial evolution. So, big promises.

-on to the abstract-

First notes:

“Antibiotic resistance is medicine’e climate change…”.       Hmmm…

Why use single quotes around ‘adaptive’? Sounds like there is a caveat coming.

Next sentence starts with “Adaptive” yet no explaination of ‘adaptive’.

Line 26 – “…of the antibiotic”.  What antibiotic? If you haven’t introduced “the antibiotic” then maybe say, “Adaptive changes can increase fitness under selective pressures such as those presented by antibiotics”.

Line 36 – This reviewer prefers to stay away from idioms such as “damned if you do/damned if you don’t”, plus I’m not sure it really applies here?

Line 49/50 – “…accelerate genotypic resistance…” This manuscript does not provide any information regarding genetic underpinnings of antibiotic resistance.

Line 67 – Is there a better way to say “invent us away from”?

Line 68 – replace “using them less” with “reduction of use” and the rest of the sentence with “which will help increase longevity”.

Lines 86-88 – “…resistance through changes in gene expression is also known as an adaptive (change in phenotype) response.” The reviewer disagrees with this interpretation of adaptive, or at least believes non-heritable acclimation by changes in gene expression are just that, acclimation. While heritable changes in genetic composition that allow for the persistence of a phenotype in a population are adaptive.

Materials and Methods
Media
The authors have used commercial herbicide formulations containing either a chlorinated benzoic acid, dicamba, or the organophosphate, glyphosate as Kamba and Roundup, respectively. Throughout the manuscript they attribute their observations to the active ingredient, however, the emulsifiers and detergents in these formulations may have profound effects on bacteria.

Plasmid constructs
The authors should use this section to better explain how the plasmids were utilized to reach their end goals. This would benefit from the addition of the plasmid maps as a supplemental figure. What did each trait allow? For example, simple addition of explanatory text such as, “which encoded for resistance to the antibiotics chloramphenicol, ampicillin, and tetracycline, respectively.” to the current sentence, “The resulting traits for this plasmid were Cam, Amp, and Tet.”, yields the sentence, “The resulting traits for this plasmid were Cam, Amp, and Tet, which encoded for resistance to the antibiotics chloramphenicol, ampicillin, and tetracycline, respectively.” This help the reader that may not be intimately familiar with the subject material to better understand the context.

Culturing conditions
Why differing seeding densities? Most likely because this allowed the best resolution of growth curves, but does seeding density have an effect on growth?

Determination of resistance levels
More information is needed here. Media concentrations are not listed.

What effects were measured to contrive the NOEL used here. Why NOEL vs. MIC as the rest of the paper?

Line 149 – Natural selection was defined as… then the author cited an article by van den Bosch et al. 2014. When the reviewer browsed the article, one of the first paragraphs that grabbed attention was, “Our findings are generalizations for fungal plant pathogens and fungicides only. They do not necessarily apply to resistance against antibiotics, insecticides, or herbicides because of the differences in genetic systems and the reproduction biologies of the species groups involved…” The authors should at least explain why they think this is a good approach in spite of the warning in their citation.

There are many other instances where this manuscript should be re-worked.


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: AGRICHEMICALS AND ANTIBIOTICS IN COMBINATION INCREASE ANTIBIOTIC RESISTANCE EVOLUTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: I found this quite a difficult paper to review as it took quite a bit of rereading of sections to get their meaning. I think the introduction needs a little more background as to the findings of their previous papers on the subject. You really have to have read those for this to make any sense. At times in the results section, it was also unclear whether the authors were referring to their previous work or to the data in the current paper.

