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 MODELED DISTRIBUTION OF CORALS AND SPONGES SURROUNDING THE SALAS Y GÓMEZ AND NAZCA RIDGES WITH IMPLICATIONS FOR HIGH SEAS CONSERVATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript represents a good modelling exercise of three important benthic taxa. The modelling approach is effective as a tool in marine management and the manuscript is well written and structured. I consider that the study is interesting and the results provided are meaningful. Figures and tables are relevant and well labelled and described. I have only minor suggestions and I recommend publishing the work after a revision following the points suggested below:

Material and Methods
Line 159 – 160. It would be interesting to provide a table with the species or taxon and number of records included in each of the Orders modelled. This could give some guidance about the species or taxon that is driving the results in each group that could be added to the discussion and useful to compare with other studies.

Line 179 – 181. This way of creating pseudoabsence records seems an interesting way to mirror the sampling bias. However, I wonder if this method will provide better results than other recently described (e.g. Iturbide et al. 2015; 2018). Perhaps this needs more discussion.

Iturbide, M., Bedia, J., and Gutiérrez, J.M. 2018. Tackling uncertainties of species distribution model projections with package mopa. The R Journal, 10(1), 122. https ://doi.org/10.32614/ RJ-2018-019

Iturbide, M., Bedia, J., Herrera, S., del Hierro, O., Pinto, M., and Gutiérrez, J. M. 2015. A framework for species distribution modelling with improved pseudo-absence generation. Ecological Modelling, 312, 166–174. https ://doi.org/10.1016/j.ecolm odel.2015.05.018

Line 195. Index of northness using a sine transformation. Should this be cosine transformation?

Line 267. It is common to use 4 degrees of freedom in GAMs to avoid overfitting. However, as the authors are including different taxa in each group, these may not show unimodal response to the environmental variables so more degrees of freedom are ok. However, I wonder why 12 degrees of freedom are chosen and not allow the method to estimate them.

Line 271. I do not understand how 501 trees ensure stabilization in RF when the default is 500. I consider that this needs to be clarified.

Line 272. Could the authors provide a reference or more explanation in how limiting the tree depth to a value of 10 can prevent overfitting to the training data? The results (line 348) show that RF models were slightly overfitting. I wonder if it could be recommended to try different values, as done with ‘mtry’ in preliminary models to assess if this parameter would avoid overfitting.

Line 290. Which threshold was used in the calculation of Kappa? Prevalence? If that is the case, how the selection of pseudo-absences could affect this calculation? Perhaps would be enough to present AUC and TSS that are both independent of species prevalence.

I suggest to present uncertainty maps associated to the predicted habitat suitability maps that can help the interpretability of the results and are very useful for conservation and management.

Results
Line 357. … and 0.952 for stony corals. Should this value be 0.973?\

Discussion
Line 506 – 513. How the lower resolution for some of the environmental variables could affect the results? It may be that it is difficult to find a relationship because the resolution is very low?

References
Some references are missing (e.g. line 60, Cordes et al. 2008; table 1 Carton et al. 2005) or need to be checked (e.g. line 65 Hill 2004; line 72 van Dover 2014)


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 MODELED DISTRIBUTION OF CORALS AND SPONGES SURROUNDING THE SALAS Y GÓMEZ AND NAZCA RIDGES WITH IMPLICATIONS FOR HIGH SEAS CONSERVATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: See below/attachment
Experimental design: See below/attachment
Validity of the findings: See below/attachment
Additional comments: See attachment for some graphics that I've used to illustrate one comment. Below are just copy and pasted.

The authors have produced species distribution models for a little studied region off the coast of South America. Their intent is to use such models to assist in the designation of areas that may contain highly suitable habitats for key benthic taxa in the form of demosponge, glass sponges and stony corals. The authors have followed appropriate methodologies for the modeling work, and the manuscript is clear and well produced. The ensemble approach used is essentially the standard one being used in most modern deep-sea species distribution studies, and the range of model types is appropriate. The quality of writing is high, and I have no major comments pertaining to the writing.

I thank the authors for taking such an approach that is well grounded in the literature. I have only a few minor comments (items 1-3) and one moderate (item 4) methodological comment that I believe the authors should at least respond to.

1. Sampling bias approach
It is great to see consideration of sampling bias in this study, and it is becoming increasingly used, so any case studies that incorporate this, over say, random background selection is valuable. I do have some questions on this, which are likely more discussion points outside of the manuscript. Basically, when I explored the supplementary figures, there were some spurious placements of pseudo-absences in some regions (see example below) and I wasn’t clear exactly how the probability grid was created, and how points were placed based upon it. Can you clarify in the methods the exact approach used, i.e. how were the 10,000 points distributed throughout the grid. As it stands, I wasn’t able to fully visualize the approach.

See attachment for images..

2. Who created the environmental data?
I was not 100% clear if  some of the environmental layers (not the topographic layers, and surface derived remote sensing layers) were created specifically in this study or were obtained from Davies and Guinotte (2011), which are freely available on Zenodo. Clearly the use of WOA13 supersedes several environmental layers created by then. It is not a critical point in terms of using the data, but the use of sensu in the attribution, to me, states that you replicated the approach and generated all the layers independently, if you used the data from the Zenodo repository where the Davies and Guinotte layers are stored or obtained it directly from Davies and Guinotte, then you should attribute them more appropriately.

3. Old environmental and bathymetric data?
One of the central requirements for species distribution modeling is to use the best available data. For the presence dataset I have no doubt that it represents the most recent data available. However, the bathymetry and the World Ocean Atlas data used in this manuscript are clearly older. These two products have moved on significantly since SRTM30 was released by Becker et al in 2009 and World Ocean Atlas 2013. There are newer products, what I do not know is how different these products are for the modeled region. It is entirely possible that in GEBCO 2020 or SRTM15, that there are new areas of high resolution multibeam incorporated. Likewise, given World Ocean Atlas is now at version 2018, but I do not know if there are new data from this region. I encourage the authors to explore these data to ensure they are using the most up to date environmental datasets.

4. Validation of environmental layers
This point is valid in either situation outlined in item 2 above. I firmly believe that validation of the environmental data is as important as the validation of the species distribution models themselves. In Davies and Guinotte 2011, extensive calibration of the environmental data extrapolation was undertaken, but this was for the global ocean. When moving down to a regional area, we cannot rely upon a global calibration exercise to show the approach is appropriate. Validation must be undertaken, and it is a fairly simple process. It does not strictly need to be independent, as all we are asking, is how well does the environmental data used fit “reality”. Reality in this case, is out best-known understanding of the main oceanographic conditions. I would really like to see this analysis done, as it is needed to provide full confidence in the models that are created.


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: NEW FREE-LIVING NEMATODE SPECIES AND RECORDS (CHROMADOREA: PLECTIDA AND DESMODORIDA) FROM THE EDGE AND AXIS OF KERMADEC TRENCH, SOUTHWEST PACIFIC OCEAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: Manuscript is devoted to the description of the free-living deep sea nematofauna that is important topic in the frame of world biodiversity investigation programs. Manuscript is well supported with materials and literature. Figures are of a very good quality.
Experimental design: Research is within aim and scope of the journal. Although line drawings are perfect there is still the lack of at least light microscopical photographs. Adding light microscopy and scanning electron microscopy photos might greatly improve the manuscript.
Validity of the findings: no comment
Additional comments: The weakest point of the manuscript is the absence of photos of described species with the exeptions of Desmodora pilosa. I believe that this disadvantage could be easily corrected.
The second main remark concerns the structure of cephalic capsule of Desmodora (line 272). Author describe the presence of numerous dense irregularly distributed pores. Using scanning electron microscopy it was shown that cephalic capsule of Desmodorids is smooth. What is seen as pores are in fact some king of rods in the median zone of cuticle that strengthen cephalic capsule and seen as pores or dots under light microscope. The question is why are they found in some species and not found in others? There are may be several explanation that needs additional studies.
Editorial corrections
Line 75-77 Repetition of previous paragraph
Line 119-120 Leduc 2020 - no reference in the reference list
Line 226 Secretroy-excterory .... present. The noun is omitted.
Line 316 Filipjev 1946 and Jensen 1991 -  no references in the reference list
Line 464 B. cephalic region; .... posterior body. C is omitted
Table 1. Line 1-4. all abbreviation is given in material and methods part.
Table 2. The same with previous.


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: NEW FREE-LIVING NEMATODE SPECIES AND RECORDS (CHROMADOREA: PLECTIDA AND DESMODORIDA) FROM THE EDGE AND AXIS OF KERMADEC TRENCH, SOUTHWEST PACIFIC OCEAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comments
Experimental design: no comments
Validity of the findings: no comments
Additional comments: Well written manuscript without any issues. Too bad there are so few specimens of Alaimella or Desmodora but it would be rather challenging to try and collect more material. I recommend to accept the manuscript, as I have no comments or corrections.


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: NEW FREE-LIVING NEMATODE SPECIES AND RECORDS (CHROMADOREA: PLECTIDA AND DESMODORIDA) FROM THE EDGE AND AXIS OF KERMADEC TRENCH, SOUTHWEST PACIFIC OCEAN
Review round: 1
Reviewer: 3

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.

