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: GEOGRAPHIC POTENTIAL OF THE WORLD’S LARGEST HORNET, VESPA MANDARINIA SMITH (HYMENOPTERA: VESPIDAE), WORLDWIDE AND PARTICULARLY IN NORTH AMERICA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is written well. There is no repetition and I found the descriptions to the point. I have pointed out some areas that need editing in my line-by-line recommendations given below. The references given are generally in line with the authors’ statements. I also found all the figures supplied to be relevant. In reference to the tables, I recommend to provide a table showing the parameters used for the dispersal simulations similar to the way the parameters for the habitat suitability modelling is provided in the first table. The table for parameters used for the dispersal modelling belongs in the article not in the supplementary information section as it is one of the main components of the modelling procedure that contributed immensely to the conclusion the authors reached regarding the potential of AGH in North America.
Experimental design: The fact that the authors used multiple scenarios of various parameter, model and evaluation method combinations is extremely commendable. The use of dispersal simulations to delineate the realized niche of the species additional to what the potential suitability is also one of the very important methodological steps the authors took. Presenting suitability studies with dispersal ability considerations gives better information for those who would like to use such studies for actual applied intervention against invasive species.
Although, I understand that the dispersal parameters not sourced from empirical data. I believe a literature review could provide an idea of how far the AGH can disperse. Therefore, it would be possible to give an idea of what one time step or “t” would represent in the dispersal scheme in the methods section. Is one time step “t” considered a given season? A year? I believe this should be specified with a caveat that the representation is not precise due to the simulation not being based on empirically derived parameter values.
The model is described in sufficient detail so that others can replicate the experiment if needed. If possible, authors should provide the occurrence locations they used to perform their niche modelling.
Validity of the findings: The study is statistically sound. Authors have reported a good level of detail for readers to understand how they undertook the modelling section of the work. The effort taken to address multiple scenarios, which usually entails considering various levels for as many factors as possible is commendable.
Additional comments: I found the study in general and the findings in particular to be timely and important considering that one of the identified families, honey bees, to be targeted by this invasive the Vespa mandarinia is already experiencing great population decline.  As I have mentioned a couple of times above, I also found the fact that the authors used multi-scenario modeling framework is very useful as the information about the uncertainty of the results that can be obtained from such scheme is very useful if such studies are to be used for applied invasive species control work on the ground. Information on uncertainty of models will allow end-users to prioritize areas of intervention based on the provided additional information about the potential suitability of different risk areas under protection.
- Include a table that shows parameters used for the dispersals simulation in the main article
- Indicate what time span “t” stands for in the dispersal simulation framework
- A rationale to why country boundary based thinning is proposed in the first place need to be given beyond giving reference to a study that has used the same method. The stark difference in the global suitability prediction between the two thinning methods is beyond what is expected from a variation of niche due to reduction in occurrence numbers (sample size). The ecologically irrelevant use of national boundaries as a reason to thin the occurrence data might have artificially reduced the importance of some sets of ecological conditions because density to an extent indicates importance of the environment.
- Both in case of agreement of suitable areas and agreement of areas with extrapolation risk it seems models using principal component based variables fared better, but I do not see this reflected in the discussion, is there a reason for this?
- S3 Table title: change to ‘predicted prevalence’ instead of ‘prevalence’
- Include occurrence dataset used for modelling, unless there is a data privacy arrangement that the authors have to honor. From their descriptions, it seems occurrence data is sourced from the GBIF, which is freely available to the public. Therefore, I believe it would be great if authors could provide the subset of the occurrence data they used for their modelling, as the actual data that people can download from the GBIF database contain far larger dataset that could have been removed from the set the authors used for their paper. Having the actual set will make replicating the experiment if needed easier.
- Include graphs of tables that show complete model evaluation results as it is difficult to obtain that information from within the body of the text when researchers refer to the publication for further modelling work on the species.

Line by line

L115: “… <50 km away from another locality” this part of the statement is confusing. What dos locality mean in this case, a place name, or another occurrence record? Please describe this more clearly.
L117: While I very much agree with the first thinning procedure the second one is not well justified. Why is an administrative boundary such as a national boundary important to be used as a unit of standardization? Is it based on the notion that the sampling schemes each country uses are different? Was an attempt made to check if sampling intensity was different for the various countries? Otherwise, simply more presence cannot be taken for more sampling effort by default.
L152: Any description why the authors used a 50:50 split for model training.
L251-L253: Is not very clear, please edit for clarity. Is this to mean not all selected models had their replicates selected? Is my understanding that each combination (scenario) has models that fulfill the criteria selected?
L271: “… as regards distribution...” insert ‘it’ between “as” and “regards”
L277: It is difficult to call predicted areas as “prevalence”. “Predicted prevalence” would better describe the proportion of the potential suitable area.
L279-L280: Being rare has a specific ecological meaning, which most certainly is associated with having a narrow ecological niche. When we have a prior knowledge that these records were not rare (relative to their methodologically downsized number -or the records left after thinning), therefore I do not think using “rarefied” is an appropriate way to describe the process. It gives the idea that the thinning process deliberately restricted the environmental range. “Spatially downsized” or “Spatially thinned” would be appropriate. I believe using the latter would be even better because that is what was used at the beginning of the article.


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: GEOGRAPHIC POTENTIAL OF THE WORLD’S LARGEST HORNET, VESPA MANDARINIA SMITH (HYMENOPTERA: VESPIDAE), WORLDWIDE AND PARTICULARLY IN NORTH AMERICA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Meets criteria
Experimental design: Meets criteria
Validity of the findings: Meets criteria
Additional comments: I read the rebuttal letter and was glad to see that the authors neatly stated replies to all of the comments I made.

Regarding methodological things with which I had had some small to medium issues, the authors do a good job in stating their view and justifying it. Whereas I do not entirely agree with their defense for all instances, it is solid and justifiable, and this is not a black and white debate. The approach they mention has its pro's and con's, as does the one I do. I now see the issue with the PCA's and lack of model discrimination when constructing them with a broad environmental domain. Regarding spatial splits for evaluation, the fact that models need to extrapolate is actually (usually) considered as a tangential benefit of this approach (see Wenger & Oden 2014; Soley-Guardia et al. 2019 Suppl docs). Nevertheless, I also understand the authors critiques, especially the difiiculty of characterizing the niche with a reduced biased dataset (especially for a species with such few recrds like this one).

My suggestion for these methodological approaches was more in the line that the ms could be enriched by mentioning their limitations and justifiyng them more explicitly (as to help more novel users and make them aware that they need not follow your approach as a cookbok). However, I don't consider these explanations as critical (there´s always the conflict with word count and keeping the ideas simple enough for the readers too).

Overall, I salute the authors efforts and consider this version ready 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: GEOGRAPHIC POTENTIAL OF THE WORLD’S LARGEST HORNET, VESPA MANDARINIA SMITH (HYMENOPTERA: VESPIDAE), WORLDWIDE AND PARTICULARLY IN NORTH AMERICA
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Congratulations to the authors, excellent work.


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

Input:
Title: GEOGRAPHIC POTENTIAL OF THE WORLD’S LARGEST HORNET, VESPA MANDARINIA SMITH (HYMENOPTERA: VESPIDAE), WORLDWIDE AND PARTICULARLY IN NORTH AMERICA
Review round: 2
Reviewer: 3

Output:
Basic reporting: This article is moving through a second review. The original article apparently was strongly written, easily interpreted, and presented sound science as submitted. The first round of reviews were also thorough, and concerns identified by the previous reviewers were mostly minor and editorial. The few substantive comments focused on model parameters; of these, they seemed more concerned with justifying the modeling decisions than with the execution and interpretation of those models per se. The rebuttal letter provide by the authors and the changes they made to the manuscript are more than satisfactory, and there is little to be gained by another round of second-guessing the modeling approach. The critical aspect is that the modeling choices are explained clearly, which I find them to be, and that the approach of this research team is thus easily compared with the other two recently published HSMs. Other than failing to acknowledge the paper by Alaniz et al, this manuscript is just fine. Please take a look at my suggestions in the manuscript - and address them :-) - but aside from those this article is ready to be read by the PeerJ audience.
Experimental design: The authors have sufficiently explained their methodological considerations and approach.
Validity of the findings: no comment
Additional comments: Hey, nice work and great job responding to the previous round of reviews! I am sad to say that I've added a few (really, just a very few) comments to the track changes version of your revised manuscript. You will see that the majority of my comments are tiny bits of word-smithing that either clarify some of the cited literature or address a few biological aspects that I think should be treated more gingerly in your narrative. The most critical missing thing is failing to address Alaniz et al.'s paper that came out at about the same time as the Zhu et al. article. The findings are not especially different (although they also present a slightly constrained potential geography compared to some of the predictions in this article), but I think you are in a position to now acknowledge and synthesize three separate analyses that have similar findings. It can actually make this manuscript stand out a little if the authors can work that in.

For the record, the suggestions I have made are to my mind fairly minor. They should be addressed, but I don't think any are significant enough to change the overall impact of this paper, and are not something that will require further review.


