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: PREDICTING GENE EXPRESSION USING DNA METHYLATION IN THREE HUMAN POPULATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article describes a method of imputing gene expression values from DNA methylation data by using two publically available data sets. However, the prediction R2 for most genes is quite low, which makes this method potentially less useful for eQTL or differential expression studies.

However, the paper fits well into the scope of peerj as the quality of the article by describing a methodologically and statistically sound analysis, regardless of subjective determinations of 'impact,' 'novelty' or 'interest'.
Experimental design: General Points

1.	Selection of publically available data: There are many more publically available data sets (e.g. from HapMap LCLs). It would be useful to test the method in more tissues, as well as perhaps having a replication cohort in the same tissue. It’s interesting that one is a very heterogeneous tissue with multiple cells, and one data set is of a purified single cell.

2.	There should be at least mention of some previous attempts to impute gene expression data from genotypes, as well as predicting methylation sites from nearby methylation sites.

3.	Could the accuracy be further improved by classifying methylation sites by location relative to the gene, position relative to CpG islands, mean methylation, as well as variability in methylation? Especially in the multiple regression model.
Validity of the findings: 4.	The author mentions that “the combination of DNA methylation and other predictors, such as genotype, may be more powerful for this purpose”. Both data sets have genotypes, it would be possible to test this hypothesis (maybe this is outside the scope of the paper)

5.	It’s interesting that immune genes come up as being best predicted - is this driven by the PMBC results? One explanation here could also be that there is a bias towards genes relevant to immune cells as there is more variability in these genes (as opposed to other genes completely switched off)
Additional comments: Specific Comments:

Line 51: Could mention CpG islands, shores. While 70% of CpGs are methylated, many of the CpGs are clustered into CpG islands, which are mostly unmethylated. The regions around the CpG islands have been found to have high variability in methylation.

Line 69: “They found that the association between gene expression and methylation is not always negative in promoter region or positive in gene body.” Need to mention where this assumption comes from, why was it expected that the gene body methylation would be positively correlated with expression? Could mention this study that look into the causal relationship of DNA methylation with expression  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3673336/

Line 73: “asthma”

Line 86: Do you mean “quantile normalized” instead of “quartile normalized”?

Line 117: How many samples remain after removing those with missing values? This means that only samples with 100% call rate were kept?

Line 125: Is this the RefGene annotation by Illumina? What are the cutoff for considering a probe as belonging to a gene, is there a cis-interval cutoff?

Line 138: what are the parameters used in glmnet?

Line 142: Have you considered using a completely separate “testing” data set to avoid overfitting?

Line 157: Multiple Testing correction?

Line 159: I think the GO enrichment studies need to be carried out using a matched permutation test instead of the whole array background (for example like it is implemented in eForge). This is because some of the probes might already be biased to come up in any kind of analysis in the specific cell types, as they have more variation, or a certain context to genes or CpG islands

Line 224: Perhaps mention that previously you just considered the R2 in model fitting, without worrying about overfitting to your data sets, so the multiple regression performs best here, whereas the LASSO model is best when considering prediction.

Line 282: “the larger genetic effect and larger epigenetic effect” - both are larger than what?

Line 283. This is interesting, it seems that the S&C “predictive power” is based around the separation of two distinct clusters. Surprising that there aren’t three cluster based on the three genotypes AA, AB and BB for each SNP - does this happen to for the S&C probes? I could imagine this also happening for some of the probes in “all” as they might have missed some in the Grundberg analysis. Is there a way to quantify how often this clustering phenomenon happens? (e.g. through running a clustering algorithm over all probes). Right now it looks like just four examples were cherry-picked.

Line 287: Again, this might not be surprising as the genes associated with GO terms relevant to fat and blood are more likely to be expressed, and thus provide more variability?

Line 314: I think a better method might be to compare the R2 in an independent validation set?


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: PREDICTING GENE EXPRESSION USING DNA METHYLATION IN THREE HUMAN POPULATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study by Zhong et al is trying to find correlation between gene expression using DNA methylation.
Experimental design: Please provide link to perlscript used in the study.
Validity of the findings: Conclusions are too strong and based on only two studies.
Additional comments: The study by Zhong et al address role of DNA methylation in predicting gene expression. The manuscript introduction is clear and Figures are relevant. The authors have done commendable job in defining the research question.  The major problem is for such a strong conclusion the authors did not provide any validation of their analysis and have only analyzed two study. I think it is very important to combine role of Histone modifications and enhancers/repressors and other gene regulatory mechanisms in addition to DNA methylation for gene expression analysis.
1.	The authors use DNA methylation of all CpG sites to predict gene expression. This is obvious since not CpG sites will lie in promoter region. It is known that CpG-dense promoters of actively transcribed genes are never methylated, but reciprocally transcriptionally silent genes do not necessarily carry a methylated promoter.
1.	The authors should discuss how MethylXcan is unique from existing softwares, such as FEM, ROADMAP Epigenomics and others.
2.	The authors indicate DNA methylation is poor predictor of gene expression but develop MethylXcan program to predict gene xpression using DNA methylation.
3.	One of the major problem of their results is lack of validation. Result from cancer genome atlas project indicate correlation between DNA methylation and gene expression (Long et al 2017). To remove any bias the authors should analyze atleast 5-6 other datasets and include the results from those analysis.
4.	Although the results described by Zhong et al are very interesting and can have profound impact on epigenetics. The authors should compare correlation between gene expression and DNA methylation at either Promoter region or on total gene body.
5.	The authors indicate immune and defense gene show correlation between DNA methylation and gene expression. However there is no information provided about role of Histone modifications, DNA binding elements, transcription factors. The author should discuss how methylation of immune and defense gene is unique and related details in detail.
6.	Figure 5 : The axis are not labelled
7.	Line 126 Please provide link to perlscript

8.	The English language usage and formatting issues in the manuscript.
a. Line 93 “Humanmethylation450”
b. Line180 “Thereinto”
c. Line210 “less than identifies”
d.Table 2 legend “andKEGG”

e. Figure 5 legend “Examplegenes”

f.	Figure 4 legend “Comparisonof”

g.	Figure 2 legend “Comparisonamong”

h.	Line 100 reference formatting error “background [9]”
i.	Line 50 “residue(You”
j.	Line 59 “2013)studied”


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: PREDICTING GENE EXPRESSION USING DNA METHYLATION IN THREE HUMAN POPULATIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article structure, figures, tables is professional.  Authors indicate that Raw data for perlcode is shared in GITHUB.
Experimental design: Research question is well defined.
Validity of the findings: Data is robust, statistically sound, & controlled.
Additional comments: I am overall satisfied with author response and revised manuscript. I do not have any further comments.


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

Input:
Title: IMPACTS OF THE INVASIVE SHOT HOLE BORER (EUWALLACEA KUROSHIO) ARE LINKED TO SEWAGE POLLUTION IN SOUTHERN CALIFORNIA: THE ENRICHED TREE HYPOTHESIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript ‘How nutrient enrichment is linked to impacts of the invasive shot hole borer (Euwallacea sp.): the Soft Tree Hypothesis’, presents a hypothesis linking nutrient enrichment (as a consequence of sewage spills) with reduced wood density and increased moisture content of two Salix spp. The hypothesis suggests the resulting ‘soft’ trees are more susceptible to attack by the invasive Kuroshio Shot Hole Borer (KSHB), providing an insight into why infestation and mortality rates have been particularly high in riparian sites in the Tijuana River Valley (TRV).
The manuscript is generally well written and well referenced.
The raw data has been made available, figures are of sufficient resolution however I have some comments:
Figure 1: A and B are missing on the figure- while the figure provides a visual representation of the hypothesis, I think the hypothesis itself is quite clear and do not see that the figure is necessary or adds value to the manuscript.
Figure 3: As with Figure 1, I don’t see that this figure contributes to the manuscript.
Figure 4: A and B missing from the figure
Figures 5 & 6: figure captions should read ‘…mean ± 1 std. dev.’
Experimental design: The manuscript presents original research within the aims and scope of the journal.
The research question is well described, relevant and meaningful, however I have some concerns regarding the rigor of investigation and conclusions drawn (addressed in section 3).

Methods lack details of the statistical package used for analysis.
Validity of the findings: Whilst I believe the hypothesis presented is valid (and I think quite a logical explanation of a situation apparently unique to the TRV), and trust the significance of results relating to wood density and wood moisture content, I have a number of concerns regarding the data supporting significant relationships between level of sewage pollution and infestation and mortality rates.