The manuscript includes description of new nematode species Leptolaimus hadalis sp. nov.. from the edge (6080 m depth) and axis (7132 m) of Kermadec Trench, Southwest Pacific with some remarks about the genus. Alaimella aff. cincta and Desmodora aff. pilosa are recorded for the first time from the Southwest Pacific region. This manuscript includes description of new nematode species provides detailed morphological comparisons between other species/populations from other localities in order to assess the status of the Kermadec Trench populations.
The manuscript contains new, original data. All tables and figures are important and contain necessary information. The description of new species is correct and clear. Drawings are photos are very good quality. A new zoological taxon Leptolaimus hadalis sp. nov, is provided the name and LSID(s) and the publication LSID.
Literature references, sufficient field background/context provided.
Experimental design: Original primary research within Aims and Scope of the journal.
Research question well defined, relevant & meaningful. The nematode fauna of these deep environments remains very poorly known, and because of this we have little information on their ecology and of patterns benthic diversity within and across trench habitats.
The manuscript provides morphological descriptions of nematode species from Kermadec Trench, Southwest Pacific Ocean. The new information in the manuscript will also help future investigations of trench ecology.
Rigorous investigation performed to a high technical standard.
Methods described with sufficient detail & information to replicate.
Validity of the findings: The manuscript contains new, original data.

All underlying data have been provided; they are robust, statistically sound.

Conclusions are well stated, linked to original research question and limited to supporting results.
Additional comments: The nematode fauna of these deep environments remains very poorly known, and because of this we have little information on their ecology and of patterns benthic diversity within and across trench habitats.
The manuscript provides morphological descriptions of nematode species from Kermadec Trench, Southwest Pacific Ocean. The new information in the manuscript will also help future investigations of trench ecology.
The manuscript includes description of new nematode species Leptolaimus hadalis sp. nov.. from the edge (6080 m depth) and axis (7132 m) of Kermadec Trench, Southwest Pacific with some remarks about the genus. Alaimella aff. cincta and Desmodora aff. pilosa are recorded for the first time from the Southwest Pacific region. This manuscript includes description of new nematode species provides detailed morphological comparisons between other species/populations from other localities in order to assess the status of the Kermadec Trench populations.
The manuscript contains new, original data. All tables and figures are important and contain necessary information. The description of new species is correct and clear. Drawings are photos are very good quality. A new zoological taxon Leptolaimus hadalis sp. nov, is provided the name and LSID(s) and the publication LSID.
I suppose, differential diagnosis for a new species of Leptolaimus hadalis sp. nov.  is not complete enough. Your differential diagnosis needs more detail. There is no comparison with the close species L. membranaceus (Wieser 1951) and L. septempapillatus Platt 1973.
Although your results are compelling, the data analysis should be improved. You should  discuss the paper Fadeeva, N. P. & Mordukhovich, V. V. (2007) [New and known Leptolaimidae (Nematoda, Chromadoridae) species in the Sea of Okhotsk and the Sea of Japan.] Zoologicheskii Zhurnal, 86, 3-15. [in Russian], where the Pacific species of the genus Leptolaimus are described.


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: QUANTITATIVE TRAIT LOCUS MAPPING ANALYSIS OF MULTIPLE TRAITS WHEN USING GENOTYPE DATA WITH POTENTIAL ERRORS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The text of the article is very neat and professional.
Experimental design: The design of simulated data can benefit from more extended explanation into its biological validity.
Validity of the findings: As pointed in general comments, the real test set needs to be expanded and a more rigorous benchmarking needs to be carried out against other existing methods.
Additional comments: In this article, the authors present a minor upgrade on previous a method on multi-trait QTL mapping. The authors first provide a rigorous mathematical definition of the problem, then provide a solution using EM algorithm. Then, first by using a set of simulated data then using a locus of real genotype data the method is being tested.

While the proposed method certainly is of real importance, I have a few questions and concerns that I think would help the manuscript to be more digestible for a reader:
1 – In the simulation studies part, presenting the results in terms of a large table is not advised. A graphical display followed with a statistical method such as ANOVA on the estimated parameters compared to the real values can be provided, at least as a supplementary figure. It is very hard to understand, from the table, the spread of deviations for different methods.
2 – The method needs to be run on multiple real QTL values from previously reported methods and benchmark against them. Using a single locus with 3 markers does not provide a sufficient amount of confidence in the performance evaluation.
3 – The study also needs to provide information on technical sides of the method such as; how many iteration EM takes to converge, memory consumption, estimated upper limits in terms of number of traints etc.


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: QUANTITATIVE TRAIT LOCUS MAPPING ANALYSIS OF MULTIPLE TRAITS WHEN USING GENOTYPE DATA WITH POTENTIAL ERRORS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article titled “QTL mapping analysis of multiple traits when using genotype data with potential errors (#62130)” by Tong L et al., proposed a new method of multiple trait and multiple interval mapping in the genetic data set with genotype errors.
Experimental design: Overall, the manuscript draft is well written and presented with good mathematical models and formulas. The statistical models are also justified.
Validity of the findings: The findings were of interest to the peers working around QTL mapping and multiple trait analysis and related studies.
The study design justifies the rationale of this work and the models were well designed. The studies were validated with statistical models and the limitations like excess amount of computation is also mentioned.
Additional comments: . As a reviewer from a reader’s point of view the below questions were made to clarify and improve the overall reach of this work.
Comments:
1.	In general, what is the overall percentage of genotype errors in a given data set? Authors may estimate the % of errors from 3 or more large data sets and estimate the genotype errors in the data.
2.	A table can be included with analysis of 3-5 data sets in which % genotype error can be determined and how successful is this newly proposed method can identify and fill the gaps
3.	Can this method be applied to large data sets including human genome data, bacterial genomes data etc.? or is there a limitation for this method?
4.	Can a computational tool/ application be developed for this method so that it can be of use for the scientists working in genome studies and other genomics works? If there is an existing website/software for the same, a link can be included in the discussion part.
Minor changes:
1.	Line 19: Change to “in a given data set”
2.	Line 20: Change to “limitations of biotechnology”
3.	Line 51: Change to “on a mixed linear model”
4.	Line 53: Change to “with the single-trait”
5.	Line 185: Change to “The analyzed results”
6.	Line 185: Change to “All the estimated results”
7.	Line 189: Change to “gave a positive”
8.	Line 190: Change to “obtained a negative”
9.	Line 190: Change to “value of the dominant”
10.	Line 199: Change to “analyze a data set”
11.	Line 212: Change to “obtaining an estimate”
12.	Line 217: Change to “QTL effect on”
13.	Line 226: Change to “adapt to much”


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: QUANTITATIVE TRAIT LOCUS MAPPING ANALYSIS OF MULTIPLE TRAITS WHEN USING GENOTYPE DATA WITH POTENTIAL ERRORS
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this manuscript the authors proposed a new QTL mapping strategy of multiple traits in the presence of the genotype errors in the genetic datasets. Within the new method, many parameters such as QTL recombination rates, covariance matrix, QTL effects and genotype error rates can be estimated, simultaneously.  In order to show the accuracy of parameter estimation, they used simulated datasets which are generated under different scenarios and a real dataset, as well. Their results showed that their method can map QTLs of multiple traits more accurately than existing methods in the literature. The study is quiet interesting and the findings seem significant enough.  I only have a few comments. Even though the main idea is clear I think the language should be improved for the intelligibility of the manuscript. There are some syntax and grammatical errors such as “i,e,....” in the line 214 and some punctuation errors such as “....; The true values “ in the line 143. Please do write the vectors in the formulas in bold. In the line 120, what does “G_....” refers to? In line 127, instead of using “.... above equation” give a number to the equation and refer it as Eq. ().
Experimental design: The research question is well defined.  The experimental design, the theory and the statistical model are explained in details.
Validity of the findings: In order to show the accuracy of parameter estimation, they generated simulated datasets and applied their strategy on simulated datasets under different scenarios. Additionally, they performed the same analysis on a real data. The results seem to be robust, scientifically sound and support the authors in their findings.
Additional comments: In order to have a clearer manuscript, improve the readability of the manuscript and to reach out more researchers, I suggest the authors to make sure that they carefully read their manuscript from the scratch and make corrections accordingly. Please use a simple and clear language so that it does not lead to any confusion while reading the paper.


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: QUANTITATIVE TRAIT LOCUS MAPPING ANALYSIS OF MULTIPLE TRAITS WHEN USING GENOTYPE DATA WITH POTENTIAL ERRORS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: Adequately explained in revision.
Validity of the findings: Might be of value for very specific tasks.
Additional comments: All the questions and comments that we posed were adequately addressed 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: QUANTITATIVE TRAIT LOCUS MAPPING ANALYSIS OF MULTIPLE TRAITS WHEN USING GENOTYPE DATA WITH POTENTIAL ERRORS
Review round: 2
Reviewer: 2

Output:
Basic reporting: None.
Experimental design: None
Validity of the findings: None
Additional comments: The authors have answered my comments and I have nothing else to add.