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

Input:
Title: GENOME-WIDE COMPARISON OF FOUR MRSA CLINICAL ISOLATES FROM GERMANY AND HUNGARY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript requires a thorough revision of English language; it is recommended that the final English edition be done by a native English speaker or a professional language editing service. Also, there are some typographical mistakes in the body of the manuscript and in tables and figures.

Several articles are available in databases that already analyze comparative genomics of Hospital Acquired- and Community-Acquired MRSA isolates, which seems to be the case of this report. The isolates selected for this work seem to fit with these features. None of those articles are cited in this work.

There is not a clear hypothesis stated or suggested by the authors. It seems a comparative genomics study of four randomly selected Staphylococcus aureus isolates. It is important to define which were the reasons or criteria to select those clones, in order to also establish a proper hypothesis.
Experimental design: The research presented fits with the Aims and Scope of the Journal.

The main question to be resolved by the research, and the hypothesis so on, are not clearly stated. It is not clear which were the criteria to select the clones analyzed, so it is not possible to generate a research question justifying the comparative genomics.

Comparative genomics is performed with enough rigor to support the results.

Methods are described with enough detail to be reproduced and are adequate to reach the goal of comparative genomics
Validity of the findings: Since the main goal of the research is to find differences among genomes of the four S. aureus isolates tested, the manuscript is mainly descriptive of the findings and differences, with little opportunity to establish correlations with pathology, genetic background or epidemiological data related to the isolates.

Genomic analysis sounds robust and well analyzed with appropriate analysis tools.

Since there is not a clear research question or a hypothesis, little or no conclusions may emerge from the extensive description of similarities and differences of genomes.
Additional comments: The manuscript presents a detailed description of the comparative genomics of four isolates of Staphylococcus aureus resistant to methicillin (MRSA) from Germany and Hungary. Although the methodology and results are well described, authors should consider several issues to improve their manuscript before it could be accepted for publication.
The title must represent the major findings or major research topics of the manuscript and be specific. For example “Genome-wide comparison of four MRSA clinical isolates from Germany and Hungary” (obviously, to make comparative genomics, whole-genome sequencing is needed; it is not necessary to indicate it in the title).
A hypothesis is not clearly stated, and that is because there is not a clear question behind the analysis. The authors should provide a clear justification for the selection of the four isolates to analyze. Why compare Germanic with Hungarian isolates? Which is the relationship between these countries with respect to the research? Was any other criterion for the selection of the isolates (particular pathologies, representatives of an outbreak, any physiological feature that makes them interesting for research)? This is important because the rationale behind the selection of the strains may direct the search of similarities and differences and establish correlations with particular features.
Authors should also emphasize which is the particular contribution of their research to the scientific community. There are hundreds of papers already published in comparative genomics of Staphylococcus aureus strains, must of them comparing few reference, well-characterized strains, or dozens of clinical isolates.
Genomic typing of bacterial isolates of clinical importance has been intended with a variety of methods. The authors should present in the introduction a brief analysis of the advantages and disadvantages of other methods (MLST, PFGE, VNTRs, spa-typing, SCCmec-typing, agr-typing) used for Staphylococcus aureus against whole-genome sequence analysis.
Biofilm is analyzed but not presented in the introduction section as a goal of the research.
Results on biofilm formation and tolerance to pH are not properly referred in Table S1.
Improve the presentation of figure S1 by specifying the name of the strains in the lanes and the identification of the amplification products, either by size or name. Make this match with the figure caption.
Avoid superlative subjective adjectives as “intriguingly, surprisingly, unexpectedly” and attach to the quantitative or qualitative description of data.
Line 259: specify which is the antiseptic agent, name, or category.
Although low similarity at the sequence level between plasmids p1G6 and pTW20_1, it is quite evident the similarity in physical distribution and content of the genes. A more detailed description and comparative analysis are necessary.
Lines 270-272. The authors should briefly describe the distribution of toxin among strains.
Lines 291-293. If genes including different autoinducing peptides were detected among the isolates, this suggests the presence of an accessory global regulator locus (agr) which encodes the autoinducing peptides. Agr locus information is also used for agr-typing, which may be determined from the sequence and included as another molecular typing method in table 1.
Lines 295-299. There is confusion in the numbers indicating proportions, which represents the number in the denominator.
Figure 4 caption should refer to the genome accession numbers with the isolates.
In order to improve discussion, authors must take advantage of the information provided by the deduced sequence typing methods, such as MLST, spa-typing, SCCmec-typing and agr-typing (not included in this analysis). Together these methods may provide a molecular epidemiology background of these isolates in relation to comparative genomics. For example, German isolates are STs included in the Clonal Complex 5 which is typical of Hospital Acquired MRSA, while Hungarian isolates also pertain to Clonal Complex 5, but ST22 background suggests its relationship with Community Acquired MRSA. The presence of some virulence genes are particularly frequent in each kind of pathology. For example, PVL toxin was commonly used as a marker of Community Acquired MRSA and is related to particular pathologies.
Discussion is more a review of literature rather than contrasting the findings of this research with previously published papers. As I commented previously, there are no literature references to other comparative genomic studies for the discussion.
Because of the lack of a solid scientific question, conclusions presented are to general that may describe any other genomic comparison between any kind of bacterial isolates.


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: GENOME-WIDE COMPARISON OF FOUR MRSA CLINICAL ISOLATES FROM GERMANY AND HUNGARY
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this work Feteke et. al. analyzed and compare the genome of four methicillin-resistant Staphylococcus aureus (MRSA). The aim and scope of the article is very interesting and relevant. However, the manuscript is not clear and unambiguous, and the writing must be improved.

The authors provide enough references and field background; however, the authors could take into account the next references:

Aanensen, D. M., Feil, E. J., Holden, M. T., Dordel, J., Yeats, C. A., Fedosejev, A., ... & Chlebowicz, M. A. (2016). Whole-genome sequencing for routine pathogen surveillance in public health: a population snapshot of invasive Staphylococcus aureus in Europe. MBio, 7(3).

Kleinert, F., Kallies, R., Zweynert, A., & Bierbaum, G. (2016). Draft genome sequences of three northern German epidemic Staphylococcus aureus (ST247) strains containing multiple copies of IS256. Genome announcements, 4(5).

The structure of the article has a standard format. The figures are relevant and help to understand the paper.

MRSA genome number accession are reported.
Experimental design: Bioinformatics and experimental findings are original and within Aims and Scope of the journal. The genome analysis of MRSA is well conducted, following high technical standard. However, some experimental data is not enough to support all the findings.
Validity of the findings: Most of the results and findings are well supported. However, there are some issues that must be clarified.

1.	 SA H27 biofilm formation was almost four times bigger than other three strains, however, this finding did not were described neither discussed.
2.	Since involved genes in biofilm formation are present in all four strains, an expression gene/protein experiment must be designed and performed to understand this point.
3.	Strain SA G6 is proposed to be more pathogenic than other three strain because of HGT gained genes; this is speculative, and an experimental approach must be carried out to prove this point.
4.	To validate HGT of SA G6 genes, a phylogenetic analysis must be performed.
5.	In the phylogenetic analysis (Figure 7C) it will be desirable to increase the number of OTUs to gain knowledge about strains origin.
6.	Phylogenetic analysis was poorly discussed; this section must be re-written.
7.	There was no discussion about the findings about SA pan-genome.

Minor corrections
•	Homogenize name plasmids in text line 258 and 260 within table 1.
•	Correct pan-genome in line 428.
•	Correct syntaxis in line 519 (but sei gene was not found in Germany isolates).
•	Correct SA H27 and SA G8 (separate acronyms) in line 539.
•	All scientific names should be italicized or underlined in figure legends.
•	In figure 4, strain names, SA G6, SA G8, SA H27, SA H32, must be showed to identification of strains.
•	Figure 7A and 16S rRNA analysis must be deleted since this tree has to low information to resolve taxonomy at strain level.
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: GENOME-WIDE COMPARISON OF FOUR MRSA CLINICAL ISOLATES FROM GERMANY AND HUNGARY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has been revised for the English style and is now more properly written. However, there are still some details in verb conjugation along the manuscript, please review grammar in more detail. Again, a professional language editing service may be useful. Authors have reviewed literature on comparative genomics of MRSA genomes and included it for introduction and discussion. Authors have also inserted an hypothesis and justification for the selection of the strains, so the comparative genomics is now justified.
Literature references were complemented and are in accordance with the results analysis and discussion.
Figures are relevant for the manuscript.
Experimental design: The research presented fits with the Aims and Scope of the Journal.
The main question has now been stated and an hypothesis has been presented, so the comparative genomics analyisis is justified.
Comparative genomics is performed with enough rigor to support the results.
Methods are described with enough detail to be reproduced and are adequate to reach the goal of comparative genomics
Validity of the findings: Differences in the genetic background of the selected strains were already known by the authors and now they have been provided to enrich the genomic analysis and the interpretation of the findings.
Genomic analysis is robust and well analysed with appropriate analysis tools.
Clear genetic background description of the tested strains is now available, and certainly helps to clarify relatedness and differences among strains.
Additional comments: The manuscript presents a detailed description of the comparative genomics of four isolates of Staphylococcus aureus resistant to methicillin (MRSA) from Germany and Hungary. Although the methodology and results are well described, authors should consider several issues to improve their manuscript before it could be accepted for publication.
Abstract: Although a hypothesis has been elaborated, this must be clearly stated in the abstract, and clear identification of the strains tested in the work must be included. The abstract content is clumpsy but it contains the information that is relevant to the research.
All of my previous suggestions on information presentation, analysis and discussion were attended, and were properly directed to improve the contributions of the manuscript to general knowledge on the topic. Besides, they included an important analysis of gene distribution in pangenome, core genome and distribution of singletons among strains that contribute to understand potential pathogenic features of the analysed strains.
Some minor observations.:
Line 34: indicate the identifier of the Hungarian isolate
Line 111. An example of the need of reviewing grammar; authors cite a previously published work and conjugate “includes” in present tense.
Lines 251 to 253. There is no reference to the table that present those results.
Lines 255 to 262. This phrase is to long; divide in several phrases and improve syntaxis.
Line 265: The term “epidemiologic characteristics” refer to the surrounding, environmental or social factors that are related to the outcome of a disease. The authors refer to molecular techniques that are frequently used in molecular epidemiology studies that provide information on the genetic background of the strains. Please rephrase to fit these concepts.
Line 285. Probably authors mean “phage transduction”.
Line 306. Table S1. Please correct the Greek letter in beta-lactamase.
Line 502. Correct the Greek letter in beta-lactamase
Line 638. “Distribution” instead of “diversity”?
Lines 685 to 687. It is not clear to me why this conclusion: “….thus HGT was not limited within S. aureus strains”. Does it mean that authors have evidence of HGT from other species or from strains outside of this study? Which is that evidence? Please clarify.


