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: ASSESSING THE EFFECT OF FISH SIZE ON SPECIES DISTRIBUTION MODEL PERFORMANCE IN SOUTHERN CHILEAN RIVERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English is good in many sections, but needs significant improvement in a few. The statistical methods are very unclear to me, however, which makes evaluation challenging.

The references to the data are generally adequate. However, I think the introduction should make reference to the possibility of differential detection/capture efficiency based on body size as a factor explaining model performance.

Figures and tables look good.

The raw data are included.
Experimental design: The research question is reasonable.

The methods are unclear as written, which makes evaluation difficult.

I think the number of species is really too small to draw any firm conclusions.
Validity of the findings: I think the authors overreach in their interpretations based on the very limited number of species. I think there need to be many more caveats, and much of this is essentially speculative and should be more clearly labeled as such.
Additional comments: The authors test whether fish body size correlates with SDM performance. This is an interesting idea, but I think the sample size (N=7 species) is too small to draw any firm conclusions; indeed, the authors find that depending on the predictor variables employed, the relationship can either be positive or negative. Therefore, I think the authors need to rewrite the abstract, the heading on line 221, and other sections that seem to claim a clear finding that body size and performance are positively related. I think the authors tend to over-interpret some of their other results as well, as noted in the detailed comments.

This is well written in many places, much less so in others (e.g., lines 97-100). There are several places where the singular is used where the plural should be employed, and vice-versa, and some instances of subject-verb agreement (e.g., line 142, should be “model that predicts”).

The statistical methods are very unclear to me as written. They need to be clarified before I can judge whether they are appropriate. For example, was the 10x resampling done for all species to maintain a ~50/50 presence/absence ratio? That’s a lot of work to deal with the prevalence issue, which other authors (e.g., Jimenez-Valverde and Lobo 2006) have shown is not such a problem if handled correctly. It’s also unclear to me how this resampling worked with the 5-fold cross validation and the 30% withholding. See specific comments for further questions.

Introduction, generally: I was surprised to see no mention of detection/capture probability as a factor involved in SDM performance related to body size. I think it’s worth mentioning that species of different sizes may have differential detection probabilities (could be higher or lower, depending on the sampling method).

Detailed comments.
Abstract: I think “overproduction” is meant to be “overprediction.” Right?
Lines 30, 34, etc: I suggest consistency in the use of “SDM” vs “SDMs.” In line 34 it appears that “SDMs” should be used.
37. “detect less food” is confusing to me. I’m not sure what is meant here. If this is all to explain that larger species have larger home ranges, consider just making that point, with a citation.
42. I don’t think “perceives” is the right term. Maybe simply state that the distribution of species with larger ranges tends to be governed by coarser grained predictors.
62. Please add a citation for this imperilment statistic.
97-100. The English needs some major attention here—it looks like this paragraph was skipped in the editing process. E.g., the first clause should read, “These species were selected because they represent…”
125. Briefly define in the text what is meant by “source of flow.”
148-149. 5-fold cross validation implies that 80% of the data were used for fitting. Is this 80% of the 70%? It may be reasonable to do both cross validation and conventional validation, but please explain the logic for doing both.
150-151. Does this sentence refer to the bootstrapping for O. mauleanum? Please clarify.
152. Is this a restatement of 147, or an additional step? This is unclear.
153. How was the consensus calculated? There are multiple methods available for creating a consensus forecast.
181-186. This is confusing to me as written. I’m not sure what the authors have done and why. Is this to secondarily deal with the rebalancing to get the prevalence close to 0.5?
210-212. This confused me. I’m not sure what this is intended to say.
228-230. It’s not clear to me why correlations between predictor variables and body size are being tested. Was this one of the research questions? I’m not sure it should be.
293-309. I think the authors are greatly over-interpreting the results of a very small number of species. I would be very careful about this kind of speculation; in particular, I would cut the paragraph at 303-309.
345-348. The English needs some attention here.


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

Input:
Title: ASSESSING THE EFFECT OF FISH SIZE ON SPECIES DISTRIBUTION MODEL PERFORMANCE IN SOUTHERN CHILEAN RIVERS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is an interesting manuscript dealing with species distribution modelling (SDM) and addressing whether SDMs performance or accuracy differ between fish species that have different theoretical maximum length. As presented, I consider that the authors need to improve a number of issues, before this manuscript can be accepted for publication.