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: NOVEL REAL-TIME PCR BASED ASSAYS FOR DIFFERENTIATING FALL ARMYWORM STRAINS USING FOUR SINGLE NUCLEOTIDE POLYMORPHISMS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The writing is clear and unambiguous in most places.  Sufficient background and citations are given.  The structure conforms to PeerJ standards. Figures are relevant and well-described, except for some problems with supplemental figures described below.  Most appropriate raw and summary data are available.   I was able to access the BioProject PRJNA645462 containing the ddRADseq and download one of the Run Accession numbers, SRX8709398.  According to lines 170-171 the reads were mapped to the published C and R genomes of Gouin et al. 2017; however there is more than one version of these genomes so the specific URLs should be given for the actual C and R genomes used.  Also, the URL of the Liu et al (2019) genome should be given.  I was able to access the TaqMan Assay Design Tool.
Experimental design: The manuscript describes original, primary research.  The research question is well defined in a limited sense, but some ambiguity remains because the assumptions underlying the existence of fall armyworm strains are not defined (see below).  Development and measurement of the TaqMan markers was rigorous and conducted to a high technical standard.  Methods are described in sufficient detail for replication by others, subject to access to exactly the same genomes (see above).  Replication by others would be improved by combining the criteria of FigureS1 through FigureS4 into a single Excel spreadsheet, with the decision rules given by formulas, and including this as additional Supplementary Information.
Validity of the findings: Although PeerJ does not assess impact and novelty, I provide some comments here.  This work provides a novel method for rapidly screening large numbers of FAW individuals, once the method has been set up.  It will have a measurable impact on the field because other workers will probably adopt the method.  The markers appear to be robust, given the data included in the supplements; however applicability to other populations of FAW worldwide is unknown and can only be tested experimentally.  The markers containing other non-diagnostic SNPs (B and D) are less robust than A and C, and future studies on other populations may also find SNPs in A and C, making them somewhat less robust.
Additional comments: 1) The authors have invested more effort into developing these markers than any other previous study, and their methods will provide a blueprint for future marker development.  However, whether the TaqMan markers provide a significant improvement over PCR-RFLP polymorphisms in the mitochondrial COI gene, and the 10 SNPs in the Z-linked gene TPI defined by Nagoshi, remains to be seen.  Most researchers will still want to know the results of these "classical" tests--why not design TaqMan assays for them as well?  Then a single platform could be used for both the old and new polymorphisms.  Also, since three of the four new markers are Z-linked, they suffer from the same ambiguity as TPI.  Why not develop one or two TaqMan markers for the W-chromosome?  These might also be strain-specific, but even if they are not, they could be used to distinguish hemizygous C females from homozygous CC males, and hemizygous R females from homozygous RR males.  This is necessary for a proper estimation of allele frequencies.

2) The genetic assumptions underlying the concept of "fall armyworm strains" are not explicitly laid out.  What level of genetic differentiation is assumed, and does this vary by chromosomal position? Is it significant that three of the new markers are Z-linked, because sex-linked TPI was used beforehand?

3) The sampling seems biased toward corn-strain habitat.  Would also sampling from rice fields have produced more robust markers?

4) It might be revealing to include information on the genomic context of the four markers.  This can be done by BLASTing the probes against the annotated version of another FAW genome in GenBank, BioProject: PRJNA590312. SNP A occurs between 7,726,842 and 7,726,792 on the Z chromosome, in an intergenic region near XM_035575061, uncharacterized protein LOC118263201.  SNP B occurs between 17,333,027 and 17,332,977, in the third position of the codon for arginine (CGG/CGC) of a protein-coding region of unknown function, SFRURICE_007604.  SNP B does not change the amino acid sequence of this protein.  The protein is a member of a large gene family that seems to be restricted to Spodoptera frugiperda.  SNP C occurs in a noncoding region between 17,573,725 and 17,573,775, in the same general vicinity as SNP B.  TPI occurs approximately between 14,025,953 and 14,026,704 on the Z chromosome, according to this coordinate system.  SNP D occurs in the third position of a codon for serine (TCC/TCG) in gene XM_035580529 annotated as a solute carrier family 25 member; it does not change the amino acid sequence of the protein.  The genomic context apparently has little relevance to the biological differences between the fall armyworm strains.  What do the authors make of this?

5) Is discordance of the autosomal marker SNP D with the others, more or less significant than discordance of one of the sex-linked markers (A, B, C) with the others?

6) In Table 4 the caption seems to contradict the headings in the table.  Are the deltaCq's reversed?  Whether VIC or FAM always correspond to R or C is not made sufficiently clear in the text; it should be stated at least three or four times for clarity.

7) FigureS1 through FigureS4, change "stain" to "strain".  For each box listing a test on the value of DeltaCq, state whether it is for VIC or FAM in each case, since it is not always obvious. "RT-PCR" usually means reverse-transcription PCR, but cDNA is not being used in this study. qPCR is the more common abbreviation for what the authors mean.


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: NOVEL REAL-TIME PCR BASED ASSAYS FOR DIFFERENTIATING FALL ARMYWORM STRAINS USING FOUR SINGLE NUCLEOTIDE POLYMORPHISMS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript provides a new molecular method to distinct genetically host strains of fall armyworm (Spodoptera frugiperda), a pest of global economic importance.

The authors bring a up-to-date tool and clearly state the practical relevance of their work regarding the population ecology and the importance for pest management strategy. For instance, reflected in the effectiveness using genetically modified insects release to control fall armyworm.

The manuscript is scientifically sound and well structured.
Experimental design: The material and methods are well described and reproducible. Nevertheless, I would like the authors to consider the improvements listed below:

1) Although the authors cite the nucleotide positions which SNP A, B, C and D are occurring, I would suggest adding a figure to illustrate the new position of SNPs (including which nucleotides are expected to be present in rice or corn strains). Are the SNPs analyzed located close to the regions (Tpi locus) analyzed by Nagoshi et al 2019 (Sci. Rep. 2019, 9, 8311) for fall armyworm populations collected in African countries?

2) Are the sequences (501 bp - line 196) used for TaqMan Assay design deposited in NCBI? What are the sizes of the PCR product formed for each SNP analyzed ?

3) Although it is clearly stated the tissue used for gDNA isolation in material and methods, I would like to suggest the authors to reinforce the importance of using a non-abdominal tissue in case moth specimens are used for the analysis. Considering that the target genes used in the TaqMan assay is a sex-linked gene and sperm present in the spermatheca could generate some result noise using such a sensitive method as qPCR.

4) Why did the authors considered using only Cq values instead of RFU?

6)The authors stated that the TaqMan assays developed have high sensitivity in detecting the different alleles at a concentration as low as 0.001 ng. However, if Cq values are used instead of final RFU as threshold for strain identification, I would question the reliability of the result using Cq values above 30, for instance NC-1, allele 1 in Table S3.

7) Line 126: were mixed sex used?

8) Line 145/242: add primers JM-76/JM-77; Tpi-632 F/Tpi-1195R in Table 2

9) Line 275: Include details of JMP 14 software used
Validity of the findings: The authors provide enough evidence to confirm the robustness of the new molecular tool proposed. Nevertheless, I ask the authorts to consider the following comment:

1) Although SNP D accurately identified the hybrids individuals, the authors also mentioned that  “for SNP D, FAM and VIC fluorescence was detected in all individuals regardless of the allele present, and the fluorescence of the off-target probe was often detected only 3 cycles after fluorescence of the target probe” (lines 322-324). Later “All four of the new SNP- based diagnostic assays were equally if not more effective at assigning strain when compared to the original COI and Tpi assays (Lines 363-365). I question here rather only using SNPs A, B and C would not be more appropriate than suggesting the use of the four assays. The limitation of a Z-linked loci could be rather overcome if the sex of individuals would be known before hand.
Additional comments: Would the SNPs analyzed in this study support the strain identification of fall armyworm populations from other geographies outside the United States? I would encourage the authors to mention in the discussion the applicability of such method in a broader geographic range, or the corresponding limitations.


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: NOVEL REAL-TIME PCR BASED ASSAYS FOR DIFFERENTIATING FALL ARMYWORM STRAINS USING FOUR SINGLE NUCLEOTIDE POLYMORPHISMS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have covered all comments raised by reviewers and changes in the manuscript were made accordingly. The revised version of the manuscript is suitable for publication.
Experimental design: No additional comments.
Validity of the findings: No additional comments.
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: EFFICACY OF 4% ARTICAINE VS 2% LIDOCAINE IN MANDIBULAR AND MAXILLARY BLOCK AND INFILTRATION ANAESTHESIA IN PATIENTS WITH IRREVERSIBLE PULPITIS: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Dear Author,
A comprehensive qualitative and quantitative analysis. The manuscript is well-drafted.
Only minor revisions/queries need to be addressed:
1) Was the review registered in any databases like PROSPERO. The importance of such registration is that it allows you to declare to the reviewers and the editor that the review protocol has been screened by PROSPERO and either there are no similar reviews or a justification is provided for the current review. Thus, I request the authors to register in PROSPERO and provide the registeration number
2) PubMed/Medline- Here PubMed included both Medline and PubMed central right? Then why mention as slash Medline?
3) Scopus database?
4) Have u included a supplementary file summarizing the search strategy employed in each database and have enlisted the articles excluded in full text assessment and the reason for exclusion?
5) Keywords and other free terms- Here what is the difference between keywords and other free terms?