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: GENOME-WIDE COMPARISON OF FOUR MRSA CLINICAL ISOLATES FROM GERMANY AND HUNGARY
Review round: 2
Reviewer: 2

Output:
Basic reporting: In this work Feteke et. al. performs a genome analysis of four strains from methicillin-resistant Staphylococcus aureus (MRSA). The aim and scope of the article is very interesting and relevant. However, the manuscript is not clear and unambiguous, and the writing must be improved since it is difficult to follow in some parts. Some examples are:

•	It is not clear the origin and reasons why the four strains were selected from the previous study (lines 110-124).
•	MGE abbreviation was not defined (line 42).
•	Results from pH tolerance assay were not referenced to the corresponding table.
•	There are incomplete ideas (e.g. line 272-274).
•	Ideas from lines 377-383, 439-442 were not understandable.
•	There is a discordance between text and figure 6A SA G6 singletons number (143 vs 220).

An all-text detailed revision should be done.
Experimental design: Bioinformatics and experimental findings are original and within Aims and Scope of the journal. The genome analysis of MRSA is well conducted, following high technical standard.

However, some experimental data is not enough to support all the findings.
Validity of the findings: Most of the results and findings are well supported.

However, there are some issues that must be clarified.

1.	Since involved genes in acidic and alkaline pH conditions resistance are present in all four strains, an expression gene experiment must be designed and performed to validate this point.
2.	It was not clear the reason to use S. aureus subsp. aureus HO 5096 0412 to compare al genomes in figure 4.
3.	A correlation between ARGs identification and antibiotic resistance analysis is mentioned (line 322), but antibiotic resistance analyses were not done in this study.
4.	Biofilm formation was widely discussed; however, biofilm formation analyses were not present in this work.
5.	It was discussed that S. aureus subsp. aureus ST228 (HE579071.1) and SA G6 addition to pangenome drastic decline of the core/pan- genome ratio, however, ¿in four strain pangenome analysis this decline was observed when SA G6 was added?
6.	To validate that singletons genes could be acquired by HGT (line 644-646), a datailed bioinformatic analysis must be performed.
7.	In the phylogenomic analysis, ST105, ST22 and ST5 are central to understand this result, however it is not clear its origin and relevance.
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: IDENTIFICATION AND ANALYSIS OF LNCRNA, MICRORNA AND MRNA EXPRESSION PROFILES AND CONSTRUCTION OF CERNA NETWORK IN TALAROMYCES MARNEFFEI-INFECTED THP-1 MACROPHAGE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Li and colleagues presented a work to study the ceRNA network in macrophages infected with Talaromyces marneffei (TM). By sequencing the TM-infected macrophages and normal macrophages, they quantified the expression levels of lncRNA, miRNA and mRNA, identified the differentially expressed genes and enriched function categories, and constructed the lncRNA–miRNA–mRNA interaction ceRNA networks. Their work revealed some lncRNA markers that may play important regulatory role in TM-macrophage interactions. Please see below for some major and minor comments.

Major comments:

1. There are quite a few  grammar issues with the manuscript. Here I just listed few examples. The author should carefully check the manuscript if it is accepted for publish.

 (1) in abstract line 55-56, “Select genes that are significantly abundant in GO analysis and KEGG pathway. The core genes…” Technically this is not a complete sentence. The “core genes” in the next sentence is also confusing in this context.

(2) line 80-82: “If not diagnosed early or appropriate systemic antifungal treatment is not performed in time,…”, maybe “Without early diagnosis or timely systemic antifungal treatment… ” would be better?

(3) Line 167-169: technically, these two sentences should not be connect with comma. Should be rephrased.

(4) Line 172-176: all the methods used the past tense other than this part. It would be better to keep the context consistent.

(5) Line 186-188: incomplete sentence without subject.

2. Line 89-91: “It has been found that the function of macrophages on TM, including phagocytosis, fungicidal activity, and phagosome-lysosomal fusion, won’t be affected with the weakened immunity.”  Is there any reference paper to support this finding?

3. Line 93-94: “The interaction between macrophages and TM changes the expression profile of cellular genes.” Is there any reference for this?

4. Line 91-94: in this part, the author mentioned the paradoxical phenomenon that TM conidia can survive in macrophages. Have there any other studies been looked into this? Did they provide any insights and if so, how would that related with this study? I would suggest the author to elucidate more details and background here, in order to state a stronger rational and motivation of this study.

5. Line 241-245: “The upper panels are based on expression values of all lncRNAs, mRNAs, and miRNAs detected. While, the bottom panels correspond to expression values of significantly changed lncRNAs, mRNAs, and miRNAs. The expression values are depicted in line with the color scale. The intensity increases from blue to red. Each column represents one sample, and each row indicates a gene.”

I would suggest move this description to the caption of Figure 2 since it’s more about how to read a plot. Also, in Figure 2, it might be better to label group a and b with specific names. The values in the heatmap should be clarified as well. I assumed it should be the z-scores of log2(FPKM)?

6. Line 272-275: “The highest peak in the Figure 3(C) is the enrichment score (ES). A positive value ES means that the gene set is enriched at the top of the list, and a negative value ES means that the gene set is enriched at the bottom of the list. The set of genes with a particularly prominent peak on the far left or right is usually the set of genes of interest.”

I would suggest move this description to the caption of Figure 3.
Experimental design: Overall, the author clearly described their experimental designs.
Validity of the findings: Pleas see below.
Additional comments: Here are some major and minor comments about the methods and findings.

Major comments:
1.	Line 162-166: “A series of filters were performed …” Did the author perform any specific filtering other than default ones?  If so, it should be clearly indicated.

2.	Line 167-169: does the “pvalue<0.05” refer to nominal p value or adjusted p value? This question also replies to all the p values showed in the manuscript.

3.	Line 182: “Differential analysis was performed using DESeq2, and differential miRNA screening conditions were: | Foldchange | ≥2 and P-value≤0.01.” The differential lncRNAs and mRNAs were identified with FC at 1.5. Is there any specific reason to use different cutoffs were used for lncRNAs, mRNAs and miRNA? Again, the author need to clarified if the p value is nominal or adjusted p value?

4.	For the GO and pathway enrichment analysis, did the author use all DE genes (up- and down-regulated) as a whole input or did the enrichment analysis for up-genes and down-genes separately?

5.	Line 205: the author used fdr at 0.25 as cutoff in GSEA analysis. Normally the fdr at 0.05 is commonly used. I wonder how would the results change with the fdr at 0.05.

6.	Line 252-254: “Based on the selected differential genes, the hypergeometric distribution relationship between these differential genes and GO or Pathway was calculated, and a P-value was returned.” The statements might need to be rephrased. The “hypergeometric distribution relationship” is not correct. It assumes that the number of genes associated with a functional category that overlap with the set of DE genes follows a hypergeometric distribution.

7.	Line 263: for pathway enrichment analysis, did the author use all DE genes or only up-regulated genes?

Minor comments:
1.	Line 188: “Briefly, all genes of this species were…” Does the author mean all coding the noncoding genes in hg38? It should be clearly stated.
2.	Line 237-240: since the differential miRNA had different cutoffs than lncRNA and mRNA, then the cutoffs stated in line 237 were not correct.