I also have specific comments on the presentation of the data - means vs medians, SEMs vs SD, but include these under the 'General comments to the author' section of the review. For those not familiar with the way the data is presented, I think the figure and table legends would benefit from a short explanation of why the data is presented that way.
Experimental design: The research fits within the scope of PeerJ, and the research question is well defined, relevant and meaningful. However, I felt that the methods were not described in enough detail and that there were important controls not included in the experimental design. For many of the experiments, there was no control of the effect of the antibiotics alone on mutation frequencies. There was also no control for whether it was the active ingredient of the herbicides that have the effect, or whether it was other components like surfactants and other ingredients. While I understand that the authors may not think those controls relevant seeing as the herbicides are used as is, as a reader I wanted to know more about what might be responsible.
Validity of the findings: I found it difficult to assess validity without the controls I described previously.
Additional comments: Line 21: Abstract – “Antibiotic resistance is medicine’s climate change: caused by human activity” – This is a really strong and powerful statement and I like the sentiment, but it is a little misleading as currently written as antibiotic resistance itself is a natural phenomenon. Suggest the authors change to something that reflects the growing crisis caused by resistance organisms rather than using the term ‘antibiotic resistance’.
Lines 66-67: I’m not sure I agree with the sentiment that “Despite over half of century of warning, neither science nor innovation has managed to invent us away from the threat of a post-antibiotics era”. This suggests that we’ve had half a century of science/innovation actively working to invest us away from the threat, but that is clearly untrue. We’ve had half a century of inaction, which is not the same thing at all.
Line 82: missing word – ‘can also dependent’
Line 146: Please define NOEL
Line 160: biological or technical replicates?
Lines 208-209. The authors state that “Genetic variants able to grow on high concentrations of ciprofloxacin after 25 generations in LB medium, with or without herbicide supplementation, arose at the same frequency.” I’m a bit confused about this statement. Does this relate to the data presented in Table 2, because that doesn’t seem to be what the data shows, at least for Kamba and S. enterica. Our have I misunderstood that table?
Line 219: should : be ,?
Table 2: Please justify use of mean over median and of SEM over standard deviation (SD), and indicate what statistical tests performed. My understanding is that the SD indicates the dispersion of individual data values around their mean, and is what is required here for the reader to fully understand the variation in the data presented. In contrast, the SEM is a measure of the variability of the means that would be expected if the study were exactly replicated many more times and is used to calculate confidence intervals so isn’t appropriate here (see: http://ww1.cpa-apc.org/Publications/Archives/PDF/1996/Oct/strein2.pdf). What is the frequency of acquired resistance to Cip alone for comparison? This table isn’t very clear to me. I’m confused by which are significantly different from each other. I wonder if this data would be better presented as dot plots of the individual data points for each independent experiment. Is the a that relates to statistically testing in the right place in the table?
Figure 2: As before, please justify use of SEM over SD and indicate what statistical tests performed. Please also include name of bacteria being tested here.
Figure 3: Please indicate if mean or median values are given. As before, please justify use of mean over median and SEM over SD, and indicate what statistical tests performed. Please also include name of bacteria being tested here.


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: AGRICHEMICALS AND ANTIBIOTICS IN COMBINATION INCREASE ANTIBIOTIC RESISTANCE EVOLUTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: I'm satisfied with the authors corrections.
Experimental design: Satisfied with the author response
Validity of the findings: Regarding my concerns about the lack of mutation rate data on CIP only treatment (point 5 -7):

In the previous version the heading was, in my opinion, formulated in a manner that would have required this assay to have been designed in a way that allowed for mutation rates on CIP-only treatment. With the new heading this is no longer a necessity. I fully understand that the LB-CIP functioned as a control in your experiments when designed like this.
Yes, the experiments answer the hypothesis that herbicide induced increase in MIC increase the likelihood of resistance development of cip resistant mutants in an above wildtype MIC cip containing environment.
It is nice that you now mention mutagenic effects of antibiotics in the discussion but I still believe should be clearer that the several-fold increase in mutation rates might be due to the mutagenic effect of cip in a cip containing environment. This is because this assay cannot distinguish between selective effects and inherent mutagenic effects. My point is that if you used a non-mutagenic antibiotic substance you might not have seen a similarly elevated mutation rate when compared to the LB or LB-Herb treatment.
Additional comments: The authors make a big point in that they are measuring mutation rates and not frequencies. However, these are still also in the new version used interchangeably in the manuscript. See line
180 - For mutant frequency experiments
255 - The frequency ranged from

legend text table 2 (Frequencies of acquired resistance…; Herbicide+antibiotic mutant frequency…

Satisfied with all other responses and corrections made by the authors.