Kindly address the above 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: EFFICACY OF 4% ARTICAINE VS 2% LIDOCAINE IN MANDIBULAR AND MAXILLARY BLOCK AND INFILTRATION ANAESTHESIA IN PATIENTS WITH IRREVERSIBLE PULPITIS: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: English Language should be revised.
References are well presented.
Experimental design: The experimental design of the manuscript Is ok, aim are clear and method are well described.
Validity of the findings: See General Comments
Additional comments: Dear Authors,
Thank you for submitting your article to this prestigious journal.
The manuscript is well presented but it presents some issues:

Abstract: Abstract should be revised, rewritten and make it more fluent and appealing.

Keywords: To ensure a properly research in medical databases, use MeSH terms to find  appealing keywords that could be helpful in finding your article.

From Line 93 to 98… I would like to propose the authors to insert the chemical formula of lidocaine and articaine as a figure to ensure a proper comprehension for the readers.
Moreover could you please describe the pharmacodynamics?
Figures are well presented.
Table 2: if it is possible make it more clear. It is too confusing.
Methods and Results are ok.
From Line 346 to 349… Please be more specific in describing the neurological patterns. Where the pain impulse is distributed in the central nervous system? What are the areas of the cortex included in the pain storage?
Discussion should be revised.
Conclusion must be entirely revised making it more appealing.
English spell revision is necessary.

Best Regards.


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: EFFICACY OF 4% ARTICAINE VS 2% LIDOCAINE IN MANDIBULAR AND MAXILLARY BLOCK AND INFILTRATION ANAESTHESIA IN PATIENTS WITH IRREVERSIBLE PULPITIS: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The revisions are to my satisfaction. The manuscript can be accepted for publication


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

Input:
Title: EFFICACY OF 4% ARTICAINE VS 2% LIDOCAINE IN MANDIBULAR AND MAXILLARY BLOCK AND INFILTRATION ANAESTHESIA IN PATIENTS WITH IRREVERSIBLE PULPITIS: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Dear authors thank you for submitting the revised version of your manuscript.
I will suggest the editor to accept it in current form
Best regards


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: LOCATING LIGAND BINDING SITES IN G-PROTEIN COUPLED RECEPTORS USING COMBINED INFORMATION FROM DOCKING AND SEQUENCE CONSERVATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this study the authors have presented ConDockSite, a method that combines docking scores and surface conservation to identify the ligand binding sites. The method is then validated using two GPCR targets with known crystal structures and demonstrated using another GPCR with no known crystal structure. Overall, the manuscript is relatively well-written and the results are clearly presented. However, the Materials and Methods should be placed after the Introduction and not at the end of the manuscript.
Experimental design: In terms of experimental design, I have a few concerns as below:

•	The choice of two class A GPCR structures to validate the method significantly limits any ability to truly evaluate how the method performs, since class A GPCRs have relatively well-defined binding sites within the TM bundle. While it is true that GPCRs are important drug targets, binding site prediction (the problem the method is intended to solve) is not one limited to GPCRs. The authors should have either use a broader range of targets, or clearly state the limited context in which their results are should be interpreted.
•	In Line 354 it is stated that SwissDock poses were manually screened for binding sites located on or near the extracellular site of the protein. This manual intervention does not allow for an objective assessment of ConDockSite’s performance since it introduces a bias (i.e. without this manual intervention would the same results have been achieved?)
Validity of the findings: •	The results obtained for the validation targets (β2 and A2A) would be highly dependent on SwissDock results. The results of the COM distance between the predicted pose and the crystal structure pose are reported, but the results obtained from only using SwissDock (without using the conservation scores) should be presented as well in order to allow readers to gauge the degree of improvement afforded by ConDockSite.
•	Similarly, for these two targets some comparison with the results of other binding site prediction programs would also be useful, since this is done for GPER but in the case of these two targets the crystal structure is available for comparison. This could be done by either describing the binding site residues identified or using their COM distance. One would expect that for binding site prediction programs the number of binding site residues identified would be larger compared to any approach that utilizes docking as part of the method, although this is somewhat dependent on the size of the ligand as well.
•	Given that the authors are presenting a new method, demonstrating the use of the method using a target GPER does not add any value, as there is no way to validate the predictions. Some comparisons with other binding site prediction tools and modelling studies are provided, but these comparisons would inherently not be precise enough to demonstrate that the results are valid. Choosing other targets with experimental data available and discussing it within that context would have been more valuable. If GPER is a target of particular interest to the authors, then some discussion in the context of any available experimental data (e.g. mutagenesis studies) would be useful. If these are not available, then the results are very speculative and should be more clearly indicated as such.
•	Line 245 – it is stated that all three methods could identify a binding site roughly similar to ConDockSite. This should be elaborated more clearly in terms of the residues involved in the binding site or the size of the binding site so a proper comparison can be made. The figure provided only allows for a very qualitative assessment of this.

Other minor comments:

•	Line 153 – What is the RMSD between homology models and crystal structures calculated based on? Backbone atoms? Alpha carbons?
•	Line 162 – the RMSD here is referring to RMSD between ligands correct? Wasn’t the results supposed to be presented as the COM distance between ligands?
•	Line 185-186 – these loops are more typically referred to as extracellular loop 2 (ECL2) and intracellular loop 3 (ICL3)
•	Line 199-201 – This is a repeat from the start of the Results and is also stated in Methods.
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: LOCATING LIGAND BINDING SITES IN G-PROTEIN COUPLED RECEPTORS USING COMBINED INFORMATION FROM DOCKING AND SEQUENCE CONSERVATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The work focus on the identification of binding sites for the GPCRs superfamily. In fact, GPCRs are one of the most import drug targets because of its implication in a huge variety of diseases and therefore I consider the work relevant for the field. Furthermore, the manuscript is very well structured and written .
In my opinion, the figures could be improved, simple changes should be done, indicating, for example, the typical numenclature for the GPCRs, and the extracellular and intercellular loops and regions.
The S.I. Fig.7 should be improved, the colors used impede to see the information of the alignment.
Experimental design: The methods strategy and workflow are appropriate.
To benchmark the binding sites found with ConDocSite the authors used receptors which X-ray structures are known and have been very well characterized, such as the Adenosine A2A. Homology models were also used to validate the methodology.
Validity of the findings: I would like the authors to address the threshold that can be used to use the method with high confidence as well as the ability of ConDocSite to distinguish between agonists, partial agonists and antagonists among the different classes of GPCRs. Can the scores be correlated with the affinity of the ligands to the receptors?
Surprisingly, the scores indicated on table two, and below on lines 3 and 5, are exactly the same for the two receptors. Can the authors justify for these results?
Table S1. ConDock predicted ligand binding sites for A2A adenosine and 2 adrenergic receptors using crystal structures.

GPCR	Ligand	Distance to crystal structure pose (Å)	ConSurf conservation score	SwissDock energy Score	ConDock score
A2A adenosine 	adenosine (agonist)	1.8	0.86	-1702.1	-1458.9
A2A adenosine 	ZM241385 (inverse agonist)	1.0	0.86	-1534.7	-1319.8
2 adrenergic 	epinephrine (agonist)	0.4	0.87	-1112.0	-970.7
2 adrenergic 	carazolol (inverse agonist)	1.8	0.78	-1534.7	-1319.8