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: IDENTIFICATION AND ANALYSIS OF LNCRNA, MICRORNA AND MRNA EXPRESSION PROFILES AND CONSTRUCTION OF CERNA NETWORK IN TALAROMYCES MARNEFFEI-INFECTED THP-1 MACROPHAGE
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no commen
Validity of the findings: no comment
Additional comments: In this manuscript, the authors constructed an THP-1 macrophage infected with Talaromyces 4 marneffei model, then high-throughput sequencing of differentially expressed mRNA, LncRNA and miRNA were performed, , finally,  constructing the ceRNA network of differentially expressed genes and some of the genes were identified. Although the study was designed clearly and the findings are interesting, there are several limitation of the study, which the author should address before publication；
1.First of all, in the line 58-59, the article stated that some differentially expressed genes were verified, mRNA and lncRNA both listed gene names in detail, and miRNA should also be written in detail.
2.In the line 213, the qRT-PCR primers for the validated genes must be listed; and in the abstract section, that 3 mRNAs, 5 lncRNAs and 2 miRNAs were verified in total (line 57-59), but 4 lncRNAs and 4 mRNAs were verified in the text of the article (line 307-308), which is contradictory, so, it is necessary to determine which genes have been validated.
3.In the line 208, it is not enough to use only one database for miRNA target gene prediction, several databases such as TargetScan、miRBase、miRTarBase should also be used to improve accuracy.


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: IDENTIFICATION AND ANALYSIS OF LNCRNA, MICRORNA AND MRNA EXPRESSION PROFILES AND CONSTRUCTION OF CERNA NETWORK IN TALAROMYCES MARNEFFEI-INFECTED THP-1 MACROPHAGE
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript “Identification and analysis of dysregulated lncRNA,microRNA and mRNA expression profiles and construction of ceRNA network in THP-1 macrophage infected with Talaromyces marneffei” described the sequencing of lncRNA, miRNA and mRNA in THP-1 macrophage infected with Talaromyces marneffei. These genes may be involved in immune response and inflammatory responserelated pathways and functions in TM-infected macrophages. Overall, this study is easy to follow and detail oriented, I have a few comments to improve it for further process.

1. Authors need to revise the use of terminologies and full terms and abbreviations. These abbreviations should be consistent and don't shift between the full terms and their abbreviations.
2. Despite the well design of current study, the writing of manuscript still needs further improvement.
3. In the RESULT part, some details need to be added, for example, in the ceRNA network part, the details of lncRNA-miRNA-mRNA need to be clarified.
4. In ‘qRT-PCR validation’ part, why there is not show the result of miRNA?
5. Some sentences are not needed in the DISCUSSION part, for example, line 317-325.
6. All figures are not clear enough. Please check and upload them again.


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: IDENTIFICATION AND ANALYSIS OF LNCRNA, MICRORNA AND MRNA EXPRESSION PROFILES AND CONSTRUCTION OF CERNA NETWORK IN TALAROMYCES MARNEFFEI-INFECTED THP-1 MACROPHAGE
Review round: 2
Reviewer: 1

Output:
Basic reporting: My questions have been addressed in the revised manuscript.
Experimental design: N/A
Validity of the findings: N/A
Additional comments: N/A


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: IDENTIFICATION AND ANALYSIS OF LNCRNA, MICRORNA AND MRNA EXPRESSION PROFILES AND CONSTRUCTION OF CERNA NETWORK IN TALAROMYCES MARNEFFEI-INFECTED THP-1 MACROPHAGE
Review round: 2
Reviewer: 2

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


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: MODELING THE BIDIRECTIONAL GLUTAMINE/AMMONIUM CONVERSION BETWEEN CANCER CELLS AND CANCER-ASSOCIATED FIBROBLASTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written in general, but in places, the presentation is unclear or lacks sufficient scientific detail. I have included comments and suggestions to the authors.
Experimental design: My one concern is the lack of demonstration of application of the results. In figure 1(a), a 'dotted' arrow is shown as potential therapy targeting the CAF phenotype. The authors should include 1-2 simulations of such a treatment, and what it's effect might be at least in the in vitro case being modeled here.
Validity of the findings: The manuscript meets these criteria.
Additional comments: Fig 1(a) – aspect ratio seems incorrect; image appears stretched horizontally

Fig 1(b) – Please make the legend more descriptive

In Lines 63, 76 etc., the year of the reference should be revisited

Lines 76-82: This feels a little abrupt. You haven’t told us what the monoculture and coculture experiments are. Also, the statement about a decline in cancer cell population feels out of place. In Line 78, ‘our experimental system of cultured breast cancer cells’ is mentioned but this is the first allusion to any experiments herein. A brief overview of the experiments you are presenting here is needed. Also, a more scientific description of the experimental set up is needed, e.g., which breast cancer cell line?

Line 85 – what dynamics are being referred to? Experimental observations? Or model predictions? Is there a figure for reference?

Lines 86 and 88-89 – Where is the evidence to back the claims that CAFs convert all Gln to ammonia but do not convert all ammonia to Gln?

Line 90 – Additions are being made to a model that we have not yet been introduced to

Equations (1)-(4) – if the target audience for this paper is both, mathematicians and biologists, then explain the ‘prime’ notation. Also, it is more standard to denote time derivatives with a ‘dot’.

Lines 95-96 – in describing the dimensionless constants c1 and c2, do you mean, for e.g., that c1 is the number of Ammonia molecules produces for each molecule of Gln consumed?

Equation (3) is confusing – What is the source of this energy? It seems to only decrease. The constant k2 is not introduced in the explanation of the equation, presumably it is the rate of energy consumption as ammonia is converted to Gln? Also, the second term on the right-hand side of (3) is a distinct process of energy consumption, unrelated to Ammonia  Gln conversion? This should be made clear. As I continue to read, I see the explanation of R(t) in the following paragraph but consider reorganizing for clarity.

Line 110 – Are these also the units of concentration for the other chemical species?  This should be made clear when introducing the model variables. Please also provide units of time.

Lines 112-113 – From looking at the equations, it is not clear that in the long run, all Gln should be converted to Ammonia. Should this not depend on the choice of parameters k1, k2, c1, c2? Do you prove this claim mathematically? Or show it via numerical simulations?

Line 115 – Figures 2 and 3 are referred to very cursorily. They need to be introduced at appropriate places within the results presentation text preceding this line. So that the reader can look at the experimental or model findings as they are talked about.

Are Figures 2 and 3 the best fits of model simulations to the data? How many parameters are being fitted? Also, the figure legends refer to Figure 2(a) and 2(b) but I see six panels.

Figure 3, bottom panel on the left – Why does the model deviate so strongly from the experiments?

Line 118 – I think the words ‘assumed to be’ are missing, i.e., the sentence should read ‘…they are assumed to be more sensitive to depletion…’. This is a (very reasonable) modeling assumption based on the experimental finding that cancer cells consume more Gln than healthy cells.

Line 123 – What are the different concentrations?

Table 3 – Should r1CC be 5.99x10^-2 h^-1?  The notation of using a dot to denote multiplication is confusing with decimals also present. Consider switching to ‘x’.


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: MODELING THE BIDIRECTIONAL GLUTAMINE/AMMONIUM CONVERSION BETWEEN CANCER CELLS AND CANCER-ASSOCIATED FIBROBLASTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a mostly well-written paper, lacking in details in some sections, that adequately describes the current state of the literature. There could be more focus on models of mono- and co-cultures and their interactions in cancer contexts, including, for example, doi: 10.1038/s41467-017-01174-3, 10.1007/s00109-017-1587-4, and 10.1371/journal.pcbi.1007278. Figure 1A (left panel) can be improved. Instead of marking individual points for triplicates, suggest indicating error bars. Raw data is supplied.
Experimental design: Overall, the experimental design is adequately described. It is unclear to me why numerical simulations were carried out in Mathematica but fitting was done in Matlab, when you would need to implement the model in Matlab to be able to solve it in an optimizer? This shouldn't impact on the results but is curious.
Validity of the findings: A clearer statement of the conclusions could be provided, the discussion reiterates much of what is said as background and context in the introduction. I question how generalizable and valid the results are given the very poor fits to co-culture experiments. Suggest that a sequential fitting procedure could be better adapted and more logical (though details of how each parameter fit was obtained are scarce).
Additional comments: 1) Lines 110-111: What is the rationale for choosing an initial concentration of 20nM? Is this due to experimental conditions or prior knowledge? Either should be stated.

2) In all tables, it appears all of the model parameters are fit. Can no prior knowledge be incorporated to reduce the number of parameters? What about using a sequential fitting scheme to fit a reduced number of parameters and then build in others? Perhaps this was done but details were not provided.

3) Lines 118-119: A reference for this statement would be good to provide.

4) Overall, I would suggest using more identifiable variables in the model (this doesn't impact on the mathematics but makes it easier for readers to follow the equations). Also the choice of e as a parameter is not a good one as it looks like an exponential function.

5) Why is no direct interaction between CAF and cancer cells assumed in the model? This seems questionable to me, and I think the lack of this interaction is related to the poor model fits to co-cultures. See, for example, doi: 10.1371/journal.pcbi.1007278. Later on lines 125-126, the authors state: "the growth rates of both cell types are allowed to change since in the co-culture it is assumed that the two cell types may affect each other" but this only modelled indrectly.