For example, it is not clear why understanding the potential impact of body size on SDM performance would be highly relevant for conservation and management planning efforts (lines 64-66), as this study address the theoretical maximum body length, not the individual body length. Also, to my understanding of Table 2, fish species studied have wider distribution than the two watersheds included in this ms., thus it appears that the theoretical relationship that the authors are considering might be biased (if the theoretical maximum body length is considered, then the whole distribution of fish species should be studied).

In any case, the introduction and background described at the beginning of the ms. (lines 28-74) is not suitable nor completely relevant to make a strong case for the research questions studied by the authors. Please add more details at the introduction section to improve the background of research questions, as some concepts are not defined in any section of the ms.: e.g. What is overproduction of biotopes? (line 25); What is a biotope? (line 73); What is variable participation? (line 232). To the knowledge of this reviewer, these concepts are very specific to SDMs, thus they should be explained thoroughly either at the introduction or mat&met.

Figures should be improved. For example, figs 1 and 5 have overlapping labels, fig 4 is confusing (why including all models for all species?). If one model was chosen for each species, it is not necessary to show the results of the other models, unless a proper discussion emerges from it.

Although some raw data (restricted to fish distribution) is supplied, it is impossible to recreate the study as ecological and anthropogenic variables are missing. Please include the raw data as supplementary material.

Finally, I appreciate the authors are not native speakers, but several phrases and paragraphs of the manuscript needed reading twice or three times in order to understand their meaning. I would appreciate if you send the manuscript for edition in order to use clear, unambiguous and professional English language throughout. Unfortunately, most paragraphs of all sections need English edition.
Experimental design: This is an original primary research within the scope of the journal, but the experimental design might be flawed. For example, 1) it is not clear how the authors selected the SDM algorithm for each species; 2) the number of occurrences (calibration vs validation) is not 5-fold (e.g. Brachygalaxias bullocki has 27 occurrences, 30% is 8 occurrences, thus the remaining should be 5x8= 40 occurrences); 3) if there were 10 models for each species, why there is no variability with consensus statistics? (fig 2); 4) correlation is not causality (lines 191-197), thus another proper statistical test should be included.

Probably, some data was not included:
Where is table S1? (line 96)
Where is table S3? (line 230)

Why comparison with prior research was done only considering studies with n>5? (line 207)

According to table 2 (description column) most variables doesn’t appear to be normal. If the authors used Box-Cox procedures to normalise data (line 217), then normality tests should be used, or at least, to analyse q-q plots of data (or histograms).

I suggest to perform a multi-species distribution modelling instead of performing separate SDMs for each species. Although the idea of including the theoretical maximum body length is really interesting, the experimental design appears to be flawed for the reasons indicated at the basic reporting section of this review, but also because correlation is not a proper statistical test to evaluate the effect of body length on TSS. This is a measure of association of two variables, and should not be used to measure the effect of one independent variable (maximum body length) onto another dependent variable (TSS).
If the authors decided to keep SDMs per separate for each species, at least should be included in a revised version:
1) how each model was selected (Eco vs Anthr vs Eco+Anthr)
2) following Allouche et al (2006): overall accuracy, sensitivity and specificity of each model (table 2)
3) another statistical test to evaluate properly the effect of body length on TSS.
4) a measure of goodness of fit for each model (is it really good a model that has TSS values of 0.11, 0.16 or 0.21?)
Validity of the findings: To my understanding, the effect of fish size on SDM performance is not assessed using correlation tests, thus, objective 1 is not fulfilled in this ms. Also, model performance in previous studies was not compared thoroughly, only were selected a number of studies. Further review should be included.
Additional comments: If the authors will stick to taxonomical standards, when mentioning a fish species for the first time (e.g. lines 62-63; 88-91, etc.), please indicate the authority who described them correctly. If the species was changed from one genus to another, the authority should be written between parenthesis, otherwise parenthesis should not be written. Please check all the species included in this study to conform to taxonomical standard.


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: ASSESSING THE EFFECT OF FISH SIZE ON SPECIES DISTRIBUTION MODEL PERFORMANCE IN SOUTHERN CHILEAN RIVERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Generally good, with some unclear sections. Greatly improved over the previous version.
Experimental design: The design is good.
Validity of the findings: No comment.
Additional comments: This article is much improved from the previous version. I applaud the authors for conscientiously addressing the comments of the reviewers. There are still a few issues to address, but they are relatively minor. Note that in the interest of providing a timely review, I was not as thorough as I normally try to be; in particular, I have not given the discussion a detailed read.