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: AGRICHEMICALS AND ANTIBIOTICS IN COMBINATION INCREASE ANTIBIOTIC RESISTANCE EVOLUTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is well-written, and clear. The authors have improved the flow of the manuscript from the first version so that it is now even more available to scientists that may not be experts in this field.
Experimental design: Data acquisition and analyses are sound.
Validity of the findings: The conclusions of this manuscript are valid and give credence to the concern that combinations of chemicals can affect the acquisition of, at least acclimative, resistance in bacteria.
Additional comments: Thank you to the authors for taking the time to revise the manuscript. This reviewer thinks that, overall, the manuscript has been greatly improved during the process.


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: AGRICHEMICALS AND ANTIBIOTICS IN COMBINATION INCREASE ANTIBIOTIC RESISTANCE EVOLUTION
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have addressed all of my queries/comments to my satisfaction.
Experimental design: The authors have addressed all of my queries/comments to my satisfaction.
Validity of the findings: The authors have addressed all of my queries/comments to my satisfaction.
Additional comments: Thanks for addressing all of my comments/queries. I'm still not sure I agree with your reworded assertion re having not innovated ourselves out of the antimicrobial resistance crisis, I'm happy to agree to disagree on this point.


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

Input:
Title: A NEW SPECIMEN OF PALVENNIA HOYBERGETI: IMPLICATIONS FOR CRANIAL AND PECTORAL GIRDLE ANATOMY IN OPHTHALMOSAURID ICHTHYOSAURS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Dear editor, dear authors,

First of all, I would like to disclose a conflict of interest in the sense that I have published recently and I am working with one of the authors, P.S. Druckenmiller. However, I assure you that it did not affect my review in any way.

The type of research and the comments I have made cannot be easily spread into the usual sections of PeerJ but rather from a coherent text. Hence, all my comments and suggestions are reproduced here.

I like this paper. It provides a very large amount of new data on an enigmatic taxon from the otherwise well sampled Late Jurassic. The analysis of coracoids is also an interesting direction to take. The descriptions are well written and quite informative (but see below). I have a couple of suggestions and points I would like to bring here in the review process. But none of them are major, nor do they require new analyses (except maybe regarding the coracoid analyses): mainly explanations, reformulations, and deletions. As a result, my recommendation is to accept the paper after a minor revision has been successfully carried on.

I list major and minor points below. Each refer to words or sentences that I have highlighted in a .pdf version of the paper to make it easier for the authors to spot.

Main points

1. While this paper provides a much-needed and thorough description, some important references are either totally missing or largely ignored, notably the works of colleagues who described ichthyosaurs that are often similar with the fauna from Svalbard. Indeed, many features are compared with a reduced array of species that are from radically distinct spatiotemporal settings: the middle Jurassic Ophthalmosaurus icenicus and the Early Cretaceous platypterygiines Sveltonectes insolitus and Platypterygius australis. While these are indeed some of the best known ophthalmosaurids, reducing the volume of comparisons with Late Jurassic ichthyosaurs is not advisable and might artificially convey a sense of ‘uniqueness’ for Palvennia, a taxon supported by a single autapomorphy (to be I do not doubt its validity but you need to compare it more thoroughly to other species).  I strongly suggest to add the following papers to the comparative description, especially for the craniodental and appendicular regions: (Romer, 1968; Fernández, 1997, 2007, Arkhangelsky, 1999, 2001, Efimov, 1999a,b; Bardet & Fernández, 2000; Arkhangelsky & Zverkov, 2014; Zverkov et al., 2015; Zverkov, Arkhangelsky & Stenshin, 2015; Tyborowski, 2016).

2. The identification/orientation of the following bones are problematic/poorly explained:
-The element interpreted as the opisthotic (e.g. Fig 9F) possesses a very unusual shape, especially the process medial to the paroccipital process. Could please you better explain this assignment and add annotations to Fig 9F accordingly?
- Fig 9H mentions the “articular view of quadrate”, while the quadrate appears to be depicted in posterior view. The articular view is posteroventrally oblique, showing the condyle.