6) The discussion can be greatly improved. Lines 152-166 don't belong in a discussion. There is a lack of focus communicating the outcome of the study.

7) Lines 181-190: As stated earlier, the model may be missing important interaction terms that could be leading to these poor fits.

8) Line 234: A better understanding of the interactions of the model could be obtained by varying the initial proportions of cells. What is the rationale for sticking to 1:1 only?

9) "Indicated time points" should be stated explicitly in the methods.

10) Line 245: The objective function should be stated explicitly.

11) To quantify the variance in the normal distributions in the error function, why use bootstrapping replicates while only fitting to the mean data? Why not also incorporate the error from the triplicate experimental observations?

12) Particularly in co-cultures, the fits are not good. How can conclusions be drawn?


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: MODELING THE BIDIRECTIONAL GLUTAMINE/AMMONIUM CONVERSION BETWEEN CANCER CELLS AND CANCER-ASSOCIATED FIBROBLASTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The basic reporting aspects of this paper are sound.  Figures are clear and readable.  Line 76 has a reference to a paper in 2027 that I am assuming is a typo.

I do have some stylistic suggestions.  One common style standard is not to begin sentences with variable names or lower case letters, to aid readability.  In line 35, "c_1 is. . ." could be rewritten "The variable c_1 is. . ." .  In line 105, "That's" may be viewed as too colloquial. The authors  may wish to write "That is".  The use of the subequations environment in LaTeX can help organize the mathematics.  The first system of equations could be labelled (1a) to (1d) rather than (1) to (4) and then referenced collectively as system (1).  Similarly with equations (5) to (9).
Experimental design: This is outside of my area of expertise as a mathematician.
Validity of the findings: "Validity" in the sense applied to experimental papers isn't relevant for a work in modeling and simulation like this.  The model in this case aims to capture the system of interest and to create an abstract object that can be used to enhance understanding and guide future experimental work.  In this sense the work is well on its way to being "valid".

The authors obtain a fair amount of data and use it to fit the parameters for their models.  Here the work seems incomplete.  It is increasingly de rigueur for ODE models in biology, which tend to have a fair number of fitted parameters, or measured parameters with wide error bars, to have a generalized sensitivity analysis done for the parameter space.  Which parameters are the most influential?  How much so?  What do they represent biologically, and what is the biological significance of them being sensitive or insensitive to perturbations?
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: MODELING THE BIDIRECTIONAL GLUTAMINE/AMMONIUM CONVERSION BETWEEN CANCER CELLS AND CANCER-ASSOCIATED FIBROBLASTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I am happy with the revised manuscript and can recommend it 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: MODELING THE BIDIRECTIONAL GLUTAMINE/AMMONIUM CONVERSION BETWEEN CANCER CELLS AND CANCER-ASSOCIATED FIBROBLASTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: In their responses, the authors state that: "the secondary source of chemical energy R is not accessed in the experiments. The choice of 20 mM is to match the order of magnitude of the other, measured, concentrations, which are between 1 and 25 mM." This should be stated clearly in the text.
Experimental design: By “sequential fitting scheme”, I meant taking parameters available in the literature, and fitting individual experiments to a reduced number of variables to reduce the degrees of freedom at each fit.
Validity of the findings: From the conclusion: "Future research will have to elucidate the precise reasons for the sharp decline of cell numbers after 48 h, and how a similar population would behave in vivo." Can the authors suggest some rationale or mechanistic explanation? The "stress" model fits are definitely improved over the original co-culture fits, and the differences between these two models should be informative to the biology underlying the response.
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: DIGITAL CELL SORTER (DCS): A CELL TYPE IDENTIFICATION, ANOMALY DETECTION, AND HOPFIELD LANDSCAPES TOOLKIT FOR SINGLE-CELL TRANSCRIPTOMICS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript presents a suite of software elements for the analysis of scRNA-seq data. It is clearly written with professional structures and figures.
Experimental design: The research question is well defined and the in silico experiment is carried out in a systematic way.
Validity of the findings: The performance of the platform the authors presented is supported by the provided data.
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: DIGITAL CELL SORTER (DCS): A CELL TYPE IDENTIFICATION, ANOMALY DETECTION, AND HOPFIELD LANDSCAPES TOOLKIT FOR SINGLE-CELL TRANSCRIPTOMICS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript written by Domanskyi et al., is well written and described with no flaws in english language.
Experimental design: This manuscript falls under the aims and scope of PeerJ. This manuscript is well defined the research question and the development of DCS toolkit would be beneficial for those performing ScRNA data analysis.
Validity of the findings: All underlying data have been provided, they are robust, statistically sound and controlled. Conclusions are well stated, linked to original research question.
Additional comments: Dear Authors,

Thank you for the well written and well described manuscript about DCS toolkit. I hope it will be highly usable for researchers performing single cell RNASeq data analysis.

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: DIGITAL CELL SORTER (DCS): A CELL TYPE IDENTIFICATION, ANOMALY DETECTION, AND HOPFIELD LANDSCAPES TOOLKIT FOR SINGLE-CELL TRANSCRIPTOMICS
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1. Currently the users do not have an input file directly created from some of the more popular softwares used in the upstream analysis. This would require some additional handling of the files before it could be loaded onto DCS. If the compatibility between DCS and other softwares can be worked out it will just expand the user base for DCS.

2.The Github repository is well well documented. However it might help to have some tutorial/vignettes along to ease the learning curve for new users.

3. The text in the figures are too small to read. Also the grey text in sankey(river) plot makes it difficult to read it well in the grey background.
Experimental design: 1. The cluster annotation is a very important step in the scRNAseq data analysis, both in terms of biological interpretation as well as evaluating the performance of the clustering. The authors here presented with a ‘voting algorithm’ the annotates clusters based on preferred marker. This methods mostly depends on a well defined maker database and assigns cell types based on absence/presence of marker genes. While this might work great with many cell types, it can get difficult when cell types with not not known specific and exclusive markers or cell states. Have the authors tested or considered the annotation based on marker identification (unbiased differential testing in one cluster vs the rest) and then using that list to identify cell types based on databases?

2. The authors also performed anomaly detection to find cells that are different from the majority of the states using isolation forest algorithm. This they also went on to show was not an effect of just technical variabilities due to experiment condition , technology or sequencing depth. Sometimes a cluster can be a mix of celltypes, this seems like a good step to verify that all the cells clustered together are indeed similar. However, clustering usually considers some significant PCs, but it could have more PCs that are not as significant but exists in the cell types (for e.g differentiation state, activity state etc). This could sometimes also be a factor of doublets or multiplets.  The AUC is a good indicator of of the model. However, to differentiate between subtle differences in state type of same cell type vs. in a mixture of 2 very different cell types, it would help if there is some stat to attached to make sense of it. For e.g Silhouette values that tells how close a member is to its own cluster compared to other clusters, or some value to signify the strength/significance of this difference.
Validity of the findings: no comment
Additional comments: The authors have presented here a python toolkit for the analysis of single cell sequencing data which performs 1. automatic cluster annotation based on predefined markers, 2. Detects heterogeneity within a cluster identifying cell anomaly, 3. Cell type identification based on a neural network algorithm - Hopfield networks. Owing to the ever increasing generation of scRNA seq data and more researchers looking for tools that provide a suite of functions in an easy to use documentation- this seems like a good solution for some of the more important and time consuming step of cell type annotation.


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: IMPROVED SALT TOLERANCE OF CHENOPODIUM QUINOA WILLD. CONTRIBUTED BY PSEUDOMONAS SP. STRAIN M30-35
Review round: 1
Reviewer: 1

Output:
Basic reporting: In my opinion, this is an acceptable manuscript for publication after corrections. In this regard, my detailed views on the current work are listed below.
- The context of the manuscript is in the scope of the journal.
- Introduction section line 32 the sentence “Add your introduction here.” should remove from the manuscript.
- The sentence in line 83 and 84 “reported that bacillus inoculum increased the plant biomass, relative water content, leaf water potential and leaf water loss, and reduced electrolyte leakage.” should have written again. Because of grammatical mistakes.
- Introduction section should have written again. Some of the sentences which are related recent examples about PGPR strain of saline soils should remove from there and please add to discussion section.
- Genus and species names should have written italic in all of the manuscript.
- There is no need for subtitles in discussion section.
-  There are some grammatical mistakes in the text. It should check and revise some professional language experts or native speakers.
Experimental design: Experimental design of article is enough and all methods are generally used in this kind of studies.
Validity of the findings: The topic of article is rationale and very popular. Conclusion is are well  explained. But language have to improved by authors.
Additional comments: In my opinion, this is an acceptable manuscript for publication after corrections. In this regard, my detailed views on the current work are listed below.
- The context of the manuscript is in the scope of the journal.
- Introduction section line 32 the sentence “Add your introduction here.” should remove from the manuscript.
- The sentence in line 83 and 84 “reported that bacillus inoculum increased the plant biomass, relative water content, leaf water potential and leaf water loss, and reduced electrolyte leakage.” should have written again. Because of grammatical mistakes.
- Introduction section should have written again. Some of the sentences which are related recent examples about PGPR strain of saline soils should remove from there and please add to discussion section.
- Genus and species names should have written italic in all of the manuscript.
- There is no need for subtitles in discussion section.
-  There are some grammatical mistakes in the text. It should check and revise some professional language experts or native speakers.