Line numbers refer to the track changes version.
103. This says “spatial distribution models” rather than “species distribution models”, which is more standard and appears in the revised title.
112. Typo here: SMD instead of SDM.
321. “designated the consensus pondering”—this is nonstandard English; I don’t know what it means.
331. Insert here a parenthetical note that this is the true skill statistic or TSS. The term TSS is used later, but is not defined in the revised ms.
368-373. This section is unclear. It says the statistical relationship was assessed, but looking at model performance statistics per se does not constitute a statistical test. Also, don’t say “metrics such as…”; the reader needs to know what was done, not examples of things that were done. Finally, variable importance needs to be defined, as there are different ways to calculate this.
376. “Betareg” sounds like a shorthand term. Is this beta regression?
383-385. I didn’t understand this.
448-449. Here and elsewhere: normality of residuals is just one of the assumptions of regression, and not necessarily the most important one; this is fine to report, but delete the final phrase “thus validating the statistical analyses.”
522-526. This is stylistic, but I suggest leading the discussion with a paragraph interpreting the main findings and discussing their significance, rather than these notes on model performance.
562. “Tend to decrease their specificity”… does this mean “tend to have lower specificity”?
562-564. I’m not following the logic of this.
640. When summarizing a result of this study, don’t cite another paper. It’s not clear why Frissell is cited here.


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

Input:
Title: ASSESSING THE EFFECT OF FISH SIZE ON SPECIES DISTRIBUTION MODEL PERFORMANCE IN SOUTHERN CHILEAN RIVERS
Review round: 2
Reviewer: 2

Output:
Basic reporting: This is my second time reviewing this manuscript. I wish to thank the authors, as this is a greatly improved version of the ms., especially concerning the English used throughout. However, some of my initial comments were not considered in this version, so I will highlight them when needed.

Regarding figures and tables, I think they can be improved. For example, text on X axis (figure 3) might be changed (species names should be in italics, 0.1. does not need to have period in-between). It seems that this figure is the direct output from an r script, one possibility is to save it as PDF and edit it in Illustrator or other software capable of editing vector format files. Figure 1 should include the percentage of variance explained by the betareg regression. Finally, please use species names consistently, is it Basilichthys microlepidotus or Basilichthys australis the species you are modelling? (check suppl. file 1, the whole manuscript and figures).

In the same figure 3, please indicate what the box-plots are showing: median, inter-quartile range and maximum and minimum? If that is the case, I do not see any difference between the occurrence probability according to predictor variable classes (riparian vegetation and within channel structures) than differences in the median/mean. If there are not statistical differences there is no need to separate each variable in two classes.

Table 1 includes some model performance descriptors, such as TSS, AUC, Acc, Sens and Spec. Although these can be understood rapidly for some readers, I suggest the authors include in mat&met a brief description of each descriptor, indicating (1) what the statistic stands for, (2) how is it calculated, and (3) when they are considered "good". Some of the readers might not be familiar with them and one of the objectives is to assess model performance.
Experimental design: The aim of the authors is to evaluate the relationship between fish size and SDM goodness-of-fit using three approaches:
(1) assess the relationship between fish size and data availability,
(2) assess the relationship between fish size and model performance, and
(3) compare predictor variable participation and patterns according to fish size.

The authors focused on two well-studied southern Chilean river basins, Bueno and Valdivia, and modelled nine native species.

According to the authors objectives, fish size is the key variable to test for. It is important to state at which ecological level the authors are working on, i.e. at the individual level (each individual has its own length measurement, with their own predictors) or at the species level (the whole distribution of every species is used). As presented, it seems that the authors are modelling at the individual level, and part of the discussion is related to individual fish length (lines 293-294), as they mention that introduced species influence fish body size. If introduced species (or any other variable) influence fish body size, is it correct to relate model performance to theoretical fish body length? I think that at least one paragraph dealing with this idea should be included in the discussion section, considering (1) the limits of this approach (theoretical fish body size will remain as theoretical, while contemporary individual fish body size will reflect model performance), (2) caveats and possible errors while using small datasets (see below).