1.	Test # 1 compares sites across a relatively broad spatial scale, with the nine polluted sites in relatively close proximity to each other, and the additional nine non-polluted sites (outside the TR flows) widely dispersed (with the exception of OUT-1, TR tributary). My main concern is that with the exception of site OUT-4 (with 9% infestation), the remaining eight non-polluted sites are entirely without infestation. Whilst this may seem a circular argument (non-polluted sites produce ‘hard’ trees which are not prone to attack), I feel uncomfortably accepting the conclusions drawn under these conditions. It is unfortunate that it was not possible to have a greater number of ‘intermediate’ sites. My concern is that the examined ‘OUT’ sites may have been ‘missed’ by KSHB, and that have 8 of the 9 sites without any sign of attack weakens the argument that infestation rates are driven by pollution. Although I’m not sure how this matter should be resolved.

2.	While determining infestation and mortality rates from ‘as many willows as could be examined in two hours’ provides a consistent method by which to score KSHB attack across sites, it results in a high variation in the number of trees assessed (ranging from 34 to 187).  No additional information has collected on stand structural traits (such as stem density, basal area and species composition) which may also have a bearing on how easily the sites may be invaded.

3.	Adding a ‘severity of infestation’ ranking at the tree level would have allowed a deeper interrogation of wood density/wood moisture content correlation with beetle attack (at the tree level rather than site level).

4.	Test # 2, comparison of sites within the TRV, test the Soft Tree Hypothesis by using correlation analysis to independently compare wood density, wood moisture content, KSHB infestation rate and KSHB mortality rate with respect to distance from the main TR channel (as proxy for the amount of sewage reaching each site). I calculated the correlation coefficients for KSHB infestation and mortality rates (vs distance from main channel) using the data provided in Tables 1 and 3. While the r and p values I calculated were consistent with the results presented for infestation rates vs distance (Line 238: r = 0.723, p < 0.05), I found the correlation coefficient for mortality rates vs distance varied to that presented at Line 239 as r = 0.696 and p < 0.05, with my calculation giving r = 0.5074 and p = 0.1344. I am not sure what has happened here, but this is a rather crucial point in terms of suggesting the data provides support for the Soft Tree Hypothesis.
Additional comments: Line 77 – The statement ‘Within a few days mated females emerge …’ is a slightly misleading statement as it seems to suggest the life cycle takes only a few days, rather than weeks to months

Line 327-328 – the suggestion is that only trees impacted by nutrient enrichment and excessive irrigation will be at risk of infestation does not take into account other possible environmental factors which may predispose trees to attack. I find the statement limiting, e.g. storm damage would also surely be among a number of disturbance factors that would predispose trees to attack

Since submission of the manuscript, delineation of the species within the E. fornicatus complex has been completed, KSHB and PSHB can now also be referred to by their scientific names E. kuroshio and E. whitfordiodendrus (Gomez et al., 2018)

Gomez, D.F., Skelton, J., Steininger, M.S., Stouthamer, R., Rugman-Jones, P., Sittichaya, W., Rabaglia, R.J., Hulcr, J., 2018. Species Delineation Within the Euwallacea fornicatus (Coleoptera: Curculionidae) Complex Revealed by Morphometric and Phylogenetic Analyses. Insect Systematics and Diversity 2, 2-2.

While overall my feeling is that the hypothesis is sound and indeed a very likely explanation of the high level of KSHB damage in the TRV, and is an important contribution with regards to where to focus further management efforts, I have some reservations around the soundness of the statistical significance of the data linking sewage levels (using distance from channel as proxy) with KSHB infestation and mortality rates.

Further experimental investigation by conducting pathogenicity trials on excised stems of various densities with the fungal symbiont would be relatively quick and straight forward to conduct. This would allow a well replicated and controlled examination of extent of fungal development in soft vs hard stems, adding wight to support the argument of faster growth of the fungal symbiont


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: IMPACTS OF THE INVASIVE SHOT HOLE BORER (EUWALLACEA KUROSHIO) ARE LINKED TO SEWAGE POLLUTION IN SOUTHERN CALIFORNIA: THE ENRICHED TREE HYPOTHESIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I have read the Boland and Woodward paper with great interest. It is known that two factors will increase the growth of the fungal and beetle population in wood, these are water and nutrients see for instance Gadd 1944 for the influence of nutrients and Bolands earlier work published in PeerJ. It also our experience when we try to grow beetles on logs removed from trees that the water condition of the logs is very important in allowing the beetles to reproduce inside the wood. So both factors are important and may very well explain the high level of infestation found in the Tijuana River Valley. However the points outside of the TRV that are used as controls to determine the influence of sewage spill on the effect of the KSHB, are also areas where the beetle infestation is of a younger age than that found in the TRV, and thus may not yet have resulted in KSHB populations that are equally high. The relative importance of water versus nutrients remains to be determined and could be easily studied experimentally. I think the name soft tree hypothesis is misleading since some of the trees that are attacked and at least loose branches following infestation are woods that are generally considered very hard, coast live oak and sycamore. So I would prefer it the authors could drop that name and replace it with something that reflects water and nutrients to explain the infestation severity. From the literature it appears that both phosphorus and nitrogen are high in the sewage coming from mexico, the relationship between these nutrients in the water, and in the xylem would influence the growth rate of the fungi and consequently that of the beetle. I did a little digging in the literature and it should not be too hard to find papers that make these links. So overall it is very likely that the highly nutrient enriched waters of the Tijuana River spur the growth of the beetle population, through the growth of the fungi, but I doubt that how hard the wood is would influence the beetle population growth.
Experimental design: You may want to clarify when exactly you took the wood samples, you state you did that in fall, is this at the time there were no more leaves on the trees, so that in principle no more xylem was pulled up? Otherwise moisture may accumulate in more heavily infested branches because the water cannot be transported to higher up in the tree, so independent of nutrients, would more heavily infested branches have a higher water content, they would also have lower wood content since part of the wood is removed by the beetles, consequently higher moisture and lower density simply determined by infestation level.
Validity of the findings: No comment
Additional comments: It is very likely that the nutrient availability together with ample water availability determined the food for the fungi and consequently how fast the beetles will be reproducing in the polluted areas. I would prefer you find another name for your  hypothesis. Some of the aspects you have brought up could be easily studied experimentally either by supplementing trees with nutrients or water, it would strengthen your arguments. The fact that a large die off has not yet happened at the other locations may be a matter of time since these rivers were invaded later. I would like you to add some literature on the relationship between Phosphorus, nitrogen in ground water, and xylem, and the relationship with growth of Fusarium fungi. I will send you a copy of the gadd paper that you may find helpful.
Line 35 remove native and agriculturally (street trees are also affected)
Line 75 trunks should be trunks and branches
Line 75 with a fungus s/b with fungi.
Line 77 Within in few days mated females emerge. You may want to rephrase that, it takes at least 3 weeks under optimal circumstances to go from egg to adult
Line 132 215-230 degrees F report in Celcius


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: IMPACTS OF THE INVASIVE SHOT HOLE BORER (EUWALLACEA KUROSHIO) ARE LINKED TO SEWAGE POLLUTION IN SOUTHERN CALIFORNIA: THE ENRICHED TREE HYPOTHESIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: see comments in my review text
Validity of the findings: see comments in my review text
Additional comments: PeerJ review of 32720

REVIEWER STRENGTHS AND WEAKNESSES
I have no expertise in tree physiology or in statistics. I do have expertise in bark beetle ecology and behavior.

GENERAL REMARKS
I really enjoyed reading this paper. It is well written and structured, and makes a persuasive argument for an original idea, the Soft Tree Hypothesis. As the authors point out, unique to this hypothesis for why some trees are attacked and not others is that it treats trees not as uniform within species but as varying within species, and explains why some trees in a given species are killed but not others.

I suspect there may be a pseudoreplication issue in that polluted sites are all in one river valley and hence not independently polluted; they share many conditions other than level of pollution. It might be countered, though, by the within-valley analysis.

Correlation is not causation, as we all know. It is important to think critically of what else other than wood density might differ between polluted and nonpolluted habitats, or along a pollution gradient (within-valley analysis). These might or might not be realistic alternative explanations:
•	Polluted = flooded (at some time), flooding damages roots and leads to oxygen stress?
•	Polluted = flooded, besides nutrients there could be substances (heavy metals? Salts?) which harm trees and weaken their defenses.
•	Increased nutrients (in polluted soils) leads not only to more rapid growth and hence less dense wood but maybe (?) increases N content of the wood, which would help ambrosia fungi get established and grow quickly and thus overwhelm trees which are trying to defend themselves.

You should say somewhere how you identified this species, given that it is very difficult.