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: IMPROVED SALT TOLERANCE OF CHENOPODIUM QUINOA WILLD. CONTRIBUTED BY PSEUDOMONAS SP. STRAIN M30-35
Review round: 1
Reviewer: 2

Output:
Basic reporting: General comments
The language/grammar used in the manuscript requires significant improvement. It is unacceptable in present format. Several statements lack of clarity, precision, and comprehensiveness. Moreover, the research work lacks sufficient novelty, for the reason that the effect of plant beneficial bacteria on plant growth under stress is known and many reports are available. The writing style is appalling and requires a major revision.
Experimental design: Experimental design is well organised, but english writing should be imporved.
I made some comments which could help to improve MS.
Validity of the findings: The results support the aim of work, and only limitation is that there is no novelty in this study.
I have suggested authors to write new findings and add more recent citations on related topic.
Additional comments: General comments
The language/grammar used in the manuscript requires significant improvement. It is unacceptable in present format. Several statements lack of clarity, precision, and comprehensiveness. Moreover, the research work lacks sufficient novelty, for the reason that the effect of plant beneficial bacteria on plant growth under stress is known and many reports are available. The writing style is appalling and requires a major revision.

Specific comments
Line 62: “Add your introduction here” remove
Lines 67-68: “The present two  popular strategies”, which strategies, describe?
Lines 102-124: Please revise and shorten the text, many repetitions
Line 127: remove, “first time”, the beneficial effect of root associated bacteria on plant growth under abiotic stresses is known.
Lines 15-133. Please shorten text and clarify the aim of study, but do not repeat
Line 139: Luria broth, please add source
Line 146: seeds were inoculated, but why after surface-sterilized?
Line 147: germinated in plates? Or pots?
Line 172: spectrophotometer model?
Line 174: Determination of root activity? Root system architecture?
Why only P concentration in plant tissue were measured, what about N,K and other minerals?
Line 221: under both the LB medium (control) and M30-35 medium, please correct, e.g. under non saline and 100mM NaCl condition?
Line 255, please explain root activity
There are many outdated references, please add recent reports on PGPR activity on plant growth under stress. Moreover, it is important to demonstrate new findings in this study. Because it is known that Pseudomonas strains increase plant stress tolerance and also modulate physiological processes.


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: IMPROVED SALT TOLERANCE OF CHENOPODIUM QUINOA WILLD. CONTRIBUTED BY PSEUDOMONAS SP. STRAIN M30-35
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this work, Cai and co-authors described the effects of a Pseudomonas sp. strain M30-35 on the responses of quinoa plants to salt stress in attempt to explain the mechanism of the increased salt tolerance by the bacterium. The comparative assays for the physiological parameters from control and M30-35 treated plants under salinity stress indicated that M30-35 treatment could increased the salt tolerance of quinoa via improving the inhibited growth and alleviating physiological toxicity of quinoa plants exposed to salinity stress. The results are simple, but they are very worth, because the findings may be helpful to generate new bio-fertilizer which may be useful to improve the growth of crops grown in saline alkaline soil. Therefore, my overall impression is positive, but some issues should be addressed before the paper may be recommended for the publication.

Major comments
1 Tons of grammatical errors occurred throughout the manuscript, so the use of English grammar and style should be thoroughly checked and corrected. It is the best to find an expert with good English knowledge to polish your text and to avoid any grammatical and syntax errors.
For example:
Lines 103-105, “This is rich in protein, amino acids, dietary fiber, vitamins, trace elements, and various beneficial phytochemicals.”

Lines 289-292, “It might be possible that M30-35 functioned on the roots through similar mediated methods, in which KM4 improved the salt tolerance in maize by promoting plant growth to improve the salt tolerance of quinoa.”
The two sentences are only the representatives of a lot of descriptions which is very difficult to understand.

2 The authors seem to choose some physiological parameters randomly, and put them in the manuscript very simply, because the relationship among them is not close, especially saponin is rarely used in studying salt tolerant mechanism of plants. If saponin is an important component in quinoa, please describe it in detail in the revised manuscript.

Minor comments
1 Why did fresh and dry weights were used together to express the biomass in the manuscript? Normally, when the change in water content is analyzed, fresh and dry weights may appear at same time. However, the relative information is not mentioned at all. So it will be enough to only use dry weight to express biomass.

2 If the growth parameter, fresh weight, is deleted, original figure 1 and 2 should be combined into figure 1. Title of the revised figure 1 should be “The effect of M30-35 on the grow of quinoa plants under salinity stress.

3 “The columns with different letters indicate the significant differences among treatments at P<0.05.” The sentence can be seen in all figure legends. Different letters were marked for some columns, but their bars of SE overlapped. Normally, if SE bar of two data overlap, the difference between two data is not significant. So please explain for these data.

4 a lot of words are not used accurately.
For examples:
Line 208, the word “estimated” should be changed into “determined”.
Line 311, the word “subdue” should be changed into “inhibit”.

Taken together, the arrangements and English style are very poor, so please check and revise carefully the manuscript for its resubmission version.


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: IMPROVED SALT TOLERANCE OF CHENOPODIUM QUINOA WILLD. CONTRIBUTED BY PSEUDOMONAS SP. STRAIN M30-35
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have addressed all issues from me.


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: RECORDS OF CTENOPHORES FROM SOUTH AFRICA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, the article is well-written and does a great job of relaying a great deal of information in an accessible way. It’s easy to imagine this being a helpful resource for people at many levels of scientific experience and interest. That said, there are a few points I would like to see the authors address that I think could improve its ease of use.

First, the article should have a Materials and Methods section. I realize that as a citizen science-based biodiversity project, the composition of such a section might be somewhat atypical, nonetheless I think it would be helpful for readers. I provide some suggestions for topics that might be helpful for such a section to cover in section 2 of this review.

Also, the clarity of the photographic advice would be improved if it were set off into its own (sub)section, perhaps with a bulleted list of key points. It might also be worth distinguishing advice for photographing ctenophores in the ocean in a SCUBA context vs photographing sampled ctenophores in holding tanks/aquaria.
Experimental design: The topic of the article is interesting from a biodiversity perspective, and it fits within PeerJ's aims and scope.

However, the article needs a materials and methods section describing how the data were collected in more detail. Some points of possible interest for such a section,

•	Examples of types of cameras/gear used by citizen scientists in this study
•	Was there a standard protocol used by citizen scientists used to record environmental data, location, etc?
•	Did citizen scientists use a web portal (such as iNaturalist) to collect/organize photos/metadata or did they correspond directly with organizers?
•	Were citizen scientists recruited specifically to help describe ctenophore biodiversity or were they participating in other, broader efforts?
Validity of the findings: The findings of the article seem valid and well-supported overall, but I have a few points of clarification,

Are there any additional photographs of ctenophores from the citizen scientists that exist but didn’t fit into the present article? If so and if they are on an online repository such as iNaturalist it would be worthwhile to provide a link to the relevant user profiles so readers can access that content.

Similarly, do more detailed data on locations/timing for observed ctenophores exist? If so, it might be helpful/worthwhile to include/discuss it, even if it is somewhat limited. It might help future citizen scientists in the area know how far out from shore to direct observations, when to time sampling relative to the tides, etc.
Additional comments: Line 77-78: Please cite a basis for this statement and be specific in the claim (e.g. "based on the molecular phylogenies of studies X and Y the affinities of ctenophores and cnidarians appear convergent and not based on shared ancestry")

The section on the ctenophore body plan should make explicit mention of the term biradial symmetry.

Line 294: Eurhamphaea sp. aboral processes/filaments should not be referred to as “tentacles” in order to distinguish them from feeding structures. I think the language of Mayer, 1912 is generally a good guide, “apical processes […] give rise to a pair of […] simple filaments” (p. 41). A similar change should be made in the caption for figure 11. Also in the caption for figure 11, I would say that the apical filaments are not a great diagnostic for this genus, as the lengths can vary quite a bit, and in some individuals, the filaments may be so reduced that they are hard to even see. It would probably be better to mention the ink spots in this caption.

Figures 4 and 5: Are these meant to be one figure with two panels, a and b? If not, neither needs a panel label.

Figure 13 caption: Which photo shows a species likely new to science? Panels a, b, d, and e should also be mentioned in the caption.

Double check citations. There are some spelling discrepancies and some citations that were cited in the text but not present in the reference section, e.g.

Line 80: Giribet and Edgecombe, 2020 not in bibliography
Line 128: Should read Deidun
Line 333: Should read Moser (1910)

Finally, if there’s any way to show the approximate observed sightings or ranges for the different taxa discussed on a map of SA, I think such a figure would add a lot to the article.