I still don't understand how the authors did the modelling for every species. In the line 159 the authors mention 2- or 3-fold cross validation scheme, e.g. in the case of Diplomystes camposensis they used 14 presences, which are divided in 5 occurrences (for the NNET modelling) and 9 occurrences for the 2-fold cross validation? Is that correct? If that is the case, then proper statistics validating such modelling approach should be included. For example, why not use the MMA data to create the models, and then use the electro-fishing data to validate it? As presented it is not clear why the authors did a field sampling campaign, how many rivers were sampled and how many fish were caught and included in the modelling. Please be more clear.

Another possibility is to use a jackknife approach to validate the model, modelling dozen or hundreds of times each species, leaving 30% of the data for validation.

Regarding statistical analysis and the statistics themselves, please include a full description, not just p-value: lm test (lines 204-25)
Validity of the findings: Shapiro test just indicate if the data (or residuals) are normally distributed, they do not validate statistical analyses (lines 213, 219). Statistical validity should be tested with an independent dataset (such as electro-fishing data), jackkniffe, or using proper statistics (TSS, AUC, etc) or using the confusion-matrix.

To the knowledge of this reviewer Habit et al (2006) is not a field identification manual, it is merely a description of the state of knowledge of fishes in Chile at that time. Please provide a proper identification manual, if it was used.
Additional comments: no comments


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS OF THE 14-3-3 GENE FAMILY IN SOYBEAN (GLYCINE MAX)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript provides a characterization of the Soybean 14-3-3 gene family in soybean (Glycine max).
As already an attempt was taken in this direction, where, Li X, Dhaubhadel S (Li X, Dhaubhadel S. Soybean 14-3-3 gene family: identification and molecular characterization. Planta. 2011;233:569–82. pmid:21120521) identified eighteen 14-3-3 genes in soybean. So further, in the current study authors have reported 22, 14-3-3 genes in soybean, and also performed phylogeny and expression analysis of these genes.

But i have some major concerns. Below are the issues that need to be addressed by the authors before the manuscript can be accepted for publication.

Poor English with many grammatical and spelling mistakes throughout the manuscript. Which need to be improved.

More details about the results and their statistical significance is required in the Figure legends.

Literature references
sufficient field background/context are not provided,
Authors didn't mention about the important work already published about "Soybean 14-3-3 gene family: identification and molecular characterization"
by
Li X, Dhaubhadel S. Soybean 14-3-3 gene family: identification and molecular characterization. Planta. 2011;233:569–82. pmid:21120521
Experimental design: Original primary research within Aims and Scope of the journal.

Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.

Rigorous investigation was performed to a high technical & ethical standard except in few cases.

Methods are not described with sufficient details & information to replicate
Like in RNA extraction and Quantitative real-time PCR (qRT-PCR)
No details has been provided about the biological amples and PCR conditions

the Authors need to specify about the biological samples taken in the relative expression analysis. Also in the material method and results sections i can not see any details about  statistical significance of the results.
Validity of the findings: Data is not statistically sound
Additional comments: No additional comments


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS OF THE 14-3-3 GENE FAMILY IN SOYBEAN (GLYCINE MAX)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language should be improved throughout the manuscript to allow the reader to understand the text. The manuscript is difficult to read and there are many expressions that need to be corrected. For example; Lines 300-301.

The Introduction, background and discussion are in general well referenced, although some literature related to 14-3-3 in soybean is missing. For example; Li and Dhaubhadel (2011) and (2012).

In general Figures are relevant and have good quality. I suggest black labels in Figures related to qRT-PCR analysis to ensure proper visualization. In addition, in Figure 5 the different colors represent transcript abundance. Are they FPKM values? This should be explained in more detail in the Figure Legend and in methods.
Experimental design: The Methods are in general well described. However some more details are needed for the measurement of transcript abundance and qPCR analysis should at least have 3 biological replicates and statistical analysis should be included to determine the significance of the data.
Validity of the findings: The results obtained are interesting; however more information can be obtained to strengthen the findings. In addition, the RT-qPCR should be repeated with at least 3 biological replicates.

Previous papers in soybean describe the presence of 18 14-3-3 genes. The authors should indicate this difference in the text and explain why they found 22.

The number of genes pairs involved in segmental duplication should be indicated in the result section.