DETAILED COMMENTS

Re significant digits in reporting results. Too many significant figures given the level of precision, I bet. Also, report SD as one more sig fig than mean: 0.40 ± 0.043. Also, sample size N, correlation coefficient r, and significance level p should be italicized, N, r, p. Standard deviation can be abbreviated SD or S.D., and it only needs to be stated once that the plus-minus refers to S.D. (you repeat this for the second willow species).
98	Study sites. The Tijuana River Valley, San Diego County, California (32o 33.080'N, 117o 4.971'W) is a coastal floodplain of approximately 15 square kilometers at the end of a 4,480  square kilometer watershed.
The precision of the coordinates and indeed the coordinates themselves don’t make sense. The coordinates give one small location of the 15 km2 floodplain.

186	Might be more effective as first sentence rather than last.

297	Importance of N levels treated also in Kirkendall 1983  and Ayres et al. 2000 (refs given below)

REFERENCES
418	Chave, J. 2005 : the link supplied did not work, but I found the pdf at http://www.rainfor.org/upload/ManualsEnglish/wood_density_english[1].pdf
425 	Fusarium -> Fusarium
432	“Eskalen A. 2018. Shot hole borers/fusarium dieback websites”: fix Fusarium

FIGURES
Figure 2, map. The blue circles are very difficult to see against the dark green background. Use another color, but not red (avoid red on green, for readers that are red-green colorblind).

Ayres, M. P., Wilkens, R. T., Ruel, J. J., Lombardero, M. J. & Vallery, E. (2000) Nitrogen budgets of phloem-feeding bark beetles with and without symbiotic fungi. Ecology, 81, 2198-2210.
Kirkendall, L. R. (1983) The evolution of mating systems in bark and ambrosia beetles (Coleoptera, Scolytidae and Platypodidae). Zoological Journal of the Linnean Society, 77, 293-352.


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: IMPACTS OF THE INVASIVE SHOT HOLE BORER (EUWALLACEA KUROSHIO) ARE LINKED TO SEWAGE POLLUTION IN SOUTHERN CALIFORNIA: THE ENRICHED TREE HYPOTHESIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript 'Impacts of the invasive shot hole borer (Euwallacea kuroshio) are linked to sewage pollution in southern California: the Enriched Tree Hypothesis' reads well and has addressed the concerns I had, it provides a valuable contribution to this  field of research
Experimental design: I am satisfied that the problem of pseudoreplication has been resolved. The inclusion of intermediate sites (while infested with PSHB rather than KSHB) add value, as does explanation of the statistical methods
Validity of the findings: I am satisfied that the authors have adequately addressed my concerns regarding validity of the findings
Additional comments: No additional comments


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

Input:
Title: ERISNET: DEEP NEURAL NETWORK FOR SARGASSUM DETECTION ALONG THE COASTLINE OF THE MEXICAN CARIBBEAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: English and style…

I do not feel qualified to judge English language and style

The authors need to review and add more information on state of the art with respect to the possible effects of such arrivals of Sargassum to the Mexican Caribbean coasts and how the application of the classifier would help the persons in charge of such work and researchers that can make use of this proposal of early warning neural network in decision making.

Professional article structure, figs, tables. Raw data shared. "It ok"
Experimental design: Even though the study is centered in the development of an algorithm for a specific classifying and detecting platform, which is one of the objectives of the algorithm and not only for developers but also for the scientific community, information with respect to the application of the algorithm or in its case the proposal of the neural network is required. Likewise, I don’t see in the manuscript the possible incorporation of other physical and biological variables that could favor distribution and abundance of Sargassum, which could be incorporated in the neural network to develop such early warning that has been proposed or better yet what is the justification for not incorporating them initially.
Validity of the findings: On the other hand, they need to incorporate more information, figures and images, with respect to the analysis of satellite images and their correlation with the specific characteristics of wavelength or specific reflectance of the Sargassum as well as making comparisons of images of the presence and absence of Sargassum. in the Mexican coasts, as well as its respective graph of reflectance values.
Additional comments: I have read the article and find the development of the classifying neural network interesting and novel with its corresponding algorithm to determine the presence/absence of Sargassum in the Mexican Caribbean since its arrival to the coasts has turned out to be a serious problem for different environments both ecologic and economic in the last years. The authors have made a great effort and contribution with the development of this neural network architecture, which shall be of great importance and application.


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: ERISNET: DEEP NEURAL NETWORK FOR SARGASSUM DETECTION ALONG THE COASTLINE OF THE MEXICAN CARIBBEAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: I am very alarmed, why the authors did not add antecedents about investigations with the same aim in the Caribbean too. For example: Jean-Philippe Maréchal, Mengqiu Wang, Chuanmin Hu, Gower J.F.R.

Nowadays this research continues but with images with finer spatial resolutions (10 meters) than MODIS images proposed in this article.

In the article the authors said that it is the first work on this topic and its not true.

Example:
https://www.sciencedirect.com/science/article/pii/S2352938516300441#!
https://www.sciencedirect.com/science/article/abs/pii/S0034425716301833#!

The methodology its a good goal but not the general aim.
Experimental design: This is not an original aim.  Since 2 years ago there are a really good articles about this research.

They do not mention in their introduction important background on the subject.
Validity of the findings: 'no comment'
Additional comments: Congratulation ...A good effort in the methods but there are already research with the same aim with satellite images with finer spatial resolutions and that also quantify the Sargassum.


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: ERISNET: DEEP NEURAL NETWORK FOR SARGASSUM DETECTION ALONG THE COASTLINE OF THE MEXICAN CARIBBEAN
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article is adjusted to the professional standards, the information included in the introduction and background are sufficient to demonstrate how the work fits the area of knowledge.
Experimental design: The research carried out by the authors is original and of high impact for the study area, and the information of the method contained in the document is relevant and significant to be followed up and can be replicated by other researchers and provide new knowledge or new methodologies for the study of sargassum.
Validity of the findings: The results are good and statistically proven, which leads to well-established conclusions by answering the main question.
Additional comments: Dear authors. The work done in the comments and corrections substantially improved the content and sense of the research, so I have no problem in recommending its publication. Good job.


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

Input:
Title: A LARGE SCALE EVALUATION OF TBPROFILER AND MYKROBE FOR ANTIBIOTIC RESISTANCE PREDICTION IN MYCOBACTERIUM TUBERCULOSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: It is written in a good English wording and clear to me as reviewer.
Referencing is partially good, I have some suggestions that provided later.
Experimental design: It falls with the scope of the journal.

I found the methods fine and enough professional for conduct such a good research in this field.
Validity of the findings: I agree with the topic but not seen enough text on rationale for this research. I am not satisfied. Please include more text to convince the readers and reviewers happy with your data  and findings,

More literature review in Scopus and Web of Science is necessary to finish the discussion with it.

Authors should have shown how they can use this predictive tool in prediction of antibiotic resistance in MT.
Is this new approach in agreement which WHO guideline in treatment of such problematic bacilli?
Additional comments: basically I like the MS but it can be considered after some minor changes in the revision.


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

Input:
Title: A LARGE SCALE EVALUATION OF TBPROFILER AND MYKROBE FOR ANTIBIOTIC RESISTANCE PREDICTION IN MYCOBACTERIUM TUBERCULOSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: In the section lines 87-94, it would be helpful to know whether the TBProfiler and Mykrobe programs were run with their respective default settings, or, if different settings were attempted (apart from allele frequency cutoffs). For example, TBProfiler offers a number of different read mappers (bwa, bowtie, minimap2) and variant callers (lofreq, bcftools) which may have influence on the number and frequencies of detected resistance markers.
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: A LARGE SCALE EVALUATION OF TBPROFILER AND MYKROBE FOR ANTIBIOTIC RESISTANCE PREDICTION IN MYCOBACTERIUM TUBERCULOSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: About methods, the Authors stated they used the command line versions of the Mikrobe and the TBprofiler pipelines. I was not able to find detailed quality thresholds, variant call thresholds, coverage thresholds and similar parameters used in the evaluation study.
Validity of the findings: no comment
Additional comments: The Authors stated despite specificity was generally high (90%), precision was found low, especially for ethambutol and ethionamide. I would suggest the Authors to reconsider this in the view of the fact that conventional phenotypic DST is an imperfect gold standard. As demonstrated by several studies indeed, (for example) liquid MGIT phenotypic DST systematically misses resistance due to specific mutations. In particular, ethambutol phenotypic DST is one of the most affected by this problem with the M306V embB mutation validated as marker of ethambutol resistance by functional genetics but testing either phenotypically susceptible by MGIT in many cases. Even worst if we consider the M306I that is causing a MIC close to the critical concentration used for phenotypic DST. This is an important point to be considered when evaluating NGS software. In this sense, discrepancies between variant calling among the two pipelines would be more relevant, compared to the performance against phenotypic DST.