3. Could you please provide a list of variable features among Palvennia as a table, this is so useful!

4. The coracoid geometric morphometrics appears as the most problematic part of the MS, by far. The methods are not well explained: we are just given the name of functions used in PAST, but without knowing the exact mathematical functions (and the reasons behind these choices). The authors should also explain why they used 8 landmarks while semi-landmarks (analysis of the outline) might have been more appropriate: indeed, the position of landmarks 3, 4, and 5 is just guesswork in the case of posteriorly rounded coracoids, i.e. that these points might not be homologous from one specimen to another. A similar issue arises for points 1, 7, and 8 in the case of anteriorly rounded coracoids (which is probably why the very well known Platypterygius australis was excluded from the analyses). Also, the dataset solely samples baracromians, not the wider clade Thunnosauria and this should be clearly stated in methods and the title of figure 21.
Also, the groups used to infer the degree of variation in baracromian coracoids are not on the same taxonomic level: one group represents the spread of a single species, O. icenicus, while the other represent the spread of a couple of species within the genus Stenopterygius. It might be better to restrict the groups to the specific level to avoid ambiguous interpretations. Finally, polygons depicting groups in morphospaces are convex hulls, which by essence, lack concavities

5. The last subchapter of the discussion. L1340-L1360: this entire paragraph does not provide anything new and no clear link is made with the rest of the paper
L1362-L1369. This entire paragraph rests on the expected link between the mediodistal length of ossified digits and the mediodistal length of the forefin. It is well known that such a link is hardly present in neoichthyosaurs; at least with the data presently at hand which is mainly based on Lower Jurassic forms. Indeed, at least some ichthyosaurs have a vast series of very small distal elements that are nearly always lost but which considerably lengthen the fin (Massare, Lomax & Klein, 2015). Also, the fin clearly extends more distal than the tip of the longest digit and this extension is quite variable (Bardet & Fernández, 2000; Lingham-Soliar & Plodowski, 2007). Thus, it is presently impossible to unambiguously speculate on the fin length in most ophthalmosaurids, making this whole paragraph conjectural. I would suggest to delete this subchapter entirely because it makes the paper end in a poorly constrained, conjectural way.

Minor points

L40-41: this makes the paper appear as very incremental and thus, of lesser interest. I would suggest to rephrase this.

L109: I do not understand this part, could you please clarify?

L129-131: as currently written this sentence is unclear and not correct: the acromion process is anterodorsal and can be captured by a 2D analysis. I would suggest the following: “in contrast, ichthyosaurian scapulae have processes arranged in orthogonal planes, which reduces the efficiency of 2D methods to capture the fine variations of their morphology”.

L138: for transparency, I would suggest to clearly list which data comes from whom/which specimen.

L139: photographs taken “directly above” do not prevent distortion, especially on the margins of the picture. Could you please rephrase?

L158: I have a hard time understanding why species that are so well known thanks to amazing material such as P. australis and B. natans are not considered, while a series of much less complete/fragmented specimens from the Slottsmoya (sorry I cannot make the proper vowel on my keyboard) member are.

L223: could you please provide the references for each of the comparisons?

L224: the text mentions the plural “autapomorphies” while I can only isolate one: “very large pineal foramen”. Again, for transparency and clarity, I would suggest to clearly state that the taxon is based on a single autapomorphy. This of course does not undermine the impressive differential diagnosis you have provided.

L251. “Based on the orientation” of what? “Landing”, I do not understand; do you mean the individual entered the soft sediment belly up?

L276. “Symphysis” applies, broadly and theoretically, for any joint of bone with some kind of cartilage in between. In our field it nearly always implies the mandibular symphysis and I’m not sure it can be applied to ichthyosaurian premaxillae.

L301: The ‘contact’ – or lack thereof – of the frontal with the supratemporal fenestra can be an important feature among ophthalmosaurids. Could you describe it and compare it with other taxa?

L415: consider removing “bone”

L418: “has been” instead of “is”?

L421: the reference after Janusaurus lundi is italicized.

L474: the reference after Arthropterygius chrisorum is italicized

L501-505: The meaning of this feature is unclear, could you rephrase? Figure 9F is not very informative either; could you please add annotations? The paroccipital process is indicated in the ‘middle’ of the lateral portion of the bulk of the bone while it should be process extending (postero)dorsolaterally. If this region is a reduced paroccipital process, what is the large (sheet? of) bone that is medial to it?

L696-698: This is the case for all ophthalmosaurids, not just Palvennia and Cryopterygius.

L698-L699: This smooth region is termed the acellular cementum ring (Maxwell, Caldwell & Lamoureux, 2011, 2012)

L706-707: All ichthyosaurs have trapezoidal sclerotic plates; the following sentence is more informative.