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: RECORDS OF CTENOPHORES FROM SOUTH AFRICA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This ms combines descriptive, taxonomic and ecological information in a report on the occurrence of ctenophore species of South African coastal waters. Unlike more conventional reports, it is based on photographs of ctenophores from citizen scientists, along with prior published information on ctenophore occurrence and systematics.
The underlying data of the paper are thus the photographic images, which have been identified by the authors, at least two of whom (Haddock, Matsumoto) are well known experts in ctenophore biology.
The authors emphasize that the delicacy of ctenophores and the difficulty of preserving specimens makes reliance on images unavoidable as a basis for descriptive studies. Presumably collection of images rather than specimens obviates any concern about proper permitting.
The Introduction established the physical context of the study region, including the influence of the Agulhas Current and the gradients in temperature, productivity and biomass that result in different parts of the South African coast.
There are paragraphs describing general ecology of ctenophores, including impacts as introduced species, and evolutionary history. A brief section on photographing ctenophores seems aimed at citizen scientist and divers who may wish to contribute future images.
A few specific comments:
Abstract:  Authors say only six species are previously described from South Africa, and that they add three more at species level and 6 at morphospecies (=genus here). But I don’t see anywhere an explicit notation of which are which in the subsequent descriptions.
Line 55:  ‘..some are planktonic others are strictly benthic..’ Really, the large majority of species are planktonic, so better to say ‘most are..’
Line 59: ‘..ensnaring prey in mucous’.  Although mucus is used by lobates and cestids to trap and transport prey, the colloblasts themselves use a glue to stick to prey, not really mucus.
Line 80: reference Giribet & Edgecombe 2020 isn’t in the references.
Line 104: Since this paper seems aimed at non-specialists on ctenophores, a definition of ‘morphospecies’ here would be useful.
Lines 106-124: These paragraphs on photographic hints should be set off with a subheading and placed at the end of this section, before the overview of classification and anatomy.
The Overview is a good addition here and should be useful to citizen scientists to understand ctenophore morphology. Figures are helpful.
Experimental design: There is no 'experiment' in this paper. It is a descriptive and taxonomic report. These comments refer to aspects of the data presentation.

Descriptive Section

The subhead here is ‘Morphospecies’ but some are described at species level and others at genus level. Previously 3 new ‘fully described’ and 6 morphospecies were mentioned, but there seem to be 11 separate species/morphospecies in this section.  This should be clarified.
The descriptions all seem accurate and clear. Presumably they will have the relevant Figures adjacent, since images are really the best way to identify ctenophores to this level.

Figure 4: the caption says Hormiphora sp., but the image and link are Pleurobrachia pileus.
Discussion
Line 358: interesting that Lampea was not found, as it preys on salps, which are often abundant in South African waters.
Validity of the findings: Conclusions
The authors cite the value of image-based data for description of morphospecies, which is sufficient to understand the ecological role and importance of the ctenophores present. This seems a reasonable perspective.

General Comments:
Even if not a rigorous taxonomic treatment, this paper provides usefully accurate new information about the presence and distribution of several new ctenophore species. Based on amateur contributions of photographic data and directed in part to that community, the paper helps fill gaps in knowledge of the plankton fauna of the region, and encourages further citizen participation in making and reporting observations. The authors all have relevant expertise and reputations in zooplankton and ctenophore biology.
Additional comments: see above


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

Input:
Title: COMMENT ON PESCOTT & JITLAL 2020: FAILURE TO ACCOUNT FOR MEASUREMENT ERROR UNDERMINES THEIR CONCLUSION OF A WEAK IMPACT OF NITROGEN DEPOSITION ON PLANT SPECIES RICHNESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general the English is clear, unambiguous and professional throughout. In a couple of areas I find the language problematic. For example: Line 20 the authors refer to Pescott & Jitlal (2020) methods as "sophisticated" and then use the term "simple" in relation to their simulation (Line 25). I find this distinction odd as both use the same types of models (one is simpler than the other but this gives the impression of suggesting that the sophistication is a contributing factor to the perceived "premature conclusions" of Pescott & Jitlal).

In Line 240, 247 and 252 the authors use the terms "their" and "our" but it is not clear who they or our are - I did not read the original paper as a direct criticism of an individual's/group's work but rather as an important consideration that all researchers in this (and other observational sciences) need to take account of (spatial autocorrelation).

After Line 165 the paper begins to read like a point-by-point rebuttal rather than a comment highlighting concerns with the interpretation of results from P&J20. Maybe structuring the comment around the main issue that are raised rather than every perceived divergence between the two groups of authors would be beneficial to the reader.

Figures - there are far too many figures to go in to the main document with legends that are not informative enough. What, for example, is the need for both Figure 2 and 3 in the main document? I would be tempted to move all the Figures in to supplementary materials except Figure 7.

Code - It is great that the code is shared.
Experimental design: The simulation design appears sound. I do have one question - the authors say that the DIC is lower in the models which include the spatial field (Line 134 to 141). I am not sure I understand this point (I am not an expert in this at all). I assume a model with a lower DIC has a higher predictive accuracy. Have I got this the wrong way around? - perhaps they can make this paragraph clearer (idiot proof!).
Validity of the findings: I have a couple of areas where I question the presentation of the findings rather than the validity (I hope it is okay for me to use this section for those). The premise of this comment is that the authors believe (and provide lines of evidence) that the findings of P&J20 are premature in that they suggest caution in interpreting observational assessments of the effect of N deposition on biodiversity. The main focus of the comment is on the failure to account for measurement error. This is a valid concern which has been highlighted in epidemiologi recently. I find it strange however, that the two sets of authors (who appear to work in the same organisation) have not joined forces on a paper using the datasets that are presented here and in the original to actually assess the effects of measurement error in combination with spatial autocorrelation. The authors state that (in Line 198) that the key test is to refit the P&J20 models with more accurate measurements of N deposition. Why has this assessment not been done? Surely this would be much more valuable for the understanding of the true effects of N deposition.
The last paragraph (Line 263 - 268) suggests that the authors feel that this is a valid point - that a "better" assessment of the causal effects of N on biodiversity are needed urgently.

I find the repeated assertation that the conclusions of P&J20 should be treated as doubtful and not proven could also equally be applied to this comment - the weight of evidence put forward is circumstantial and does not unequivocally prove that the findings of P&J20 are incorrect. I would have preferred to see an actual assessment of the data taking in to account the measurement uncertainty which is demonstrated in this comment. What are the reasons why this has not been done - is it that the data are not available (if yes - then this should be highlighted here as an important next step), or is there another reason(s)?
Additional comments: Line 81 states that the original authors overlook measurement error but line 149 states that they do address it - they dont include it in the model and suggest a reason for this. These two statements need to be reconciled.

Line 51 states that causal modelling in ecology is tough (yes it is!). The authors highlight the problems around these but then disagree with P&J20 who cast doubt on the size of the effect of N on biodiversity previously reported after trying to empirically account for some of these problems. Do the authors disagree that prior modelling of the effect of N deposition on biodiversity has been problematic (the true effect has not yet been determined) or that the true effect has been determined and this is reflected by previous work despite the problems that they highlight. This is not clear throughout the comment and I think a clearer statement is needed.


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: COMMENT ON PESCOTT & JITLAL 2020: FAILURE TO ACCOUNT FOR MEASUREMENT ERROR UNDERMINES THEIR CONCLUSION OF A WEAK IMPACT OF NITROGEN DEPOSITION ON PLANT SPECIES RICHNESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: See below.
Experimental design: See below.
Validity of the findings: See below.
Additional comments: Note that because the paper is a response to a paper that I authored, I do not provide a full response to the substantive points of Smart et al below, I only comment on aspects where it seems to me that the authors misrepresent Pescott & Jitlal (2020). I do this because I may respond to the interesting and substantive aspects of Smart et al. in an actual paper. I have just selected "minor revisions" in order to submit my review. However, as I am reviewing a rebuttal, I leave it to the editor and other reviewers to make decisions about revisions, publication etc.

Given the situation I leave it to the editor and the authors to decide whether they wish to make any amendments in response to the below points given, but I may comment directly on any article paragraphs that are misleading once the article is published if they are not altered. For these reasons, I also see no point in my re-reviewing whatever revision is submitted.

Taking the above into consideration, the below points are by line number in the PeerJ reviewing PDF.

##Overal conclusion
Smart et al. provide a useful example of a situation where uncertainty and error in space-for-time Ndep impacts modelling can mask the truth. However, their model merely portrays one possibilty, and many others are possible, including situations where unmeasured confounders lead to incorrect conclusons, and where finer-scale Ndep estimates still support a conclusion of small impacts. Moreover, fine-scale Ndep estimates would still require measurement error modelling (predictions from the FRAME model at 1km are obviously not the truth), so it seems likely that estimated coefficients will still exhibit important uncertainty in such situations. Overall then, the work of Smart et al. supports one of the key points of Pescott & Jitlal, that clearly and accurately reporting impact estimates with all associated uncertainty (including that which is not covered by model outputs, but which is related to having chosen one model out of numerous possible models in the first place) is essential to move this field forwards. In my opinion Smart et al. could make more of this point, acknowledging the many issues with much of the previous work in this field that P&J raise, rather than just putting objections to one aspect of P&J front and centre in their piece, and using this language to insinuate that everything about P&J is somehow unreliable. The lack of any discussion of estimated effect sizes, whether with or without the spatial field (which doesn’t actually make that much substantive difference) is also conspicuous in respect of this.