The Authors found very different performance characteristics compared to previously reported studies. One of the reason could be that previous studies were performed on "controlled" samples, whereas in the current study - except for the ReSeqTB datasets that ensure a level of data curation - could not check for phenotypic DST accuracy in the metadata. I would suggest to mention this possibility among the reasons of such differences. It would be interesting have a quick insight on this by taking the list of markers of R reported in Miotto ERJ 2017 and looking how many isolates harboring such mutations have been reported as phenotypically S in the dataset.

Could the Authors exclude the presence of duplicate isolates in the dataset?

The question about the lineage-specific performance needs to be some adjustment. First, is the dataset excluding highly-related isolates (e.g. <12 SNPs of difference on a distance matrix)? If the dataset is including strains belonging to an outbreak, this could effect the analysis about the performances on different lineages. Second, it is well known that the frequency of some mutations is depending and varying upon geographical region; since lineage distribution is also geographical-dependent, a given mutation can show different sensitivity accordingly. The extreme case is the S450L in rpoB that is working very well worldwide, except in Swaziland (eSwatini), where the most (and nearly solely) rpoB mutation causing RIF-R is at codon 491. I would suggest to mention the complexity of evaluating lineage-specific performances in the manuscript. Also, whereas it is relevant to evaluate performances against different lineages, I would suggest to revise the sentence at lines 368-370 in the conclusions, as because we would need a clear frequency distribution among lineages before defining a confidence level as the one suggested. In addition, this is again depending upon the list of mutations used for interpreting sequencing data.

ReSeqTB raw sequencing data are provided according to specific terms and conditions. Please clearly state if the datasets used are publicly available and/or any specific permission has been approved.


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

Input:
Title: A LARGE SCALE EVALUATION OF TBPROFILER AND MYKROBE FOR ANTIBIOTIC RESISTANCE PREDICTION IN MYCOBACTERIUM TUBERCULOSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I have already reviewed the paper and the authors made my corrections. I agree with positive decision for this paper.
Experimental design: it is fine by me
Validity of the findings: valued
Additional comments: nice job


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

Input:
Title: A LARGE SCALE EVALUATION OF TBPROFILER AND MYKROBE FOR ANTIBIOTIC RESISTANCE PREDICTION IN MYCOBACTERIUM TUBERCULOSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment - reviewers' questions addressed
Validity of the findings: no comment
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: CLINICAL CHARACTERISTICS AND PROGNOSIS OF BASALOID SQUAMOUS CELL CARCINOMA OF THE LUNG: A POPULATION-BASED ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript entitled “Clinical characteristics and prognosis of basaloid
squamous cell carcinoma of the lung: a populationbased analysis” has analyzed the clinical features and prognosis of basaloid squamous cell carcinoma of the lung and constructed a nomogram to predict the prognoses of patients. However, it poorly quotes literature and lacks explanation on several fronts.
Experimental design: Fine
Validity of the findings: Fine
Additional comments: This study needs a major revision. My specific comments are:
1)	In literature basaloid SCC has been shown to have a poor survival compared to Non-basaloid SCC. However, Authors conclusion is just opposite? (Reference: DOI:10.1158/1078-0432.CCR-14-0459 Published November 2014, DOI: 10.1183/09031936.00058507). Please explain in detail in light with the above quoted literature.
2)	There is a huge difference in patient number between SCC (around 89000) and Basaloid SCC (425) which could affect survival curve to get biased.
3)	Are the patient classified under Basaloid SCC, pure or mixed type?
4)	Nomogram has not been explained in detail to emphasize its importance. More introduction and relevance should be added in the manuscript to conclude data.
5)	“Worldwide in 2018, it accounted for 2.1 new cases and 1.8 million deaths”: Is it 2.1 million new cases?


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: CLINICAL CHARACTERISTICS AND PROGNOSIS OF BASALOID SQUAMOUS CELL CARCINOMA OF THE LUNG: A POPULATION-BASED ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The figures and tables are clearly described.
References include the most that are relevant. The authors do not miss any major referencing.
The article is clearly written.
Experimental design: •	The classification of BSC as a subtype of non-small cell lung carcinomas (NSCC) is currently accepted by WHO organization but there is uncertainty on the nature of BSCs. Currently, BSCs are described to overlap features of both squamous cell carcinoma (SCC) and large cell carcinoma (LCC). Yet, it is not clear why authors focus their comparative studies to SCCs only. For a comprehensive analysis, BSS analysis must also be compared with LCC data.
•	BSC is now regarded as a subtype of NSCC. In addition to the individual comparisons of BSCs with SCC and LCC, BSCs must also be compared to a cumulative set of NSCCs. It is essential to understand the prognosis and correlative features of BSCs within the NSCC category.
•	Moro-Sibilot D et al (2008) reported that, BSC patients are older than non-BSC patients. The results here do not report a difference in median age. Is this a reflection of higher number of patients enrolled in this analysis compared to the previous?
•	Include the difference in the prognosis on a subset of these tumors: 274 poorly-differentiated BSCs v/s 32,307 poorly-differentiated SCCs described in Table 1. This might help to address the discrepancies discussed in line 137 under the discussion section.
Validity of the findings: The manuscript summarizes the epidemiological analysis of clinical parameters on a rare set of basaloid squamous cell carcinoma of the lung (BSC) and describes the prognosis and correlations. The authors have used a previously available database, conduct the well-described statistical analyses and propose that, BSC have worst prognosis, display distinct correlations with tumor stages and suggest that their observations are clinically useful. The presented data is of high quality but the author’s interpretations are generalized and limits the findings of this manuscript to its epidemiological analyses and does not explore the biology of the rare tumors.
Additional comments: None.


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

Input:
Title: CLINICAL CHARACTERISTICS AND PROGNOSIS OF BASALOID SQUAMOUS CELL CARCINOMA OF THE LUNG: A POPULATION-BASED ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Fine
Experimental design: Good
Validity of the findings: Fine
Additional comments: Authors have incorporated all the suggested changes. Manuscript can be accepted.


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

Input:
Title: CLINICAL CHARACTERISTICS AND PROGNOSIS OF BASALOID SQUAMOUS CELL CARCINOMA OF THE LUNG: A POPULATION-BASED ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised manuscript has fixed the previous typos. The new manuscript is easy to read and the figures and tables are well organized.
Experimental design: No comment
Validity of the findings: The prognosis findings and clinical correlations is novel but the pathological analysis of the tumors is limited.
Additional comments: The authors have attempted to address the reviewer’s concerns and included additional figures, tables and text as requested. Additional data and explanations offered by the authors addresses the clinal prognosis and correlations of the basaloid squamous cell carcinoma of the lung (BSC) and strengthens the original observations presented before. The authors have also attempted to correlate their findings to the previously published articles and offered explanations where they observed an inconsistent finding. Due to the limited sample availability, the authors do not add new data to describe the pathological characteristics of BSC tumors, but the epidemiological analysis alone may of be interest to the researchers and clinicians.


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: CAPTIVITY CAUSES TAXONOMIC AND FUNCTIONAL CONVERGENCE OF GUT MICROBIAL COMMUNITIES IN BATS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	Basic Reporting

a.	Clear/unambiguous English:

The English should be improved for clarity and comprehension throughout the manuscript; example areas that require improvement are lines 22-23, 48-49, 273-274. Current phrasing is awkward and makes comprehension of certain points difficult. The title also could use improvement; the authors did not raise the bats from pups, so the title should be amended to something like “Captivity Causes Taxonomic and Functional Convergence of Gut Microbial Communities in Bats”.

b.	Literature references, sufficient context:

Line 42: Consider also that the social behavior of animals that live in close proximity, such as bats, can drive gut microbiota structure in the wild (Tung et al. 2015; Moeller et al. 2016; Kolodny et al. 2018). This has especially been demonstrated in bats roosting together. As such, you should add a sentence explaining whether or not you housed bats in the same enclosure during the controlled feeding portion of the study.

Line 41-42: Rather than saying “host-specific”, you should expand this part of the introduction to discuss that in addition to diet, host evolutionary history has a strong influence on gut microbiome composition (Ley et al. 2008; Phillips et al. 2012; Ingala et al. 2018).

Line 45-47: Specify that the taxonomic composition of gut microbial communities converge in the references you cited.

Line 57-58: You should qualify what you mean by “herbivores are only a portion of mammals” by specifying what percentage of mammals are herbivorous. You might also state that insectivory is thought to be the ancestral feeding condition for placental mammals (O’Leary et al. 2013), as well as for bats (Dawson and Krishtalka 1984), which makes understanding the microbiome of insectivorous animals important from an evolutionary perspective.