L719-720: A similar feature has been described in Grendelius/Brachypterygius by (Zverkov, Arkhangelsky & Stenshin, 2015).

L758-760: This is one of the difficulties of comparing outlines; the coracoids of Sveltonectes insolitus are very strongly thickened medially for example; which does not appear to be the case for Palvennia.

L762: The reference after “P. autralis” is italicized.

L785-786: could you please explain this sentence a bit more? All neoichthyosaurian humeri have a mid-shaft constriction.

L800: The reference after “Ophthalmosaurus icenicus” is italicized.

L800-801. Are these similar to the bulb-like process found in Sveltonectes and some other ophthalmosaurids (notably the Late Jurassic ‘Macropterygius’ material pers. obs. MJML [the Etches collection])

L807-808: and Maiaspondylus lindoei and Grendelius (Druckenmiller & Maxwell, 2010; Zverkov, Arkhangelsky & Stenshin, 2015).

L812: The reference after “Gengasaurus nicosiai” is italicized.

L813: please add “in” after “unlike”. Platypterygiine ophthalmosaurids lack this deflection of the ulnar facet as well (those with an intermedium facet on the humerus have a similar but non-homologous situation though).

L837-838: this region of the holotype of Gengasaurus is very poorly preserved and the ulna might actually be incomplete.

L843: Could you please describe the posterior margin of the ulna more thoroughly, notably whether the ulna is posteriorly tapering or not; this can be an important character.

L848: The reference after “Cryopterygius kristiansenae” is italicized. Also, this condition is seen in many other ophthalmosaurids.

L955: Given the fact that most ophthalmosaurids humeri are known, could you please add more comparisons?

L980: similar comment.

L991: similar comment.

L1034: the correct affiliation is now NHMUK I think.

L1035: The reference after “Platypterygius hercynicus” is italicized.

L1073: Same comment as for the other indet specimen above: please add comparisons, especially with other Late Jurassic taxa. Otherwise, these specimens have very little value in the present version of the paper.

L1085. I would advise against abbreviating here, as there are some other generic name starting with a P.

L1035: The reference after “Sveltonectes insolitus” is italicized.

L1171-1175: the number of exoccipital foramina is a feature that is known to vary intraspecifically in ichthyosaurs (Maisch, 1997).

L1096-1097: as in?

L1182: The reference after “Keilhauia nui” is italicized.

L1182-1184 (and the rest of the paragraph): all the features listed here also apply to Ophthalmosaurus, Baptanodon, Arthropterygius, and, depending on how you quantify what is a “relatively small deltopectoral crest”, Cryopterygius spp., and Acamptonectes.

L1189: not sure if “relationship” is the right word here, could you please clarify?

L1215-1216 and the rest of the paragraph: this is actually known to vary in Ichthyosaurus (Vincent et al., 2014). It is also a likely ontogenetic feature among plesiosaurians (e.g. Fischer et al., 2018).

L1221: or “organ” instead of “eye”?

L1229: the use of “variable” leads to confusion as it might be interpreted that this is an (intraspecifically) variable feature. May I suggest something along the lines of: “it is clear that this feature is restricted to a single species of the SML ichthyosaur assemblage”.

L1237: The reference after “Opthalmosaurus icenicus” is italicized.

L1248: Ibid.

L1259: I might be biased here, but this subdivision is a valid result from a valid analysis. The fact that it represents a genuine cladogenetic event is what is tested and questioned by the accumulation of novel data. Given the high homoplasy in the evolution of Thunnosauria, we expect a series of reversals to take place, and this might or might not affect the architecture of the ophthalmosaurid tree. In all fairness, I also think that the story is more complex that a simple subdivision in two clades, but you need new phylogenetic results to back such kind of claim.

L1262-1263. There is an issue in the fraction of variance explained by the axes. As you surely know, the total of all axes = 100%. I think you mean that axes 4, 5, 6, 7, etc. each explain less than 13% of the variance. I would suggest to provide a table with each value.

L1263: “increasing values of PC1”. I do not understand; could you please clarify?

L1267: Latin names of taxa cannot be used as adjectives.

L1289-1290: this should be tested using proper clustering methods rather than a PCA, which in essence, just a visualization tool.