The authors could include a Figure or table showing the segmental duplication pairs, the % of identity, and their expression patterns in different tissues/organs and abiotic stress.  The fact that similar expression patterns are present in the duplicated pairs is interesting and could be shown more clearly.

The expression patterns of some members of segmental pairs during abiotic stress are missing. What were the criteria to use 19 and not 22 genes?

The expression pattern of the 14-3-3 from M. truncatula could also be included since expression data is available and the results could contribute to the discussion.

In addition to ABA responsiveness, some other cis-regulatory elements are also overrepresented in the 14-3-3 gene members. This could be discussed in more detail.

Minor comments
Please change “gene replication” for “gene duplication”
Additional comments: In this manuscript, the authors analyzed the 14-3-3 gene family in the soybean genome, compared their phylogenetic relationship with 14-3-3 from Arabidopsis, rice and Medicago truncatula, and classified them according to conservative motifs and gene structures. They show the occurrence of segmental duplication events and the evolution of the soybean 14-3-3 gene family comparing them with Arabidopsis and M. truncatula. Using the publicly available RNA-Seq data, the authors analyzed the expression patterns of this gene family in different tissues and organs. The authors also analyzed the expression pattern in response to abiotic stress, including drought, salt and cold stress. The results show that 14-3-3 soybean genes are higher expressed in vegetative organs compared to reproductive organs and that they are differentially regulated during abiotic stress. This manuscript would be of general interest. However, there are major comments that need to be addressed.


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

Input:
Title: IMPROVING THE PREDICTION ACCURACY OF RIVER INFLOW USING TWO DATA PRE-PROCESSING TECHNIQUES COUPLED WITH DATA-DRIVEN MODEL
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper develops an integrative AI technique to predict inflow pattern. Thus, it is part of the large body of literature related to hydrological and environmental researches. It is commonly known on the field. The most relevant aspect of the paper is the application of a new technique of 'hybrid' model. It is novel enough and seems technically sound and should be considered for publication however, the paper in its present form is still far of being mature enough to be published. The paper needs to improve on organization, elaboration, documentation of procedures and discussion as well as in the English language
Experimental design: I can not comprehend the actual proposed modeling procedure. You need to report a clear flow chart presenting the adopted learning process. Cite appropriate references for the methodology phase. Revise all the notation and formulations. You need to report carefully the the historical data construction for the prediction process. Clearly state the data division.
Validity of the findings: First of all, you need to state the performance formulations. The mathematical expressions. Readers need to be familiar with those metrics. Cite the following references for those indicators:
https://www.sciencedirect.com/science/article/pii/S0022169418307819
https://link.springer.com/article/10.1007/s11269-018-2038-x

Based on the reported statistical metrics. Merely a marginal enhancement are obtained. hence a validation with similar researches picked up from the literature is recommended.

Scatter plot evaluation Taylor diagram are needed to be generated for the attained results.

The discussion phase is most likely a statistical display. I did not see a hydrology interpretation. You need to focus on this. Bear in mind, this is highly essential for your research record.
Additional comments: i. The significant of inflow modeling
ii. The main engineering problem associated with the inlfow problem.
iii. What are the basic methodologies introduced and what are their drawbacks?
iv. What kind of newly developed machine learning models as an intelligence models for inlfow forecasting.?
v. What are the main limitation of these AI models emphasis the hybridization with bio-inspired optimization algorithms?
vi. I do expect the authors report a comprehensive literature review on this. River flow forecasting has been conducted using over 200 scientific researches.
Literature review is very much poorly presented. Hence you need to recall some valid researches and try to discard the conferences papers. Here I suggest you to consider those researches for your literature.
https://www.sciencedirect.com/science/article/pii/S0022169419302471
https://link.springer.com/article/10.1007/s10489-018-1232-0
https://www.sciencedirect.com/science/article/pii/S0378377418311831
https://link.springer.com/article/10.1007/s11269-018-1909-5
https://link.springer.com/article/10.1007/s11269-016-1408-5
https://www.sciencedirect.com/science/article/pii/S0022169418309545
https://www.sciencedirect.com/science/article/pii/S0022169417306029
https://www.sciencedirect.com/science/article/pii/S0022169416305893
https://link.springer.com/article/10.1007/s12665-018-73768
https://www.sciencedirect.com/science/article/pii/S0022169419302513