The distance between ConDock and crystal structure poses presented on S.I. table 2 are very large.
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: LOCATING LIGAND BINDING SITES IN G-PROTEIN COUPLED RECEPTORS USING COMBINED INFORMATION FROM DOCKING AND SEQUENCE CONSERVATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: -	Line 49: according to GPCRdb the experimental structure has been solved for 70 class A GPCRs, 10 class B, 4 class C and 3 class F.
-	Line 92: I suggest characterizing the binding sites as hypothetical.
-	Line 126: what does the author mean with “proxy”?
-	Lines 124-125 and 126-128 tell almost the same
-	Line 150: rephrase with something like “by excluding their known X-ray structures from the templates”
-	Lines 163-164: please, reformulate because the sentence is not clear.
-	Line 177: repetition of “we used”, delete the second one
-	Lines 199-202: better to move the explanation of ConDockSite at the beginning of the Results section, where the technique is introduced for the first time.
-	Line 228: replace “bound by” with “surrounded by”
-	Line 239: replace “is neutralized” with “makes ionic interactions with”
-	Line 332: substitute “prep” with “prepared”
-	Lines 333-337: move those lines referring to SwissDock at the end of the paragraph, around line 346, where SwissDock is explained in more detail.
Experimental design: -	Lines 101-102: “We discuss how the ConDockSite-predicted binding sites provide a basis for G1 and G15 binding specificity” this statement is misleading. The research question about binding specificity is not addressed and answered in the manuscript.
-	Lines 122-123: I am not able to find in the results the mentioned comparison between the residues within 3.5 Å of the top scoring poses and the crystal structures.
-	I understand that the purpose of the project is not the prediction of accurate binding modes, but the manuscript would benefit by mentioning the RMSD between the predicted poses and the crystallographic ones.
-	Line 137: the authors mention the PDB ID of one of the employed structures (4LDO) just for beta2. I suggest including the PDB IDs for all the structures that were used in the manuscript.
-	Lines 136-138: I suggest including in the text the Consurf score for epinephrine as done for the other ligands.
-	Line 152: which templates were used by I-TASSER to build the models? Further details about A2A, beta2, mu opioid and 5HT2B models generation are lacking in the Experimental section.
-	Line 153: which are the atoms used to compute RMSDs between crystal structures and models? Ca carbon atoms? Backbone atoms? Heavy atoms?
-	Line 154-155: why were 2RH1 and 5K2A used as reference to compare beta2 and A2A AR models? In the case of 2RH1 for beta2, resolution could be the reason, but I cannot understand the choice of 5K2A for A2A AR. Please explain, and in case provide RMSD to some more valuable experimental structure.
-	Line 162: the authors mention a “RMSDs of 3-4 Å between the predicted and crystal structure ligand poses”, but in table 2 the 3-4 Å is related to the distance between the ligands’ centers of mass. The authors should correct this incongruence, and, in addition, I suggest adding the ligands RMSD in addition to centers of mass distance, also in this case.
-	Line 163: “in this RMSD range”: what do the authors mean? And again, is this RMSD or centers of mass distance?
-	Line 165-166: again, is this RMSD or distance?
-	How did the authors choose the sequences for the alignment and ConSurf calculation?
-	How did the authors generate the multiple sequence alignment? Why just ortholog genes were employed?
-	Line 182: which are the other 9 GPCRs structures employed by GPCR-I-TASSER? How were they used?
-	Line 184: avoid the use of “excellent” to describe a result. The authors should instead mention how the models’ phi and psi dihedral angles fit the favored regions of the Ramachandran plot, and/or how many outliers are present.
-	Line 203: can the authors explain what they mean with “outscored”? Which cutoff was employed?
-	Line 198: does “we then ranked all ligand binding sites” mean that the poses located throughout the receptor and considered “nonviable” (line 193) were considered in the evaluation? Did they present a low ConDockSite score enabling the discrimination from the poses in the putative orthosteric site? Reading line 354 it seems that poses far from the extracellular site of the receptor have been manually removed, and this would distort the accuracy of the method, given that its aim should be the effective prediction of binding sites.
-	Lines 226-227: the hydrogen bond with Asn310 is not visible from the image, please, remake the image to make in clear. Moreover, what does “long hydrogen bond” mean? Long?
Validity of the findings: -	According to what reported in the paper DOI: 10.1038/NCHeMBIO.2266, “phylogenetically unrelated GPCRs frequently share ligands” and “homology-related receptors frequently differ substantially in their ligand selectivity”. Moreover, looking at the multiple sequence alignment, a high percentage of residues show high conservations, so I would expect a leveling of the ConSurf scores, because it does not seem rare for a ligand to interact with highly conserved residues. In other terms, I think that the major shortcoming of this work is that there is no proof of the advantage in merging SwissScore and ConSurf, because a comparison with these two single scores is lacking. I suggest, as a major revision, to include this kind of analysis as a control, and compare the pose rankings obtained with SwissScore, ConSurf and ConDockSite. Which poses would have been selected for the test cases (crystallographic A2A and β2) if just the SwissScore or the ConSurf score were used? Without this control, the advantage in using the product of the two scores, and the sentence “Incorporating sequence conservation information in ConDockSite overcomes errors introduced from physics-based scoring functions and homology modeling.” at the end of the abstract, are not supported.  The only comparison with different methods has been provided in the case of GPER, but there is no experimental data making this test case a true positive, and, in addition, among the methods that have been compared there is no pure SwissScore nor ConSurf.
-	Lines 167-169: which are the “serious errors in homology modeling” the authors refer to? The RMSD between model and template reported in Table S2 is just 1.8Å for 5HT2B receptor, comparable or even lower than more successful cases like mu opioid and A2A AR. How can the authors explain the 7.3Å distance (or RMSD) between predicted and crystallographic ligand? Which are the “serious errors” in the model? Maybe the RMSD between model and crystal structure that was employed is not sufficient to explain 5HT2B model limits? Any thoughts? Or is the depth of the binding pocket the problem? In that case, it would be better to reformulate the sentence.
-	Why did the authors employ beta2 and A2A AR for the cross-docking validation, and add mu opioid and 5HT2B just for the homology modeling validation stage? Given the limits of mu opioid and especially 5HT2B models, the authors should add these receptors to the cross-docking validation stage. In this way it will be clear if the problems related to mu opioid and especially 5HT2B are model-related, or instead receptor/binding pocket-related.
-	Lines 169-171: “Nevertheless, our results with ConDockSite are consistent with benchmark modeling results that show that GPCR homology models of modest accuracy from templates with low sequence identity are still useful for docking and virtual screening” How can the authors state this? The authors have just shown that the 5HT2B model is for some reason poor (even if it is not clear why) and do not provide good docking results, so how can it be useful for docking and VS? I see some discrepancy between what just observed by the authors and what stated citing the literature.
-	Lines 245-248: what does “more detailed and of higher resolution due to the information from chemical interactions from ligand docking” mean? I cannot get what details and resolution are in this context.
-	Lines 248-249: I also cannot understand this point. What does it mean that the other methods are not able to differentiate among sites for different ligands? This statement is not supported by any observation/result.
-	Lines 291-292: from Figure S6 it does not seem that “the other proposed site mostly involved surface-exposed loops”
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: LOCATING LIGAND BINDING SITES IN G-PROTEIN COUPLED RECEPTORS USING COMBINED INFORMATION FROM DOCKING AND SEQUENCE CONSERVATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is well-presented.
Experimental design: The authors have not made any changes to the initial experimental design, which potentially limits the applicability of the method to Class A GPCRs. However, this is now more appropriately acknowledged in the manuscript, allowing readers to consider the findings in context.
Validity of the findings: The authors have added some results and discussion that adequately address the previously-raised concerns.
Additional comments: The authors have improved the manuscript based on the previous comments. While some limitations do remain, the manuscript now acknowledges them more readily and provides better context for the potential domain of applicabilty for this method.


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: LOCATING LIGAND BINDING SITES IN G-PROTEIN COUPLED RECEPTORS USING COMBINED INFORMATION FROM DOCKING AND SEQUENCE CONSERVATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article does not address all the questions asked and, in my opinion, the authors claim results that are not fully proven.
Experimental design: Techniques are ok but the pipeline could be improved.
Validity of the findings: In my opinion the results are not robust and it needs to be benchmarked.
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: DIETARY EFFECTS ON FITNESS IN CAPTIVE-REARED HAWAIIAN TREE SNAILS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: Some minor additions to Methods are suggested in the General Comments to Authors section.
Validity of the findings: Conclusions are well-stated, but I think there could be some further exploration regarding why the cultured fungus only treatment was so much less preferred to the leaves and fungus combo. I've elaborated a bit on this in the General Comments.
Additional comments: Summary:

The authors use a "less endangered" species of Hawaiian tree snail to try to optimize the captive diet of Hawaiian tree snails in their captive breeding program. This is of critical importance because so many achatinellid species have already gone extinct. The authors are under particular pressure here to find a diet that enhances snail survival and reproduction, as well as one that could potentially be replicated elsewhere in the world, since otherwise they are stuck continuing to breed these snails in one isolated locality that is prone to the potentially devastating effects of power outages due to hurricanes and storms, as well as disease or toxicants (which they specifically mention) that could come in on the wild vegetation they are currently using to feed their snails. Overall this is a straightforward study with straightforward results, that suggest that that magical food solution that would allow them to raise endangered tree snails outside of Hawaii, has not yet been found. The snails barely move or grow if raised on cultured fungus alone.

This research is important because of its subject matter (highly endangered snails) and these snails' current precipitous decline (to only a few individuals of certain species; here again I would mention this with specific examples-- you can't assume all of the readers will have heard about the species SEPP has or the numbers left. It adds to the weight and importance of the work). This study also would provide peer-reviewed evidence that these snails differ from the partulid tree snails, another group of highly endangered Pacific island snails that, in contrast, have been able to be raised in many different localities on diet that humans can concoct easily with a published recipe. (Along these lines, I think it would be helpful for the authors to mention these partulid breeding programs and a few citations, because it would help show the general interest in the study, as well as help contrast with the authors' results. This is the only similar Pacific island land snail breeding program of equivalent size out there.).

The paper is clearly written and interesting, particularly to researchers, conservation managers and zookeepers focused on increasing numbers of understudied, small-bodied endangered species through captive breeding programs.

Specific comments by line number:

Lines 48-49: Use of "radiation" too vague here: The 750-plus snails are the result of multiple radiations. But it is definitely true that the resulting diversity is among the greatest on Earth! (also I would suggest changing "world" to "Earth" in this radiation context, just because world tends to refer to human matters.

94-95: Suggesting elaborating slightly on this statement to say that this is problematic because it could affect the security of critically endangered species (e.g. if a hurricane wiped out the SEPP lab).

101: Remove "drastically." Unnecessary.

106: Redefine the "SEPP" abbreviation and where it is located. Or if you don't want to do it twice, maybe save this explanation for Methods section. (Also not sure why this was done at line 119 instead of earlier.)

108: By "sister subfamily" I am assuming you mean phylogenetically speaking? If so, a citation is called for, especially since later you are talking about the fact that you use Auriculella as a proxy _because_ it is closely related.