L1297-1298: this is unclear; do you mean that both are congeneric, i.e. that Cryopterygius is a junior synonym of Undorosaurus?

L1304: same comment as for line 1267.

L1305-1307: This might actually result from a bias your analysis: you mix specific and generic taxonomic levels when discussing the morphological disparity; see also main point 4 above.

L1312-1313: In all honesty, who ever doubted that coracoids are shaped by phyletic heritage rather than being a purely convergent element??

L1321: May I also suggest to cite the work who coined the name? Asking for a friend… (Fischer et al., 2013)

L1329: In Acamptonectes, it is probably ontogeny; such source for the intraspecific variation in important to highlight.

Table 1: Specimen S2: “sp.” is italicized.

Good luck,

Valentin Fischer

References mentioned in the review

Arkhangelsky MS. 1999. On a new ichthyosaur from the Callovian stage of the Volga region near Saratov. Paleontological Journal 33:88–90.
Arkhangelsky MS. 2001. On a new ichthyosaur of the genus Otschevia from the Volgian Stage of the Volga Region near Ulyanovsk. Paleontological Journal 35:629–634.
Arkhangelsky MS., Zverkov NG. 2014. On a new ichthyosaur of the genus Undorosaurus. Proceedings of the Zoological Institute RAS 318:187–196.
Bardet N., Fernández M. 2000. A new ichthyosaur from the Upper Jurassic lithographic limestones of Bavaria. Journal of Paleontology 74:503–511.
Druckenmiller PS., Maxwell EE. 2010. A new Lower Cretaceous (lower Albian) ichthyosaur genus from the Clearwater Formation, Alberta, Canada. Canadian Journal of Earth Sciences 47:1037–1053.
Efimov VM. 1999a. Ichthyosaurs of a new genus Yasykovia from the Upper Jurassic strata of European Russia. Paleontological Journal 33:92–100.
Efimov VM. 1999b. A new family of ichthyosaurs, the Undorosauridae fam. nov. from the Volgian stage of the European part of Russia. Paleontological Journal 33:174–181.
Fernández M. 1997. A new ichthyosaur from the Tithonian (Late Jurassic) of the Neuquén Basin (Argentina). Journal of Paleontology 71:479–484.
Fernández M. 2007. Redescription and phylogenetic position of Caypullisaurus (Ichthyosauria: Ophthalmosauridae). Journal of Paleontology 81:368–375.
Fischer V., Appleby RM., Naish D., Liston J., Riding JB., Brindley S., Godefroit P. 2013. A basal thunnosaurian from Iraq reveals disparate phylogenetic origins for Cretaceous ichthyosaurs. Biology Letters 9:1–6. DOI: 10.1098/rsbl.2013.0021.
Fischer V., Benson RBJ., Druckenmiller PS., Ketchum HF., Bardet N. 2018. The evolutionary history of polycotylid plesiosaurians. Royal Society Open Science 5. DOI: 10.1098/rsos.172177.
Lingham-Soliar T., Plodowski G. 2007. Taphonomic evidence for high-speed adapted fins in thunniform ichthyosaurs. Naturwissenschaften 94:65–70.
Maisch MW. 1997. Variationen im Verlauf der Gerhinnerven bei Ophthalmosaurus (Ichthyosauria, Jura). Neues Jahrbuch für Geologie und Paläontologie, Monatshefte 1997:425–433.
Massare JA., Lomax DR., Klein A. 2015. A large forefin of  Ichthyosaurus from the U.K., and estimates of the size range of the genus. Paludicola 10:119–135.
Maxwell EE., Caldwell MW., Lamoureux DO. 2011. Tooth histology in the Cretaceous ichthyosaur Platypterygius australis , and its significance for the conservation and divergence of mineralized tooth tissues in amniotes. Journal of Morphology 272:129–135.
Maxwell EE., Caldwell MW., Lamoureux DO. 2012. Tooth histology, attachment, and replacement in the Ichthyopterygia reviewed in an evolutionary context. Paläontologische Zeitschrift 86:1–14.
Romer AS. 1968. An ichthyosaur skull from the Cretaceous of Wyoming. Contributions to Geology, Wyoming University 7:27–41.
Tyborowski D. 2016. A new ophthalmosaurid ichthyosaur species from the Late Jurassic of Owadów-Brzezinki Quarry, Poland. Acta Palaeontologica Polonica 61:1–13. DOI: 10.4202/app.00252.2016.
Vincent P., Taquet P., Fischer V., Bardet N., Falconnet J., Godefroit P. 2014. Mary Anning’s legacy to French vertebrate palaeontology. Geological Magazine 151:7–20. DOI: 10.1017/S0016756813000861.
Zverkov NG., Arkhangelsky MS., Pardo-Pérez J., Beznosov PA. 2015. On the Upper Jurassic ichthyosaur remains from the Russian North. Proceedings of the Zoological Institute RAS 319:81–97.
Zverkov NG., Arkhangelsky MS., Stenshin IM. 2015. A review of Russian Upper Jurassic ichthyosaurs with an intermedium/humeral contact – Reassessing Grendelius Mcgowan, 1976. Proceedings of the Zoological Institute RAS 319:558–588.
Experimental design: The type of research and the comments I have made cannot be easily spread into the usual sections of PeerJ but rather from a coherent text. Hence, all my comments and suggestions are reproduced in part 1 Basic reporting.
Validity of the findings: The type of research and the comments I have made cannot be easily spread into the usual sections of PeerJ but rather from a coherent text. Hence, all my comments and suggestions are reproduced in part 1 Basic reporting.
Additional comments: The type of research and the comments I have made cannot be easily spread into the usual sections of PeerJ but rather from a coherent text. Hence, all my comments and suggestions are reproduced in part 1 Basic reporting.