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: IMPROVING THE PREDICTION ACCURACY OF RIVER INFLOW USING TWO DATA PRE-PROCESSING TECHNIQUES COUPLED WITH DATA-DRIVEN MODEL
Review round: 1
Reviewer: 2

Output:
Basic reporting: The most relevant aspect of the paper is the application of a new hybrid technique for river inflow prediction. This research is not so technically sound in methods and analysis. The results are not fairly presented. There are several factors which detract the quality of the manuscript. I cannot recommend publication of this work in its present form. The authors need to address the queries and incorporate the suggestions given below.
Experimental design: 1. The authors claim to develop several ensemble or hybrid models for the task of river inflow prediction on daily time scale. They adopt several signal processing methods namely, Variational Mode Decomposition (VMD) based on a Singular Spectrum Analysis (SSA); Empirical Bayes Threshold (EBT); Empirical Mode Decomposition (EMD); and Ensemble EMD for pre-processing of river inflow data. However, it is to be noted that, the manuscript lacks a detailed methodology on how all these techniques were implemented and used for river inflow prediction using SVM.
2. The paper suffers from many technical errors in using the SSA. I have a serious concern on the choice of SSA. It seems like, the authors have decomposed the complete time series at a stretch. It is important to note that performing VMA based SSA decomposition on time series must be independent for each partition periods (training, testing phases) in order to avoid incorporation of information from future data (that is to be used in the testing period) in the training phase.
3. I strongly recommend the following paper for consideration of the authors which gives details on the incorrect usage of SSA. Du, K., Zhao, Y., & Lei, J. (2017). The incorrect usage of singular spectral analysis and discrete wavelet transform in hybrid models to predict hydrological time series. Journal of Hydrology, 552, 44-51.
4. How detailed is description of methods is a key aspect on writing scientific papers. First, note that in the title and in the introduction there are references to novel hybrid modelling and in the section 2 (Lines 123 – 245) there are details related to the mathematical formulations of different methods.  In the framework of any novel research that has been carried out, a detailed explanation about what a hybrid modelling is? should be provided, including its origin and the logic to use it. In respect of the link between methods and results the way hybrid modelling is selected is a little bit dark. The paper seems weak in terms of hydrological discussion on the results. The paper needs to improve on organization, elaboration, documentation of procedures (EMD) and discussion.
Validity of the findings: 1. If I am not wrong nowhere in the text before is told about this parametric characterization of SVM models. Information cannot be placed so out of context. The authors need to shed light on how the SVM parameters (Cost, Epsilon and Kernel parameter) were optimized?
2. I recommend the authors to use advanced model evaluation metrics such as Gross Performance indicator, Taylor diagrams etc. to present the model results.
3. Clarity of Figure 1 needs to be improved.
4. Tables 3 & 4, mention the units of MAE, RMSE and MSE metrics.
Additional comments: The contribution of manuscript is too weak which unfortunately convinced me to reject the 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: IMPROVING THE PREDICTION ACCURACY OF RIVER INFLOW USING TWO DATA PRE-PROCESSING TECHNIQUES COUPLED WITH DATA-DRIVEN MODEL
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors revised the manuscript accordingly.
Experimental design: The methodology is clearly reported.
Validity of the findings: The finding of the research support the main research set-up.
Additional comments: The authors revised the manuscript accordingly.