Line 59-60: What is interesting about this study compared to (Kohl et al. 2014) is that you are studying three bat species that are members of different families (Hipposideridae, Rhinolophidae, and Vespertilionidae). Kohl et al. 2014 studied two closely related woodrats (Neotoma spp.). You might better frame your research question more like this: “Given the phylogenetic distance among hosts studied here, it is unclear whether homogenization of diet/environment or host evolutionary history has a stronger impact on microbiome community composition.”

Line 77-78: In the materials and methods, the authors describe performing functional prediction on the bat microbial communities, but do not contextualize why this is important or relevant in the introduction, nor do they hypothesize about what they expect to find. I suggest further developing the introduction by discussing that taxonomy and function are decoupled in microbial ecosystems (Graham et al. 2016; Louca et al 2016; Inkpen et al. 2017). There is also a study in insectivorous bats which showed that among three different bat species, microbial community taxonomic composition varied but all were functionally convergent (Phillips et al. 2017). This context would help the authors articulate a hypothesis about the functional assays.

Line 81-94: There are several ways to sample gut microbiota (swabs, guano, intestinal sections). You should justify why you chose to sample feces only. A helpful reference is (Ingala et al. 2018), which shows that signal of diet in the microbiome is more detectable in fecal samples than intestinal samples.

Line 90: You specify that you collected fecal samples from individuals “after more than two months”, which makes it sound as if you collected two month old fecal pellets. Rephrase for clarity and state that you kept the bats on a homogenous diet for two months, and collected fecal pellets from captive animals X hours/days after defecation.

Line 110-111: Please cite the appropriate reference for primers 338F and 806R.

Line 132-134: Did you use open- or closed-reference clustering method? Please specify

Line 145: Please state the version of QIIME you used, and please cite individual R packages when you mention them later in this section, including the version (i.e. phyloseq v 1.1-2, McMurdie & Holmes 2013, etc.). Version of PICRUSt is also missing.

Lines 206 & 208: KEGG should be capitalized (Kyoto Encyclopedia of Genes & Genomes)

Line 273-281: It is interesting that you found microbial diversity increased after bats were taken into captivity. It would be good to provide some hypothesis or speculation as to why this might be; for example, is it possible that the bat species were exposed to one another’s microbes (thus explaining taxonomic homogenization), and so the overall community becomes more diverse from sharing among species? Alternatively, could contaminating laboratory bacteria be the cause?

c.	Professional article structure, raw data shared:
Article structure appears standard and professional. Raw data is shared as supplementary files, and clicking through a few, the downloads work successfully. Raw data was also uploaded to NCBI. You may also consider making your R code available for easier replication.

d.	Self-contained with relevant results to hypothesis:
Paper is self-contained and results follow hypotheses logically.
Experimental design: 2.	Experimental Design

a.	Original research within aims and scope?:
The manuscript is within the aims and scope of PeerJ and meets criteria for original research

b.	Research question well defined, relevant, meaningful?:
The research question is somewhat well-defined, but it would be more compelling if the authors addressed several contextual comments made above. In particular, they should better introduce the functional aspect of the study, which isn’t introduced to the reader until the methods section.

c.	Rigorous investigation performed to high technical/ethical standard?:
All ethical standards seem adequate, and appropriate permissions were obtained for the fieldwork. Investigation was performed using techniques that are standard and accepted in the microbiome field.

Line 149-151: Please check the assumptions of the Student’s t- test; this test assumes a normal distribution of data and homoscedasticity of variance. Please check that your Unifrac distances do not violate these assumptions, in which case the non-parametric Wilcoxon test would be more appropriate (Xia and Sun 2017). In addition, in Table 2 it appears you use the Bonferroni post-hoc correction method, but you do not mention this in the Methods. Please add to methods for clarity.

Your results regarding the functional convergence are very interesting, but I am curious why you only tested presence/absence of functions and not relative abundance of functions. For example, all captive bats may converge on the same functions in terms of presence/absence, but the relative abundance of functions may differ substantially among host species. See such an example in Phillips et al. 2017 (Integrative & Comparative Biology):  “Given that comparisons were made among insectivorous host species, frequency differences for metagenome functions pertaining to macromolecule metabolism may reflect host lineage-specific metabolic fine-tuning.” Please explain if any of the hosts differed in terms of relative abundance of functions.

Figure 3: In the figure legend, you indicate the shape of symbols correspond to the “host order”, but all bats belong to the Order Chiroptera. I think what you meant here is “host family”. In any case, there is no clear indication of what the families are because the legend shapes are just the Species names. Please correct the legend to show the appropriate host families (i.e. Red circle = Hipposideridae, green square = Vespertilionidae, blue triangle = Rhinolophidae)

d.	Methods described with sufficient detail & information to replicate?:
Thank you for describing the methods in good detail. Metadata and raw sequences are provided so that analysis can be replicated.
Validity of the findings: 3.	Validity of Findings

a.	Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged?
The authors encourage replication of their study. Results seem overall compelling and deserve future replication.

b.	Data is robust, statistically sound, & controlled
The data was robust, and treated in an overall statistically sound manner. As far as controls, the authors do not mention whether a negative extraction or PCR control was sequenced alongside their fecal samples. Many kits have bacterial contaminants (Salter et al 2014) that can be filtered from microbiome samples in silico. The R package “decontam” can be used to filter out potential contaminants using either (a) a negative control sample, or (b) the initial concentrations of each library.

One analysis missing from the manuscript is a bar plot of overall microbiome composition. It would be helpful as an additional control to have a supplemental figure of sample by sample microbiome composition, and a brief discussion if the community compositions are consistent with previous work on bat microbiomes (Phillips et al. 2012, Carrillo-Araujo et al. 2015, Phillips et al. 2017, Ingala et al. 2018).

c.	Conclusions are well-stated, linked to research question and limited to supporting results
Conclusions drawn from the analyses are easy to interpret and directly relate to the research questions and results.

d.	Speculation is welcome but should be clearly identified.
Speculation in the discussion is limited and appropriate.
Additional comments: This manuscript seeks to answer whether homogenization of diet and environment leads to convergence of gut microbial communities in bats. Overall, the manuscript is interesting, technically sound, and uses appropriate methods to answer the question. After substantial revision of the English language, incorporation of some needed context and references, and addition of a few critical elements in the results, this manuscript will be in good shape.


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: CAPTIVITY CAUSES TAXONOMIC AND FUNCTIONAL CONVERGENCE OF GUT MICROBIAL COMMUNITIES IN BATS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Whilst I appreciate that English is likely not the first language of the authors, the standard of English throughout the manuscript requires significant attention. The authors should consider having the manuscript thoroughly revised by a colleague who is a native English speaker or consulting a professional editing service.

In general, I find that the manuscript is well supported by reference to prior literature. The figures are also of a good standard, although the legends should be made more informative if the manuscript is revised. However, there are some structural issues that I believe need to be addressed.

Lines 49 – 57 of the introduction discusses the findings of Kohl et al (2014) at great length. Whilst obviously relevant, this work could be summarised much more succinctly to improve the flow of the manuscript.

The methods section which relates to sample collection (lines 81-94) lacks detail on potentially important aspects of the study. Most of the information can be gathered from table 1, however it should be at least mentioned in the main body of the text for clarity. For example, from how many sites were the samples for each bat species collected? (Since there are likely site-specific gut microbiomes within each species group). At what time of year were the samples collected (Since bat diet and feeding ecology vary seasonally).
Experimental design: The hypotheses that this work seeks to test are well defined, however the experimental design is weak.

My largest concern is that given the methodologies as they are currently presented, what the authors have categorised is the faecal microbiome as a whole, rather than the host-associated gut microbiota of the sampled bat species. Aside from an abundance filter, there is little attempt to ascertain what detected bacterial species are truly host-derived and which are transient environmental bacteria or commensals of the prey animals. Equally, despite mentioning inter-species variation in diet and intra-species seasonal variation in diet composition in the introduction, the authors seem to have given little thought to the actual composition of the diet of the wild bats that were sampled. Given that the structure of the host gut microbiota cannot be teased apart from the faecal microbiome, the authors cannot exclude the possibility that observed divergence in faecal microbiome structure between wild bats and observed convergence between the faecal microbiome structure of captive animals are not entirely due to the absence of different environmental and prey-derived transient microbes in the captive setting. It is probably not possible to address this issue completely without undertaking a much larger study incorporating dietary classification via metabarcoding and classification of the microbiome of the invertebrate prey.

The project would undoubtedly benefit from some further sequencing to help address these concerns. However, if this is categorically not an option then the authors should add extensive caveats to the discussion that interpret the findings of the paper in light of this uncertainty.