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

Input:
Title: A NEW SPECIMEN OF PALVENNIA HOYBERGETI: IMPLICATIONS FOR CRANIAL AND PECTORAL GIRDLE ANATOMY IN OPHTHALMOSAURID ICHTHYOSAURS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I have added my comments to the authors in the 'General comments to author' section, below.

Clear and unambiguous, professional English used throughout.
   - Yes.

Literature references, sufficient field
   - Yes.

Professional article structure, figs, tables. Raw data shared
   - Yes.

Self-contained with relevant results to hypotheses
   - Yes
Experimental design: Original primary research within Aims and Scope of the journal.
   - Yes.

Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap
   - Yes. However, I have expanded upon this in the comments below.

Rigorous investigation performed to a high technical & ethical standard.
   - Yes.

Methods described with sufficient detail & information to replicate
   - Yes.
Validity of the findings: Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated
   - There study will be of interest to anybody working on ichthyosaurs. Specifically, for anybody interested in the Spitsbergen fauna and interested in the phylogenetic importance of the ichthyosaur coracoid.

Data is robust, statistically sound, & controlled
   - Yes.

Conclusion are well stated, linked to original research question & limited to supporting results
   - Not quite. All of the research is supported, but there is no conclusion section that really summarises the entire paper.
Additional comments: 22.06.2018
Reviewer: Dean Lomax

Review PeerJ manuscript #28469

A new specimen of Palvennia hoybergeti: Implications for cranial and pectoral girdle anatomy in ophthalmosaurid ichthyosaurs

Firstly, I would like to congratulate the authors on a great paper. The descriptions of the specimens are quite excellent. Well done to you all. I do, however, have some issues with the paper, although most of these things are very minor, which are included in the marked-up pdf document, but I wanted to elaborate on a couple of these below.

1. Testing variation in the SML ophthalmosaurid coracoids
I really like what you have done with this. However, with the SML specimens you are, of course, limited to a small sample size, which can have its own issues (see below). You have also only used x6 Stenopterygius specimens, of which thousands of specimens are known, so you don’t capture the full/bigger picture. In total, you only use 30 specimens. In order to really grasp an understanding of this level of variation it’s always better to include a much greater sample size. Of course, I understand the limitations of this and I do not suggest you go and examine 100+ ophthalmosaurid coracoids and 100+ Stenopterygius coracoids, but this would be another (bigger) step to test the PCA and to explore the phylogenetic importance of the coracoid, which could also be extended to include even more taxa. Instead of doing this as part of the present study, you must include something along these lines otherwise you run the risk of being criticised for not using more specimens.