113: "auriculellines" should be lower case. You only need upper case for the full subfamily name.

120: What tree species?

121: Since people reading this might be more familiar with just about any other animal besides tree snails (i.e. animals with larger home ranges for individuals), it might be worth noting through a citation the fact that most of these tree snails likely do not leave their home trees. I don't know it that is yet known for Auriculella in particular, but still you can give some scientific basis for the reason you took the multi-tree approach, based on another achatinellid species.

137: Add something about this 12:12 photoperiod being chosen to mimic Hawaii day/night cycle.

146: What species comprise the "wild collected vegetation"? Are these all native species? Is this a secret for a reason or can you share at least some of this. For depressing practical reasons, if this species declines or goes extinct later, it is important to have a record of what species you were finding it on. And do the species tested differ from the species you typically find this snails species on? If so, why?

156, 158: I think the better way of writing "2.4x" would be "2.4 times" just because it could get confused with equation notation in other papers.

161: How sad!

177: I have a question about the idea of "bulk" in the snail diet suggested here. I am not sure that this conclusion necessarily follows from the Results. Or, you might just need to talk more about to what extent having leaves or branches of leaves (should also specify in Methods), increases the surface area for fungi/microbes. I am wondering about your result showing that snails raised on cultured fungus alone, just appear inactive and don't eat much. Could it be that the winning combo of leaves plus cultured fungus has as much to do with snails being stimulated to eat through the presence of their natural leafy habitat, than it does with the cultured fungus being a poor food? I am just wondering if what snails are getting out of the leaves plus cultured fungus diet is not just about quantity-- and perhaps suggesting that it is hard to say. It just seems strange that the snails in the cultured-fungus only tank didn't really seem active at all-- so it wasn't just that they were forced to be eating less because of lack of microbe quantity. For example, it is also possible that the leaves/branches provide so many more natural-feeling perching sites, that snails are bound to be more active, and perhaps cross over and through many more microbes just as a result of tasting familiar foods on their branches, and then happening by the cultured fungus when they are on their feeding route. I suppose that still could be a matter of survival and repro. "success due to bulk"-- but the reason for it could be less about the bulk itself, and instead some other x-factor that the presence of leaves/branches provides.

184: I think you are partially getting at my comment above here with the mention of the "stress response." But I think you need to go into more detail about what you mean. A vet could assess signs of stress in an animal, even a snail. Sometimes this involves retraction into the shell, etc.-- it depends on the stimulus causing the stress. So it might be more helpful to explain the typical activity level of these snails, and how the behavior you noticed differed from that, a bit more in the Results, and/or add any references about stress response or adaptation to e.g. dry periods in snails. Could the lack of leaves/branches in the terraria also have reduced humidity to the point where the snails would not want to be active? And thus they might not have eaten for that reason?

185-189: Suggest moving this idea to the top of your Discussion, since it helps underscore the general importance of the study. You aren't just testing a bunch of diets for fun, you are actually trying to save some species. Also I would suggest breaking this up into two separate sentences, so the 3-5 year thing is separated out. The link between the two ideas in the sentence isn't so closely tied together that it all needs to be in one sentence. This might come off as more convincing if you break it up.

195: "Our" study, rather than "this" study, so it is clear you aren't talking about one of the referenced studies.

199: Can you give some examples of pathogens, parasites or toxicants that you know are occurring and/or that are a problem now, or could be more so in the future? Also if forests become drier due to climate change, could such contaminants become more concentrated on the leaves?

215: Not necessarily here, but maybe elsewhere in the discussion, could you give some examples from snails or other organisms where such dispersed breeding efforts have proven more successful than breeding in just one location, and why? It would be good to explain the increased risk involved by having all individuals of a critically endangered species in one place. Also, presumably decreasing or mitigating such risk is also part of the USFWS species management plan (cite)?


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: DIETARY EFFECTS ON FITNESS IN CAPTIVE-REARED HAWAIIAN TREE SNAILS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	I would like the introduction to give a broader contextualisation of the literature concerning raising snail in ex situ facilities. I have provided a few links to some studies that aren’t cited, but there are more. In particular the Partula literature. I know that grazing strategies in Partula are somewhat diverse, and some of the results of the Tahitian snail studies are old (but successful), and the more current references (last 2 -3 years) are in the “grey” literature. However, there are instructive scientific publications that bolster some of the authors more speculative points (Gouveia et al. 2011 https://bit.ly/3kJQW0a, and https://bit.ly/3kDYl0M). Of the literature that works with A. diaphana, I think that Holland et al., 2017 (https://bit.ly/33VRNnB) should at least be cited, which uses the same response variable and same snail species in a similar experiment. However, I think the authors would make their publication stronger if they argue that they are extending on the findings of the Holland et al., publication.

2.	A large portion of the Introduction and Discussion place the motivation for addressing diet as being due to the risk of introducing pathogens on branches/leaves that are periodically added to the terraria for the snails to graze epiphytic microbes from. I think that this might be too speculative for the Introduction, as a pathogen has not been identified. In L88 “Recent mortalities above baseline levels in laboratory populations of Hawaiian tree snails were attributed to a possible pathogen introduction or exposure to an environmental toxicant (Price et al. 2015; Sischo et al., 2016)” the cited papers only really speculate on the cause of population crashes, and are population genetic studies, not epidemiological. Price et al. 2015 refer to an unsuccessful search using microscopy to look for Microsporidia as a cause of mortality, but found none. I agree with the authors that pathogens are a likely explanation of population crashes amongst different species, but I think that other less likely causes are possible too and shouldn’t be ruled out without evidence.

3.	I also think that there are many other routes of pathogen transmission other than wild leaves – the ex situ facilities aren’t high-level containment facilities. If they had UV treated hepa-filtered air, directional airflow away from the incubators, controlled human access and strict PPE, then perhaps the inference that the only path a pathogen could be introduced is through the leaf cuttings would be more convincing. There is strong evidence that Partula snails that are fed artificial diets also have crashes and problems with pathogens (Cunningham & Daszak 1998: https://bit.ly/360w0xF; also https://tinyurl.com/yxoypj5u), and to my knowledge, Koch’s postulates are almost impossible to satisfy with endangered taxa, and suspected pathogens are routinely found in healthy animals (Cunningham et al 1996, https://bit.ly/2RTwn4J). These points all make definitive attribution of pathogen mediated disease difficult. I agree with the authors that bringing cut foliage into the facility has a lot of problems. However, I don’t think that this particular inference, that they harbour pathogens, has sufficient evidence.
a.	I wonder if the pathogen angle is needed. Perhaps it is sufficient motivation that the experiment addresses whether culture can be improved? I am fine with some level of speculation in the Discussion, but with some caveats.
Experimental design: 1.	Egg production vs egg viability. Currently the number of “eggs produced” is used as the independent variable in the ANOVAs. It would really improve this study if some measure of the condition of the eggs was used as a response variable instead. In particular, did the authors record how many eggs hatched? Or even a crude measure, such as egg size?

If hatching or egg condition has not been recorded, then this limitation should be acknowledged. Also, the authors should acknowldge that a 10 week study has limited scope and an ideal study would track survival and reproduction of offspring.

2.	A problem which would be easy to remedy is that the authors use an ANOVA (L 152), but they should use a repeated measures ANOVA because they are resampling the same terrariums over time. That is, without using a repeated measure design they violate the statistical test’s assumption of independence and creates a ‘batch’ effect because the measure at each timepoint is not of an independent batch.

3. The choice of a box and whisker plot compounds this problem. It would be preferable to plot egg production as a function of time as a line for each terrarium (using 3 different colours, one for each treatment), that way we could see if production is constant, goes up or goes down, across the course of the experiment both within and between treatments.

4. The species of tree cuttings need to be identified, as well as when they were collected (i.e., were they fresh when put in?) and from where. This is for at least four reasons that impact the replicability of this study. 1. Wilting occurs at different rates for different plant taxa, just like a vase of different flowers, some plants will wilt in a day, and others might appear to hold their structure for the full two weeks before the plants are changed. This could change the behaviour of snails, it is also likely to change the microbial communities that depend on different aspects of the living plant host’s physiology and metabolism. 2. Rotting will likewise occur at different rates with different plant hosts, changing the relative proportions of microbes associated with decomposition and differentially liberating various nutrients and micronutrients from the different species of decaying plants. 3. Snails seem to prefer particular host tress and might behave differently if they are on a preferred host (Price et al. 2017). 4.  Similarly, Holland et al., 2017 (https://bit.ly/33VRNnB), find the highest egg production for A diaphana occurs on native host plants (interestingly, both this study and Holland et al. 2017, report similar levels of egg production).

5. Humidity and temperature loggers were deployed in the experiment to check that these variables were identical across treatments, but the results are not presented. These could be relevant in interpreting if treatment effects were confounded. For example, Hadfield Holland and Olival 2004 (https://bit.ly/33QMxl7) claim that humidity measurements are lower in terraria that lack leaves (page 23), which would explain non-feeding behaviour in that treatment group. Did the authors add any substrate to the terrariums such as paper (following the Partula protocol https://tinyurl.com/yxoypj5u)?