My secondary concerns which should be addressed subject to correction of the first are;
•	While common place in the literature, the binning of OTUs based on universally applied cut off of 97% identity is likely inappropriate for some of the detected species and somewhat antiquated in light of newer clustering algorithms. The authors should consider using DADA2 to define bacterial “species”.
•	Why did the authors decide to use UniFrac distances over other distance metrics? A brief discussion of this in the methods would be helpful.
Validity of the findings: Given my primary concern under the experimental design section, I believe that the findings of this paper as they are currently presented are invalid.

To reiterate, I believe that what the authors have measured is the composition of the faecal microbiome of wild vs captive bats. Whilst they have found that the composition of these microbial communities converges between species fed on a uniform diet, the authors cannot conclusively say that this convergence is due to changes in the composition of the host-associated gut microbiome rather than a much larger shared component of the faecal microbiome based on a completely shared diet comprising of meal worms and their commensal bacteria.

If the authors cannot address this uncertainty through additional sequencing, then they should incorporate extensive caveats and discuss their results in light of this uncertainty.

Finally, there is no mention that the data generated by this study has been or will be made publicly available.
Additional comments: In this paper Yanhong Xiao et al. seek to use a metabarcoding approach to categorise and compare the gut microbial communities of three bat species both in captivity and in the wild. Unfortunately, I have several concerns regarding the methodologies of the work and the quality of the resulting manuscript that must be addressed before the manuscript can be accepted for publication. However, should the authors comprehensively review and improve the manuscript then I believe the study could act as a starting point or useful reference for others working in similar areas.

This review focusses on the over-arching problems which I believe require attention rather than specific issues line by line.


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: CAPTIVITY CAUSES TAXONOMIC AND FUNCTIONAL CONVERGENCE OF GUT MICROBIAL COMMUNITIES IN BATS
Review round: 2
Reviewer: 1

Output:
Basic reporting: My comments regarding the standard of the English and the flow of the introduction have been satisfactorily addressed.

However, some small errors remain and I have highlighted all of these that I noticed in the attached version of the tracked changes document.

The methods section is improved for the additional details provided

A few additional comments

On lines 67 - 68 the authors suggest that captive animals are usually housed in a uniform manner. Could this be clarified, and if it is the intention of the authors to suggest that different species are kept under uniform conditions at zoos etc could this be backed up with evidence? I would suggest that most responsible zoos endeavor to mirror the conditions of the native habitat of their captive species as closely as possible in many instances.

The section of the introduction between lines 96 and 107 is very methods heavy and any information contained within this portion which in not already in the methods section should be moved there instead.

The first section of the methods (lines 127 to 159) is very difficult to follow and should be split into separate "Field" and "Methods" sections.

The final section of the results (lines 287 to 310) is also very difficult to read and should be simplified or clarified to improve comprehension.

I also believe that the manuscript potentially contains far to many figures for a manuscript of this type and length. The authors should consider keeping 2 or 3 main figures and relegating the rest to supplementary material.
Experimental design: I am disappointed that the authors did not deem it possible to address some of my major concerns regarding experimental validity. However, I find the inclusion of extensive caveats an acceptable solution in this instance.

I would also suggest to the authors that just because something is the most widely used example of a given tool, does not mean that those who are aware of problems surround that tool should continue to use it and so prevent the collective forward progression of the field. In future studies, a move away from arbitrary 97% cut off OTU clustering would be beneficial to all.
Validity of the findings: No comment
Additional comments: The extensive revisions that you made to the manuscript have greatly improved it for the last version. However I believe there are still some minor issues that need to be addressed prior to publication. Please see my above comments and attached version of the track changes document.


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: QUALITY OF LIFE OF OLDER CHINESE ADULTS RECEIVING PRIMARY CARE IN WUHAN, CHINA: A MULTI-CENTER STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The introduction provides background information and a rationale for the study.  But please see my comments in the Edits section.  Relevant literature is cited.
Experimental design: The study reports original research findings that fit the aims and scope of the journal.
A research question is stated, which is relevant and meaningful.
The investigation appears to have been conducted rigorously.  Methods are described with sufficient information to be reproduced, but please see my comments in the Edits section.
Validity of the findings: The data is statistically sound.
The conclusions are appropriately stated and are connected to the original question investigated.
Additional comments: Edits
The following edits should be made to the manuscript and are listed based on where they appear in the manuscript.
1.	The revised title should read: Quality of life of Chinese older adults treated in primary care in Wuhan, China: a multi-center study
2.	Throughout the entire manuscript, please refer to older adults as older adults and not as OA. OA stands for osteoarthritis.
3.	Throughout the manuscript replace no exercise habit with not physically active or physical inactivity (depending on the sentence structure).
4.	Page one, line 33 should read no living adult children.
5.	Page one lines 36 and 37 should read:  Addressing psychosocial problems in Chinese primary care settings could be helpful in improving QOL in Chinese older adults.
6.	On page 3 (the manuscript does not appeared to be numbered.  But counting from the title page) please provide a clear sentence or a paragraph that links QOL to health status.
7.	Page 3 line 97 should read: All participants provided informed consent prior to the interview.
8.	Page 4 lines 108 and 109 should read: Participants who regularly participated in physical activity were defined as being physically active.


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: QUALITY OF LIFE OF OLDER CHINESE ADULTS RECEIVING PRIMARY CARE IN WUHAN, CHINA: A MULTI-CENTER STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Some grammatical mistakes throughout the text and suggest to seek language editing service to improve on the English and expression. Some revision and comments are included in the annotated pdf file.
Experimental design: Research question defined, knowledge gap identified and statements were made to show how the study would fill the gap. Statement on ethical approval included. Informed consent obtained.	Methods were described with sufficient information.
Validity of the findings: Statistical analysis is appropriate. Conclusions drawn were supported by the results, well stated and answer the original research question.
Additional comments: •	Authors should standardize the use of terminology and use of English (British Vs American English)
•	Discussion is sometimes a bit superficial, especially for paragraph line 201-212. Suggest more in-depth discussion by explaining the meaning of the findings and why they are important, and comparing the findings with other similar studies.
•	Strength: Multiple linear regression analysis with reasonable sample size from multi-center, weaknesses were clearly noted by the authors
•	Weakness: English language, meanings of some of the expressions are a bit vague and thoughts were not connected


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: QUALITY OF LIFE OF OLDER CHINESE ADULTS RECEIVING PRIMARY CARE IN WUHAN, CHINA: A MULTI-CENTER STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revisions are fine.
Experimental design: The revisions are fine.
Validity of the findings: The revisions are fine
Additional comments: Thank you for you revising your manuscript. I am satisfied with the revised manuscript.


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

Input:
Title: A CITIZEN SCIENCE APPROACH TO EVALUATING US CITIES FOR BIOTIC HOMOGENIZATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Throughout I think there could be better reference to the literature. State explicitly in the introduction how this study fits into the broader field of homogenisation and urbanisation e.g. Clergeau et al. 2006 Biological Conservation, La Sorte et al. 2007 Global Change Biology, McKinney 2006 Biological Conservation (I only include these as suggestions, there is plenty of other relevant literature out there). There is also a wealth of literature on the role of traits in homogenisation in urban areas, so it would be good to at least acknowledge ecological traits within the discussion.

The results mainly rely on the interpretation of NMDS plots, of which there are a substantial number, and leads to a very qualitative description of the findings. Some of the plots could be put into supplementary material (perhaps the plant only Fig. 3) to make the paper more succinct. Clearer interpretation of the PERMANOVAs I think will improve the clarity and flow of the results.

Throughout the results, but e.g. line 164, when you refer to species diversity I assume you mean species richness. I think this term should be used throughout.

You use the term cosmopolitan (line 177) to represent species that are found in the majority of cities but are these species actually generalists? I.e. they are found everywhere. I think you could make it clear somewhere that cosmopolitan species are not necessarily exclusively found in high intensity land cover types.

I have a number of issues with Figure 5. Firstly, the legend could be clearer, adjust your ggplot code so that you just have dots, not dots overlaid on ‘a’. In 5b, I can’t distinguish Red-winged blackbird from Mallard, are they overlapping?

Overall, 5a and 5b are difficult to interpret. Were the slopes of the CAM and ARM with intensification always significant? The analyses that lead to the creation of Figure 5, as well as the interpretation of this figure could both be expanded upon to improve comprehension. What statistical tests have been done to generate this? What is it actually showing? Is there an overall relationship?
Experimental design: I’d like to know what taxa fell into the ‘other’ category. Can you give a couple of examples? (Line 114)

Add reference to justify use of 0.20 as stress cut-off in NMDS e.g. Quinn and Keogh 2002 Experimental design and data analysis for biologists. – Cambridge Univ. Press

Design is relatively clear but interpretation of how this reflects homogenisation could be stated more explicitly within methods. For example, how do city accumulation metric and averaged ranking metric reflect biotic homogenisation? To my knowledge these aren’t commonly used measures, and it took me a few reads to get my head around what they mean. Although they are detailed in the supplementary material, more detail on their interpretation is necessary within the main text.