#Title/Abstract
This is somewhat misleading. P&J said that the modelling approach they had taken indicated small and ambiguous effects, and that previous estimates were likely to be have been overestimated etc. Reading this title and the abstract you would imagine that P&J were absolute in their conclusions, and that these conclusions were stated to be independent of the models they used.

L19 It’s interesting that Smart et al. refer to false positive results here, whereas the entirety of P&J was about effect sizes and uncertainty. I just note this in passing as an example of how pervasive the focus on P-values often is.

L26 The simulation study presented by Smart et al. is useful and interesting, but it is a simulation of a possibility, not the truth, so it seems odd to say that the conclusion of P&J is flawed. A more accurate and less rhetoric-laden statement would be that Smart et al. have demonstrated one situation in which the model used by P&J may be misleading.

It is also misleading that Smart et al. state that P&J “failed to recognise” that covariate measurement error could affect our results. What we actually said was:

“Other uncertainties relating to our conclusions pertain to the fact that the broad-scale spatial field used here may be accounting for information that, if known, would change the size of the Ndep impact regression coefficient. This could be in the form of additional covariates, or more highly resolved estimates of the Ndep load that a location has actually received. The Ndep estimates used here (and by the original studies) are resolved to a grain size of 5 x 5 km, and this additional uncertainty could have attenuated our estimate of the regression coefficient (the measurement error in explanatory variables problem; e.g., see Fox, 2016), even if there is no systematic bias relating to the association of 5 x 5 km-estimated Ndep levels with particular types of vegetation.”

Obviously we did not explore this in detail, but it is inaccurate to say that we completely failed to recognise this issue.

L38 I agree that the conclusions of P&J were “not proven”. Can Smart et al. point to an observational study using inductive logic that is absolutely proven and not merely a point on a journey to fuller understanding? Do they perhaps consider that their earlier studies on Ndep are all proven and that none were “premature”, whatever that means? More rhetoric.

L59 This is confusing, as you use the word “significance” vaguely, which conflates P-value threshold-based conclusions with the normal usage of the word. It’s also worth noting the analyses of P&J that exactly replicated the model of MEA10 (i.e. the one without the spatial field) did not exactly show a large effect size, so some elaboration on how this statement about our conclusion of “weak effects” actually differs from MEA10 would be useful. After all, our main criticism of MEA10 was the failure to report any effect sizes from the multivariable model run (and, indeed, the replacement of these with a plot featuring a univariable regression instead).

L62 No, the main conclusion is that space-for-time studies of Ndep have either not used appropriate models and/or have not reported effect sizes; spatial auto-correlation was part of this but was not the only or main conclusion. I would say that the main points of our paper were about whether or not it is worth collecting more of these type of data and running these types of studies, and about better reporting standards (uncertainty, effect sizes, not relying on P-value thresholds to detect effects, acknowledging model-dependency of results clearly).

L64 Given that P&J conclude by saying (in their abstract) “we suggest that a greater focus on clearly reporting important outcomes with associated uncertainty, the use of techniques to account for spatial autocorrelation, and a clearer focus on the aims of a study, whether explanatory or predictive, are all required.” perhaps Smart et al. could clearly state what they mean by “the central conclusion of P&J”.

L71-74 Overall this is misleading, because, for SEA04, the inclusion of the spatial field was not the only difference. If you are focusing on spatial autocorrelation, please clearly separate this from the issues of P-value-based SVS exhibited by SEA04 (and also the lack of random effects, and the wrong support that used for the model, i.e. Gaussian rather than Poisson or some other distribution suitable for count data). As with an earlier point above, it would also be useful to have more clarity around what a “weak effect” (L66) is in relation to the issues with spatial autocorrelation. Without a spatial field P&J estimated a median effect for MEA10 of -2%, which would have been reported by MEA10 if they had reported any effect sizes from their multivariable model. The same estimate for SEA04 (vascular plants, all variables, monad RE only) is -1.5% (this can be verified from our R code, although we did not present this result in our paper). (Recall that this is compared to -1% under the model with the spatial field). Our point here is not that the model of Smart et al. is not a possibilty, but that even the models without the spatial field do not indicate what we would consider to be large effects. This means, of course, that the criticisms here also apply to SEA04 and MEA10; some clearer acknowledgement of this would be valuable, rather than just stating throughout that P&J are somehow wrong in isolation.

L77 Not true. Our paper gives numerous reasons for reanalysing the data of SEA04 and MEA10, the central reason and initial motivation was just to get an effect size that was absent from the literature after 16 years+ of research on this topic!

L81 Not sure how we overlook it given that we discuss it in the Discussion. See the quote above.

L104 Not sure how this was implicit given that we discussed it.

L104/105 Again, interesting how you elide SEA04 and MEA10 here even though they used quite different approaches. The effect of this statement is to overlook the issues with PSVS and implicitly endorse that approach. Do you?

L150/151 This is misrepresentation. We do not write it off as “too difficult to assess”, what we said was (feel free to quote us) was:
 “Whether or not the potential for these biases is more serious for inference than the absence of covariates that are unavailable, such as historic land management events (e.g., Rackham, 1986), is difficult to say.”

The simulation conducted by Smart et al. applies to one particular model of reality, we do not know whether that model is true in respect of Ndep impacts on richness. Other simulations involving unmeasured confounders with different types of spatial structure are possible (e.g. historic land management events etc.) What we essentially say in our quote is that it is difficult to know the truth of the relative importance of measurement error in independent variables and unmeasured confounder variables. It would be easy for me to say, in line wth L111 of Smart et al., that the assumptions of Smart et al. could be false in this respect.

L180 What proportion of the SEA04/MEA10 samples are actually in these high ratio areas?

L189 Maybe, but they didn’t assess whether this was successful, or consider the impacts of it not being completely successful on their inferential strategy.

L192 Maybe, but no-one in the statistics literature seems to think that it is a good idea to use PSVS to do this. See some of our references.

L241 is it a “likely” flaw or a “possible” flaw? At the end of day Smart et al. have asserted that if we had access to better estimates of Ndep at the sites (and there really are no important unmeasured confounders) we would get larger effect sizes; we still do not know that this is the case. Moreover, there is still the issue of whether 1-2% drops in richness are to be considered a large or small effect.

L254 I don’t know whether Cramer et al.’s concept of “detection and attribution” relates to P-value cut-offs, but it would be good to be clear about what the aims of the area of space-for-time Ndep studies actually are. To me quantifying effects and their variation is the most illuminating thing to do; merely “detecting” an effect at some P-value cut-off is not as useful in my opinion.

L246 Rather than airily dimissing unspecified “philosophical points”, it would be more useful to discuss these issues. All the points made by P&J have been raised as important for actual estimates of effect sizes from observational data (this is why they were included and clearly referenced), and some of them may be particularly relevant for attempts to make causal inferences about effects from space-for-time data (e.g. proxy variables).

L259 Are they potentially flawed or flawed? In other places you just say that they were outright flawed.

L261 Again, hard to know what Smart et al. consider “importance” to mean here. All of the effects we reported were in the 1-2% loss of richness ballpark (even if I run a Poisson GLMM without the mesh or 1km square RE on the SEA04 data with Ndep as the only covariate the effect is only -2.2%). How does this relate to the effects of management, climate change etc.? How do these effect sizes relate to the “widespread loss” claimed by MEA10? Or the “linear function” claimed in the abstract of SEA04? One gets the impression that effect sizes, or partial effects, have been ignored in this field, and “significance” focused on, precisely because the detectable effects are relatively small.


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: COMMENT ON PESCOTT & JITLAL 2020: FAILURE TO ACCOUNT FOR MEASUREMENT ERROR UNDERMINES THEIR CONCLUSION OF A WEAK IMPACT OF NITROGEN DEPOSITION ON PLANT SPECIES RICHNESS
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is a much improved manuscript and most of my concerns have been addressed. I have enjoyed being "on the side-lines" of this debate that appears to be far from settled. The article is much clearer and I appreciate the effort the authors have made to clarify their language and to add explanations where needed.

I find the section "other points raised" (line 272 - 283 in the marked-up word document) redundant. I think the ms gains nothing from this paragraph. The authors do not address the philosophical points raised in P&J here (which is that stated aim of the paragraph) but rather just state that they are qualified scientists who understand the study system (which is evident from the rest of the ms and from their other publications which they refer to). Removing this entire paragraph will improve the ms greatly and I urge the authors to do so.
Experimental design: The simulation study is repeatable using the code provided and the methodology is sound.
Validity of the findings: This version of the ms reads much less like a point-by-point rebuttal and highlights some key points for debate and objectives for future research in this field.
Additional comments: Line 37 in the abstract needs a comma I think: "Until then, the conclusions of..."
Also commas need here:
Line 82: "However, ..."
Line 118: "For brevity, we..."
Line 201: "Thus, across Britain..."
Line 216: "Hence,..."
Line 289: "However,..."

Comment on style:
Lines 210-213: Three consecutive sentences start with "We" - maybe reorder or reword these to make it more readable.

Line 303: Delete "be" in the sentence: "...in large observational studies will vary depending on the inclusion of other..."