6. I think it might be helpful for the authors and journal if they identify the protocols that were in place to ensure the snails in the fungus on PDA trial that stopped feeding were not impacted in the long run.
Validity of the findings: 1.	The (easily addressable) major criticism that I have of the manuscript is that it draws very specific, but underdetermined, inferences from the experiment. This is an experiment that appears simple, comparing the presence or absence of two things, and would therefore be expected to have a simple interpretation. However, they -- are very heterogenous things, which makes the interpretation a little messy, which I think comes through in the Abstract which talks about microbial diversity on the leaves (L30) or energy in the agar/fungus supplement (L32). Discussion, especially in the inference that “the likely need for both microbial diversity and bulk in snail diets, and the potential benefit of calcium supplementation (L176-L177)” as well as lines 191-195 concerning diversity.
I think that a lot of these points are plausible, but the experiment only tells us that a combination of native leaves and a fungus monoculture grown on a carbohydrate + calcium rich agar produce more snail eggs than on leaves alone. The resolution of the experiment has no finer scale of information or gradient regarding microbial diversity, energy, micronutrients or bulk.
I do like the agenda-setting sentences on lines 200-206, and maybe it is sufficient to list the follow up experiments that need to be performed.
Additional comments: First of all, I like the general approach of trying to understand the ecology of endangered snail species experimentally by using snails that have a life history strategy that makes them more resilient to population fluctuations. By using such proxy species, the research on imperilled snails is able to move away from speculation and actually experimentally test hypotheses. I do have some criticisms of this particular piece of research, but I feel that the research project as a whole is very important and I hope that my feedback can help the authors make the most of the work they have done.

Some jargon is treated as synonymous when, although terms have overlapping meaning, they do not mean identical things. I am thinking of the interchangeable use of the words: egg production, growth, reproduction, fecundity, fitness. In this study, I think egg production is the best term to use.
L26 give t and p values for significant associations
L33 Replace “growth and reproduction” with “egg production”
L38-and elsewhere: It is conventional to italicise latin phrases
L90 I think toxicant is more often used for an artificially introduced toxin, would “toxin” be a better word?
L106 I don’t think the snails were observed for 16 weeks. Below it says they were held for 3 weeks acclimation (L127) and experimentally observed for 10 weeks (L146)
L112 I think Price et al 2015 is supposed to say Price et al 2017
L112 neither of these two studies establish that A diaphana are abundant species in the wild. Some of that information is in the grey literature. And, neither Price et al nor O’Rorke et al establish that their diets overlap. The best reference for comparing the diets of snail species is still possibly Pilsbry, Hyatt and Cooke 1912. O’Rorke et al 2015 does look at the diet of two Auriculella ambusta individuals, but not A diaphana. If the authors want to be more quantitative O’Rorke et al 2017 (https://go.nature.com/3iXmP4P) do sequence microbes from the feces and phyllosphere of A diaphana and these, along with the sequences from the  O’Rorke et al 2015 and Price et al 2017 study are publicly available, so the authors could be more thorough and compare the diversity of these. However, this isn’t necessary, just an option.
L120 What were the host trees - Cestrum nocturnum? Price et al 2017 suggests the identity of the host might be relevant.
L129 What species of plant cuttings were put in the terraria? Was it the same as the snails were collected from or different? If Different, what species? Was the foliage sourced from the same location for the duration of acclimation and experimental timeframe?
L148 Were all terrariums in the same incubator?
L175 “These data suggest that a diet consisting of diverse microbes, like that occurring on native vegetation, may be important for snail reproduction.” I think the authors should add that other factors might be responsible for this though – eg habitat rather than food cues.
L176 What about the possibility that the habitat induces/facilitates egg production?
L177 I’m not 100% sure what “bulk” means here. Is the inference that the “plant only” terrarium might be food limited?
L182 “. . . the length of the study period may not have revealed long-term differences in survival among treatments”. The authors currently only refer to the long term impacts of aestivation. Personally, I would expand on this point about expanding the trial to last for longer. For example, although the snails provided with fungus on PDA laid more eggs, we know that these snails come from an energy poor environment and therefore providing them with a starch+sugar rich substrate (PD agar) has unpredictable outcomes for their long-term health, resilience and survival. However, I don’t insist on this as I understand that the authors might disagree with my point of view.
L185 Or starvation?
L192 I’m not sure it can be said it is “optimal”, it just appears to be the best set up for egg productivity. There might be an experimental set up that is as yet untested that is optimal. Also, long term mortality and reproductive fitness might be better in another treatment.
L200-206 I like this agenda setting paragraph. Possibly the authors should add the need to test short/long term components of an artificial diet given L204
L205 I would prefer “secondary metabolites” or even “toxin” to “environmental toxicants”. The latter term is frequently used to refer to exogenous toxins, rather than microbial metabolic products.


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: DIETARY EFFECTS ON FITNESS IN CAPTIVE-REARED HAWAIIAN TREE SNAILS
Review round: 2
Reviewer: 1

Output:
Basic reporting: yes
Experimental design: yes
Validity of the findings: Yes, but also please see specific comments in the "general comments to author" section. Thank you.
Additional comments: 96-98: There is not necessarily a cause-effect relationship here. Yes, when snails are at high densities the potential for disease spread would be greater. But the issue of a disease first being brought in from the wild is a separate issue, that would be a problem whether or not the snails are at high densities in the lab. I suggest separating out these different factors into different sentences.

106: Suggest removing "very." "Successful" is enough-- especially since with Platydemus and E. rosea encroaching in the wild, it is probably already a stretch to say that extinction prevention has been fully successful, unfortunately.

109-111: This miss-states what Gilbertson et al. (2019) say, and/or maybe the statement appears misleading because it follows a mention of Clarke's manufactured diet. Gilbertson's diet by contrast 1) isn't manufactured, 2) it isn't really a captive diet in the sense you are talking about in the paper, and 3) it definitely isn't perfected. Gilbertson et al. refer to wild leaves being brought in, with some other stuff to supplement. So really it is closer to the Hawaiian tree snail lab's original diet of leaves brought in from the wild periodically. Again: unfortunately. Gilbertson's study focuses mainly on which wild species are better, in terms of which leaf species the snails grow best on.

120: It is unclear what "pathogen" and "toxicants and toxins" refer to here. Are these compounds produced by the wild fungi and trees/plants naturally? Are they chemicals that you worry might have been sprayed onto the wild vegetation, or introduced through rainfall or air pollution? Are the pathogens tree diseases or snail diseases? Since Ohia lehua diseases have made the news recently, this might be especially confusing. After reading O'Rorke's reviewer 2 comments and your responses (e.g. response to #3) I was surprised that this part about toxins or pathogens was left for readers to wonder about. People are already chemical-averse, but it doesn't mean that any toxin you bring in isn't something the snails also already experience in the wild and might therefore already be adapted to. It would really help I think to give some specific examples that you think might be issues for the snails, and then also note that this is speculative. I just think that someone who hasn't worked in your lab and talked to the people you are talking to about these snails every day, might have no idea what the real or perceived concerns are with respect to "toxicants" and "pathogens." There are bacteria and viruses all around us, even when we try our best (as you have) to remove them. Perhaps the problem is with impaired immune response in captive snails, more so than the fact that contaminants are coming in. That might even make sense in a crowded terrarium with rotting vegetation and lack of wind exposure (not a criticism here-- that would be true of any captive breeding setting for snails!). Again, quite speculative here, but I think it is important to say so when you really don't know. That is OK by me. Honestly, even your speculation would be helpful, because it gives potential avenues for future study. And sometimes it is better to keep the discussion broad so that alternatives aren't accidentally missed (and that is with the caveat that yes, with 30 years of research in the rearview, I understand that you feel like the conversation has exhausted many possibilities already).

204: Replace "less than" with "fewer than"

211: The result that the addition of cultured fungus is interesting in light of Reviewer O'Rorke's comment regarding PD agar, in his reference to L182. While I do understand that achatinellids in the lab have been fed wild leaves/branches, fungus, and the agar on which the fungus grows for 30 years-- I do think it is still worth a sentence or two speculating in the discussion along the lines that O'Rorke has mentioned, and perhaps then saying exactly what you said-- that the PD agar has been an important part of the diet in captivity for a long time. Readers might not realize that snails eat the agar as well as the fungus growing on top. They might also not be aware that rainforests are nutrient-poor environments. Maybe there is no reason to suspect that snails directly consuming agar over decades in captivity is a problem-- but for readers 20 years from now reading your paper, and wanting to disentangle different effects of food types, as we become more advanced in captive rearing of invertebrates, I think this would be helpful. O'Rorke: " although the snails provided with fungus on PDA laid more eggs, we know that these snails come from an energy poor environment and therefore providing them with a starch+sugar rich substrate (PD agar) has unpredictable outcomes for their long-term health, resilience and survival."
I would also add that even with 30 years of experience, this is still but one example, out of fewer than five, ten? major endangered land snail captive breeding efforts in the world. So anything you can say, even if a hunch, or even if you disagree, can be very useful for the next study in another land snail group in another setting. I would urge the editor to be a bit lenient in allowing you to speculate and/or post disagreements to this reviewer's comment in the Discussion, which I think is preferable to eliminating possibilities without strong evidence. I don't think it would detract from your paper, it would just help to ensure that it provides a balanced view, given the results and the difficulty in interpreting them.