Further, in line 142-143, it would be good to have a brief explanation/example on the assumption that urban specialists are contributing to homogenisation.
Validity of the findings: I am not convinced that you can use this data for community composition and then calculate Bray-Curtis. In line 105 it is stated that differences in collector effort are addressed in the analyses, but I don’t see how. I understand that the ARM accounts for recorder effort in the species level approach, but how is it accounted for in the community approach. If the observations are used to build the Bray-Curtis dissimilarity matrix, then are you assuming that number of observations = relative abundance? If recorder effort has been accounted for, I think this needs to be made much more explicit.

The statement in line 203-204 that city explained more than land cover type needs to be justified numerically in the text. Is this based on the R2?
Additional comments: Overall I think this is an interesting piece of work, but the presentation of the study could be improved.
Theories underlying the paper could be fitted better within the current literature. The reference list is by no means exhaustive so more detail about how the current approach compares to alternative (and more common) approaches within the literature and how their results fit into the wider discussion of urbanisation effects on community homogenisation.

Some minor comments:
Keep use of abbreviations consistent e.g. line 152
Check grammar e.g. lines 198, 238 and 265-266
Lines 232-236 This seems to be more methods than results
Use e.g. instead of ex. throughout
Splitting the results and discussion would make the paper easier to follow and better put the results in the context of the current literature. It would also fit the format of the journal. Also add conclusions section to abstract.


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

Input:
Title: A CITIZEN SCIENCE APPROACH TO EVALUATING US CITIES FOR BIOTIC HOMOGENIZATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: A great contribution to the urban homogenization literature. Clearly citizen science and iNat can be a huge source of data. The problem is the bias factor but your discussion correctly identifies potential influences. In this case, your finding that relative abundance may play a big role (e.g., decline of natives) is really interesting and often neglected but could be strongly affected by the fact that citizens tend to photograph large, colorful or otherwise conspicuous species so that abundance is inflated in those species. Nevertheless, this study is a great first step in describing the visible patterns. Your finding that urban composition is driven by a few cosmopolitan and many local/regional species adapted to humans is compelling and critically important to our basic understanding of urbanization impacts on biodiversity. Your analysis is well conceived, implemented and easy to follow.


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

Input:
Title: A PREVALENCE OF ARTHROPTERYGIUS (ICHTHYOSAURIA: OPHTHALMOSAURIDAE) IN THE LATE JURASSIC—EARLIEST CRETACEOUS OF THE BOREAL REALM
Review round: 1
Reviewer: 1

Output:
Basic reporting: Dear editor, dear authors,

I consider this manuscript as a real tour de force, bringing an impressive amount of novel data from a series of spatiotemporal settings, to better understand the early evolution of ophthalmosaurids ichthyosaurs. I really like the thoroughness of the morphological analyses, the expanded phylogenetic dataset and the idea to use PCA. I am in certain this will make a great paper.

I am thus totally favorable for publication of this work in PeerJ, but after some revision. Indeed, I feel that the present version of the manuscript is problematic in its (i) tone, (ii) structuration/argumentation (Basic Reporting section) and (iii) taxonomic decisions (Validity of the findings section). I detail these three points in each section. I have indicated in General Comments, a series of line-by-line minor comments. I am confident that the authors can work these issues out and so I suggest that the paper requires a *moderate revision*. This is not an option in PeerJ, so because "minor" requires no additional revision, I had to select "major".

(i) Tone
I think that the text is generally unnecessarily critical of some colleagues, using words and verbs suggesting that most of them were quite profoundly mistaken: “misinterpretation”, “misleading”, etc. (e.g. L66 and in Materials, L244, L324-336). By reading the text, it appears that nearly all authors who have been working on similar taxa were mistaken:  e.g. Delsett (many instances), Roberts (many instances), Maxwell (L575), Druckenmiller (L683), Kasansky (L1048). Only one scientist, the Russian geologist and palaeontologist A.P. Pavlov is regarded as “outstanding” (L100). While this might be the case and that our knowledge is filled with a series of errors and mistakes, remember that in most cases, you are providing re-interpretations (which, indeed are probably very good!) rather than the absolute truth. I think this helps putting things in perspective. I am totally certain that this is not intentional.

Similarly, the burden of proof is on your side; you cannot mention current knowledge as "alleged" prior to detailing your results. I would suggest to carefully re-read the text and rephrase some sentences, first sticking to what is currently known (or believed) and then acknowledging that you have a different interpretation than previous authors.

(ii) Structuration of the text and argumentation
There are two things here, the first being that in the present version of the paper, it is stated upfront that profound changes in the taxonomy are made, but the reasons are disseminated in the entire (fairly long) text. Given that important taxonomic changes proposed, I think it would be better to first describe the new remains and re-interpretations, then discuss the taxonomic possibilities, and then place a Systematic Palaeontology section, towards the end of the paper.

The second issue is that the text appears to frequently mix facts, interpretations, and opinions (I have indicated several instances in the line-by-line comments below). This has the (surely undesired) effect of confusing the reader. Similarly, in some instances, results are presented without any argumentation or sensible/tangible data (e.g. L20-21, L150 and after and to a lesser extent the whole biostratigraphic data, L166-167, L237-238, L324-336, L418-421, L451-453), making them opinions rather than testable results and trying to get certitude out of incertitude. It is difficult to propose a clear-cut solution here, but rather a thorough re-read of all the paper’ sections.
Experimental design: I do not see obvious flaws here as methods have been carried on with great care, but I have a couple of questions on PCA, which I discuss on the next section?
Validity of the findings: (iii) Taxonomy
There seem to be a distinction in the taxonomic relevance of morphological features throughout the manuscript. Many features listed by previous authors as differing between Arthropterygius and other genera are said to be misinterpretations (nearly all authors who have been working on similar taxa were seemingly mistaken, as mentioned above in these comments). Other differing features are dismissed right away as variability (e.g. L504).

But at the same time, all previously established species are retained, sometimes based on rather minute differences (e.g. A. volgensis) and no variation is invoked in these cases. This difference in the taxonomic importance (generic VS specific VS population) of morphological features appears at times puzzling from the reader’s perspective and would benefit from a more rigorous treatment throughout the paper.

Indeed, no clear arguments is provided for or against another and simpler possibility:  that Arthropterygius, Janusaurus and Palvennia are valid genera belonging to a well-defined clade of ophthalmosaurids. The results of the morphospace analyses suggest the same, with Arthropterygius spp. having a wider spread (and thus disparity) than entire ophthalmosaurids subfamilies, with a much larger time span. Don’t get me wrong, the simplicity of this solution does not mean it is the correct one, but at least it should thoroughly and fairly evaluate, along with more taxonomically complex solutions.

The inference (L1253) that a series of ichthyosaurs had assymetric limbs appears quite unlikely and most probably an effect of diagenesis. From my experience, all the humeri I have examined were very similar left-right (not totally similar not certainly not massively dissimilar); this result actually call into question the influence of diagenesis on the results, which is not discussed in the text. Indeed, the authors state that humeri of Arthropterygius can be recognized quite easily, but at the same time, mention that there are large in vivo intra-individual differences (added to the result mentioned above that the disparity of the humeri of Arthropterygius exceeds those of Ophthalmosaurines and Platypterygiines).
Additional comments: Line-by-line minor comments

L14 and L53: “heretofore” might be a bit awkward here, how about “hitherto”?

L15-16: you should briefly argue why (i.e. phylogenetic position with respect to ophthalmosaurid subfamillies)

L49: this might be a strecht to say Aegirosaurus is *well* known... Same for Undorosaurus. Why do you omit Ophthalmosaurus? Do you specifically refer to the taxa abundant in the Kimmeridgian-Tithonian instead of the whole Late Jurassic?

L54: Awkward sentence construction; could you please rephrase?

L65: poorly known “in” instead of "for"

L73: “allow us *to* substantially”

L100: Consider removing 'outstanding', it is an opinion

L108-110: I would suggest to remove this sentence, as the main message is explained in the next sentences.

L128: Geological setting. I would suggest to produce a couple of figures with the specimens mentioned that have a stratigraphic importance. Indeed, this is scientific data that should be analyzed, commented and criticized by others just like in other data in the paper. This might be especially important in future, notably regarding the hot debates surrounding the Jurassic-Cretaceous boundary.