Bearing this in mind, and the reason why I wanted to bring the above to your attention, I would recommend you have a read of Massare and Lomax (2018), which discussed this sort of issue with respect to hindfins in Ichthyosaurus. We found that: fewer specimens = appears to show ‘distinct’ species characters; BUT more specimens however = show that this is a continuum of variation among specimens and that many ‘unique’ characters are simply the result of this variation. For example, in a smaller sample size, some of these features might have been considered as representative of a new species.

2. Results of landmark analysis
This and the following sections on the pectoral girdle seem out of place, or at the very least need to have an introductory couple of sentences and/or a sub-heading to explain this. It seems out of place because you go from the description of the material (importance of the material) to ‘Results of landmark…’

3. Ontogeny of PMO 222.669
You have not provided a distinct section on the ontogeny of PMO 222.669 like you have for the other described specimens.

4. Consistency
Although the MS is very well written, there are some inconsistencies, e.g. use of meter Vs metre Vs m. Or Slottsmøya Member Lagerstätte is used but sometimes SML.

5. Coracoid of PMO 222.669
You say that the coracoid is markedly longer than wide but I disagree. Your measurements are 260 vs 220, so not that much of a difference. Instead, would be better to say slightly longer than wide.

6. Conclusion.
I feel that you need to have a conclusion at the end of the manuscript, to really emphasise: a. the importance of your new specimen; b. importance of the indet specimens; c. your PCA and the findings therein; d. future studies that can be built from your study – i.e. as above, more taxa and coracoids added to the PCA may reveal even more important phylogenetic information.

References mentioned in the marked-up pdf
	- Apologies that several of the following are papers authored or co-authored by me, but I feel they will assist with your study, as outlined in the marked-up Pdf.

Danise S, Twitchett RJ, Matts K. 2014. Ecological succession of a Jurassic shallow-water ichthyosaur fall. Nature Communications.

Lomax, D. R., Evans, M. and Carpenter, S. 2018. An ichthyosaur from the UK Triassic–Jurassic boundary: A second specimen of the leptonectid ichthyosaur Wahlisaurus massarae Lomax 2016. Geological Journal, 10.1002/gj.3155.

Lomax, D. R., De la Salle, P., Massare, J. A. and Gallois, R. 2018. A giant Late Triassic ichthyosaur from the UK and a reinterpretation of the Aust Cliff ‘dinosaurian’ bones. PLOS One, doi.org/10.1371/journal.pone.0194742.

Lomax, D. R. and J. A. Massare. 2015. A new species of Ichthyosaurus from the Lower Jurassic of west Dorset, England. Journal of Vertebrate Paleontology.

Lomax, D. R. and Massare, J. A. 2017. Two new species of Ichthyosaurus from the lowermost Jurassic (Hettangian) of Somerset, England. Papers in Palaeontology, 3, 1–20.

Lomax, D. R., Larkin, N. R., Boomer, S., Dey, S. and Copestake, P. 2017. The first known neonate Ichthyosaurus communis skeleton: a rediscovered specimen from the Lower Jurassic, UK. Historical Biology.

Massare, J. A. and Lomax. D. R. 2017. A taxonomic reassessment of Ichthyosaurus communis and I. intermedius and a revised diagnosis for the genus. Journal of Systematic Palaeontology, http://dx.doi.org/10.1080/14772019.2017.1291116

Massare, J. A. and Lomax, D. R. 2018. Hindfins of Ichthyosaurus: effects of large sample size on ‘distinct’ morphological characters. Geological Magazine, doi:10.1017/S0016756818000146.

McGowan, C. 1993. A new species of large, long-snouted ichthyosaur from the English lower Lias. Canadian Journal of Earth Sciences, 30: 1197–1204.

Wahl, W. R. 2009. TAPHONOMY OF A NOSE DIVE: BONE AND TOOTH DISPLACEMENT AND MINERAL ACCRETION IN AN ICHTHYOSAUR SKULL. Paludicola, 7, 107-116.

Wahl, W. R. 2011. Hyoid structure and breathing in ichthyosaurs. Journal of Vertebrate Paleontology 31: 183A. [Poster presentation]

I look forward to seeing this paper published.

Best regards,
Dean Lomax

Visiting Scientist
School of Earth and Environmental Sciences,
The University of Manchester