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: DIETARY EFFECTS ON FITNESS IN CAPTIVE-REARED HAWAIIAN TREE SNAILS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The manuscript is well written
Experimental design: See below
Validity of the findings: See below
Additional comments: This is a topic where any new data is very useful. The authors have either made changes or given reasons why they won’t change the manuscript, which I am happy with and I think the manuscript is fine for publication except on two points. However on those two points, I would strongly recommend that the authors reconsider their approach given the reasons that I raised earlier, but I’ll more fully explain why this is below.

First, the choice of statistics for the experimental design. I still think the ANOVA and t-test are inappropriate tests because the data violates assumptions of those tests. Namely, the response variables are not independent of each other (and it would be worth viewing the distribution of the residuals). By using an ANOVA they are treating each level as though there are 15 independent results for each level, when there are only 3. Nested inside each of those 3 is a repeated measurement of the same snail cage. At the very least this needs to be treated as a blocked design. I think it is a repeated measures design. I understand that authors disagree with this because they aren’t interested in time, but it is still a component of the experimental design and needs to be accounted for. Considering their response, I think the best approach would be a mixed effects model, then the authors can perform an AIC to see if it is fair to remove time from the model. Alternatively they could (maybe) do a test for variation with time and a blocked design test sequentially. Previously I suggested a repeated measures ANOVA because conceptually it isn’t too different from what the authors have done. They just need to check the distribution of the residuals whichever approach they choose.

Some examples of why it is important to test if time should be included/excluded from the model: although egg production is generally higher in the plant-cutting + fungus_&_agar treatment this productivity goes down in at the end of the experiment in one terrarium. In contrast, in two out of the three terraria that contain cuttings the egg production is zero or less than ten for the initial four or more weeks and then counts shoot up to over ten. These look like a temporal pattern, and if it is, it needs to be accounted for. The third of these terraria dries out, so that compounds this problem.

For the graph, for the same reason, I don’t think the authors can use the box and whisker plot of all the data binned into three explanatory treatments. A line graph of egg production vs time with three colours (one for each treatment) with one line for each snail enclosure shows ALL the information of the experiment in just nine lines. The current graph obscures a lot. Also, I think the y-axis label needs to be checked.

Second, lines 91-93 “Recent mortality events above baseline levels in laboratory populations of Hawaiian tree snails were attributed to a possible pathogen introduction, or exposure to toxicants or environmental toxins (Price et al. 2015; Sischo et al., 2016, Sischo et al., in prep).”

I am happy with the rest of this paragraph, the gist of which is that plant cuttings from numerous wild trees increase the risk of pathogens entering the facility. I strongly agree that this is possible. However, I think that it is misleading to state that snails deaths have been “attributed” to a possible pathogen, toxin or toxicant and then cite two population genetics studies that do not test for pathogens or toxins. Price et al. (2015) includes a pers com that microsporidia tests had negative results, but that is all. I think this sentence should be removed. I worry that future generations of scientists will read it and maybe not explore a new hypothesis that could transform snail conservation because of the confidence with which this sentence is written and that it is backed by citations that don’t back it up at all.

The Sischo et al., in prep, result sound very useful, but as non-peer reviewed literature need to be moved to the discussion. Furthermore, if referred to, then the nature of this work needs to be elaborated on a little (given that we can’t read it elsewhere yet). I don’t think the authors need to give the results – it sounds like it was a lot of work and it is fair that it stand as its own manuscript. But the reader needs some vague indication of what the study involved, eg “histology of prematurely dead specimens showed lesions not in other specimens” (I think  that is what the study is about, I’m guessing from the authors comments) and tell us if known pathogens were isolated that might cause morbidity and if Koch’s postulates were attempted or not and whether they were satisfied.  Then cite (Sischo et al., unpublished)” or a BioRxiv preprint reference. It sounds like there is some really positive stuff to come.

Otherwise, I am happy with the manuscript, as I said,  all information on this topic is welcome. The above comments are long, but that only reflects that this is a reply to my earlier feedback and I am trying to make my reasoning more clear. Some minor points below:

Line 221 “may increase reproduction” I’d say “have unknown effects”. Increased nutrient and energy has massively variable effects on invertebrate egg production and fitness.

Line 245 change “optimal” to “better”, there isn’t enough evidence yet to say what is optimal

L246 “and survival” I’d delete this, survival is outside the scope of this experiment

L251 “beneficial” to “beneficial for increased egg production”


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: C3 PLANT ISOTOPIC VARIABILITY IN A BOREAL MIXED WOODLAND: IMPLICATIONS FOR BISON AND OTHER HERBIVORES
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: Good reporting of experimental methods.
Validity of the findings: No Coment
Additional comments: This work presents an isotopic survey of the ecology of a C3 ecosystem, and a discussion of the relevance of the findings for herbivore ecology. I especially like the finding “seasonal changes in ... isotopic compositions do not necessarily indicate changes in foraging strategies, but can result from isotopic changes within the plants themselves.” This is indeed an under-appreciated aspect of many herbivore isotope studies, so I welcome a more integrated and cross-disciplinary approach.

I have a few comments and suggestions, which I hope you will find useful.

Line 22-23. “These results suggest that (1) herbivores eating these different types of C3 plant can have identical tissue isotopic compositions”. Whilst the Abstract is generally clear, this concluding sentence is a little confusing to me, because it comes immediately after the sentence (line 21-22) which states that there is seasonal variability in δ13C and δ15N.
If I have fully understood the intended meaning here, perhaps a more precise phrase would be: “(1) in this ecosystem, dietary preference for C3 plant type should have little effect on tissue isotopic compositions of herbivores, within a particular season and habitat” or something similar? Then, go on to the effects of seasonality and habitat (point 2). Unless you are directly testing this against herbivore δ13C, it might be better to make it clearer that these are predictions – not findings?

As it is currently written, the possible implications for herbivores are a major focal point of the paper, but the paper is a bit short of studies of intra-tooth herbivore and bison δ13C (i.e. data). Therefore, if the conclusions and suggestions are to remain, I think that the work would benefit from a greater incorporation of previously-published Bison intra-tooth δ13C data into the discussion (i.e. how do the findings transfer into herbivore C-isotope space, with tissue-diet enrichment, and explain certain trends in previous data?). This would reinforce the present emphasis on implications for higher trophic levels. Some very nice references are briefly mentioned in the Introduction (lines 41-47), but they are not mentioned again. Fleshing these refs - and data therein - out a bit in later discussion subsections will help to tie the author’s predictions to the paper.

It is interesting that leaves are more negative for δ13C than seeds/flowers. The author might find the recent paper of Vogado et al (2020) of relevant to this finding, as it presents a physiological reason for this effect (e.g. incorporation of older C-pool during leaf aging), and these authors found a difference of around 2 per mil. Vogado, N.O., Winter, K., Ubierna, N., Farquhar, G.D. and Cernusak, L.A., 2020. Directional change in leaf dry matter δ 13C during leaf development is widespread in C3 plants. Annals of Botany, 126(6), pp.981-990. This reference could be added to that of Lowdon & Dyck (1974), in Line 59.

Lines 253-254 “Previous studies have also found no isotopic differences, or variable differences, among C3 plant life-forms in different environments (e.g., Barnett 1994; Chen et al. 2005; Funck et al. 2020; Tahmasebi et al. 2017).” Bear in mind that this might be true amongst particular C3 plant groups for terrestrial systems, but is not true between C3 aquatic/marine systems, or between major macro-evolutionary C3 lineages (in different environmental conditions). See Porter et al (2018) GCA, Leavitt & Newberry (1992) Plant Physiol., also Hare & Lavergne (2021) GCA, and Pedentchouk et al. (2008) Organic Geochem.

Lines 77-78 “This is especially true in mid- and high-latitude woodland environments, where botanical remains tend to degrade rapidly due to high soil acidity.” As far as I understand it, the opposite is true; that preservation is generally better in high-latitude contexts, because the effect of (lower) temperature predominates diagenesis and respiration (in soils). Preservation is worst in mid-latitude contexts, because of very high soil acidity and high temperatures, and higher soil respiration. Perhaps a reference could help clarify things here?

Line 101 “All grasses in the park utilize C3 photosynthesis”. Unless there is physical evidence to support this, I think I would omit this sentence. Even in C3 temperate ecosystems it is extremely rare to have absolutely no C4 photosynthesis (e.g. some seasonal, highly localised pockets of grasses will almost certainly be C4), especially if summers are moderate. This will have little effect on herbivore δ13C.

Lines 276-277. I’m wondering here what proportion seeds will contribute to the overall herbivore isotopic composition, given that seeds are generally much tougher, and much less digestible than leaves? In fact, many plants use animals for seed dispersal – implying that they are generally undigested. Perhaps the “poop literature” might have some insights?

Table 1. For future work, another useful resource for cold-temperate plant isotope data could be found by looking through the compilation of Cornwell W. K., Wright I., Turner J., Maire V., Barbour M., Cernusak L., Dawson T., Ellsworth D., Farquhar G. and Griffiths H., et al. (2016) A global dataset of leaf delta 13C values. Scientific Data