L150= "almost" and "unambiguous" do not go well together most of the time.... I would suggest to rephrase and to provide arguments for this (e.g. only X m below the start of such biozone etc.)

L195: synapomorphies are usually applied to suprageneric clades; I would suggest to use "autapomorphies" to avoid confusion.

L224: I suggest to make a table with the different features and their presence/absence/NA in the species merged into Arthropterygius. That would be a good companion to the text.

L237-238 and throughout: such sentence, somehow forcing the reader into an opinion, should be avoided; I suggest to delete it.

L244: "misleading" is not appropriate here; I would suggest "questionable"

L246: consider replacing "say something"

L264: lacks *an* obturator foramen

L254-266: the sentence is unclear; could you rephrase?

L324-336: again please watch out for tone and the difference between fact and opinion

L338: Description: would it be possible to add more comparisons with other ophthalmosaurids?

L418-421: Again, an opinion is voiced without any argumentation. This needs to be thoroughly checked in the entire text.

L451-453: I suggest not to estimate jaw length when the entire dentary is missing.

L639: "h" missing in Arthropterygius

L788-790 The meaning of this sentence is unclear; could you rephrase?

L807: too many "th" in Arthropterygius

L1136 a series of "unambiguous" synapomorphies are indicated, some of which being labelled as "non-unique" (thus ambiguous). Could you clarify?

L1247-1249: this is well known (and did not require a PCA to be noted by previous authors ;) )

L1364: I do not understand the meaning of "aerials" in this sentence.

L1398 "lagerstätten"

All the best and great job!

Valentin


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

Input:
Title: A PREVALENCE OF ARTHROPTERYGIUS (ICHTHYOSAURIA: OPHTHALMOSAURIDAE) IN THE LATE JURASSIC—EARLIEST CRETACEOUS OF THE BOREAL REALM
Review round: 1
Reviewer: 2

Output:
Basic reporting: General comments

Throughout the manuscript, it is confusing which specimens are referred to which species. Please add a table to make this very clear for readers, that might not be familiar with the previous specimens and publications. When describing a species with multiply referred specimens, please include which specimen you are referring to in the description (this needs to be really clear). This is to avoid confusion for readers in which specimen preserves what. Please fix this throughout the description
In general, the level of English in the paper is high, however there are a few things that should be changed. I have suggested a few wording changes in the document, one of these that should be searched and replaced are the words “thanks to”. This is particularly scientific language and should be changed throughout the document. Examples of words they could be changed to: “due to”, “as consequence to” ect. Other sentences/words that should be rephrased are highlighted.

Figures:
The figures are of a good standard and quality, figure 1 in particular is very detailed and visually appealing. However, I have not seen any evidence of figure captions, where are they? Some of the figures of cranial elements should be edited. The lines bordering the different bone elements follow, in some cases, taphonomic cracking/breakage and do not represent a suture. These should be shown either by a dotted line or pointed out on the figure. On the PCA figure, one of the names seems to be incomplete (just PMO). This should be changed.
Experimental design: I insist on changes made to the emended diagnosis as it is currently confusing and should include which features which are present in which taxa/specimens and which features it shares or differs with other taxa. This will make the diagnosis more useful to other researchers and will avoid misunderstandings on which features are present in which species.

The palaeobiogeography section can be shortened, as much of it just repeats previous work, which is well summarised in Zverkov et al. 2015. Please just refer to this work and add in what the new data tells us about the distribution of Arthropterygius.

Other comments on methods and analyses- see annotated pdf
Validity of the findings: From the text I cannot clearly understand why you have referred PMO 222.669 to A. chrisorum and not A. hoybergeti. This needs to be clarified. There is no evidence of a large pineal foramen in any of the other Arthropterygius specimens referred to here, which was one of the main reasons to referring PMO 222.669 to Palvennia.

The new combination referral of the juvenile KSU 982/P-213 to Arthropterygius volgensis lacks sufficient justification. Basing a species purely on the morphology of the humerus is not acceptable, as mentioned in your own PCA analysis, the is even variation in the morphology of some of the traits in individuals. This specimen should be referred to Arthropterygius indet. This problem is also the case for the referral of UPM 2442 and YKM 63548 to A. hoybergeti.

The section on “Ontogenetic changes and variation in Arthropterygius chrisorum”, requires significant revision. Using a few incomplete specimens of different species (which could vary in size), is not sufficient to estimate ontogenetic change. This either needs to be confirmed using methods such as histology (or μCT scanning), or played down significantly. There are multiple papers by the Sander lab looking into the histology of marine reptiles. I can provide these if the authors need them.
Additional comments: I very much enjoyed reading the paper and I think it fits the angle for the journal sufficiently, I recommend acceptance with major revisions. There are numerous changes that need to be made to the manuscript before publication. These are available in my edited word document and some of them are highlighted in my comments. hope my review of this manuscript will be useful to the authors and editor and I look forward to seeing the paper published after these changes have been made.


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

Input:
Title: A PREVALENCE OF ARTHROPTERYGIUS (ICHTHYOSAURIA: OPHTHALMOSAURIDAE) IN THE LATE JURASSIC—EARLIEST CRETACEOUS OF THE BOREAL REALM
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I commend the authors for their extensive and detailed description, which is the fruit of years of fieldwork, personal observations and detailed analyses of the collected data. The result is an undeniably convincing and well exposed hypothesis that attempts to revise the morphology, taxonomy, systematics and palaeogeography of an important ichthyosaur genus.

The manuscript can be improved in some respects – which I consider marginal compared to the amount of effort and work already done.

The language should be improved to ensure that an international audience can clearly understand your text. The manuscript contains a number of grammatical mistakes that should be addressed. In some cases, these imprecisions make it unclear the meaning of individual sentences - but it rarely affects the overall meaning of the text.
Amongst these:
- 	There is a recurrent misuse/omission of articles “A” and “THE” (see attached PDF).
-	In several cases sentences are unnecessary long, full of subordinates – often exceeding 3-4 lines. They should be simplified to improve the flow and clarity of the text (see attached PDF).
-	Use of odd sentences words (e.g. Lines 108-110; 115-116). While the meaning of the sentence is clear by context, it is not immediate. These sentences/words should be replaced simplified. These and other examples can be found in the attached PDFs.
-	The whole description should be considerably simplified as it is currently difficult to follow.
-	There is no consistency in the verb tenses used through the text.

My biggest concern is that TOO much details are referred to other papers (most worryingly to one that is “in press” and was not provided with the manuscript – thus that information is unverifiable).
Description
Generally, the authors report well supported arguments – in places the authors should tone down the text (e.g. “In our opinion, this is…” rather than “This is”.
In places the authors should instead providing better evidence for their claims, especially in the initial part of the manuscript where they argue against a number of researchers’s observation (e.g. paragraphs before and beginning with line 324).
Unfortunately some of the authors’ arguments are not sufficiently justified and they offer an alternative interpretation without supporting it (a simple reference or observations on other specimens would be enough).
A lot of the statements in the descriptions are referred to “Zverkov & Efimov, in press”. As I do not have access to this manuscript, I cannot assess any of these statements/observations.
In the description section there is very limited comparisons with other ophthalmosaurids (and also put in a phylogenetic context as it is very well done in some paragraphs: e.g. prefrontal (beginning at line 373) and basioccipital (beginning at line 422) and basisphenoid (beginning at line 422). This paragraph should be used as an example for all the other descriptive paragraphs). This issue should be addressed especially for those elements that had not been previously described or for new species.
Finally the authors should more clearly underline the similarities between species they refer to the same genus, especially where the newly available materials show them.
Multivariate analysis of ophthalmosaurid humeral morphology
This whole section is accessory to the manuscript – and the authors may want to consider moving this in the supplementary section. It is not vital – and a really minor point. I understand the desire of the authors to include it in the manuscript.
I would agree if the authors decided to keep it in the main text, but if they do, the relevance of the analysis for the manuscript should be more clearly stated.
Biogeography.
Overall, very interesting section. It would greatly benefit from inclusion of some missing references and comparisons with other groups (see PDF). The importance of the Hispanic corridor is likely dismissed too quickly and the dispersal pattern of other marine reptile groups should at very least be mentioned.
Supplementary information
There is an array of several different fonts and font size in the supplementary material.
Please review it and make it consistent through the text and with the main manuscript.
Figures
Beautiful and very clear figures. Well referenced within the text.
Additional clarifications on the stratigraphy should be probably added.
A comparative plate of overlapping Arthropterygius cranial and postcranial elements should be added (similar to Fig. 17 but with additional diagnostic features and overlapping elements).
Additional details and comments are included in the PDF.