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: DYNAMIC CHANGES IN PRESCRIPTION OPIOIDS FROM 2006 TO 2017 IN TEXAS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article has potential, but it needs much work.
Footnote 1 cites a survey. I believe the correct survey should be the National Household Survey, Footnote 3 refers to areas with large military bases. I have never seen this article and wonder if there are not more robust research on the increases in population using pills. Suggest using local NSDUH or poison control data to be more meaningful. For footnote 5, you might find data showing that the number of methadone programs and population in treatment has been low, in comparison to NSSATS numbers. Footnote 6 refers to minorities. What proportion of heroin admissions were minorities and did NSDUH give any data on use by minorities. First paragraph should reflect the actual situation in Texas and not use data that does not seem relevant.
The second paragraph is incorrect. The State’s Prescription Monitoring Program was housed in the Department of Public Safety until September, 2017, when it was transferred to the State Board of Pharmacy. The Texas PDMP is now evolving a true PDMP. Suggest you reframe that paragraph to explain that the numbers until 2017 are describing an operation that really was not a PDMP.
The discussion about uninsured population is really not relevant. What about the numbers for states such as West Virginia? Be relevant.
Where did the numbers on methadone in Texas come from? I suspect the federal funding for opioid treatment was the driver of the increases in numbers,  What is the source of the numbers in Figure 1B? Contrary to your statements, Texas has had a long history of not providing MAT. Provide numbers on how many methadone programs started because of the federal money, the increase in patient levels, how many physicians became qualified to prescribe buprenorphine, and the number of pharmacies that were dosing buprenorphine before and after the law changed.
Please describe how you accessed the ARCOS data. Did you have access to the ARCOS data set? If not, where did the numbers come from? How did you get the estimates for the various counties?Also, please make sure that statement cited n footnote 5  is a correct, given Texas did not have a true PDMP.
Do comment on the use of the federal funding to provide additional treatment. I seriously doubl that it would have happened without the federal initiative.
Figure 3 intrigues me. The counties that have historically had the highest use of hydrocodone and other opiates show decreases in Figure 3.  I have followed some of those counties for years and been unable to fighre out the high use of opiates in them. Figure 4 raises questions because some of the areas with the highest “heat” already have active methadone program Other areas which did not have methadone now show high increases in buprenorphine.  Can you cross-validate the increase in number of physicians now approved to prescribe buprenorphine with the increase in the drug?  Use of NSSATS may help explain this finding.
This article takes a number of statements out of context with no relationship to the drug scene in Texas. It is pretty apparent to this reviewer that your knowledge of drug trends in Texas is limited. Your idea for a paper is interesting, but this paper is not relevant to the situation in Texa sand it did not provide me with an understanding of why some opioids decreased and others increased.
Experimental design: see above
Validity of the findings: see above
Additional comments: My comments on #1 include my concerns about the overall article.


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

Input:
Title: DYNAMIC CHANGES IN PRESCRIPTION OPIOIDS FROM 2006 TO 2017 IN TEXAS
Review round: 1
Reviewer: 2

Output:
Basic reporting: •	In this article, opioid use in Texas is discussed which is of great importance as it is one of the States with the lowest opioid use and misuse. The introduction gives a good picture of opioid use in Texas however it would be interesting to consider people reading the article that may not well understand the American health system. For example, when discussing the lowest insurance rate (I guess we are talking health insurance here), it is important to highlight what it means in terms of Americans accessing the health system. In addition, a better link between sentences would benefit the introduction.
•	Line 133: The sentence “Hydromorphone doubled while the increases in oxycodone was double the population” can be rewritten to increase clarity. I would suggest maybe add the number to the text to make it clearer, i.e.: Hydromorphone doubled from 2006 to 2017 while the increases in oxycodone of 46.6% was double the population change (+21.1%).
•	Figure 1 – It is not clear to me why in the figure 1B oxymorphone and buprenorphine were not included.
•	Figure 2 – I suggest mentioning that meperidine MME is too low to be visualised in the graph (it is colour-coded but not shown in the graph)
•	Figure 3 and 4 – Is it possible to use different color schemes in these figures? It would be easier to visualise what the heat map means if decrease was shown in a cool color and the increase in red (as it is).
•	Line 163: “…due to concerns about methadone and NTPs…”  for clarity may be changed to “…due to safety concerns about the use of methadone in NTPs…”
•	Line 165: “The rescheduling of hydrocodone…”, a further explanation of how was it rescheduled would help readers starting to work in the opioid field or not used to the scheduling system in the US.
•	Line 176: The sentence “Prescription opioids were the first opioid…” should be rewritten for clarity and note that the ¾ claim is for 2000s data (it seems to have a shift since 2010). This data may be better presented discussing the shift from heroin to prescription opioids as the first opioid of abuse since the 1990s.
•	Line 190: What does PMP (prescription monitoring program – add acronym to line 187) and CFR (Code of Federal Regulations?) stand for? 42 CFR part II is important to protect patient confidentiality. I am not sure that the way the sentence is written reflects the importance of the code. It sounds like it is disadvantaging the patient as they are “denied the benefit” of PMP, is it true? If so, can you further explain? Or is it disadvantaging data collection?
•	In relation to Figure 3A, further discussing the difference in opioid use and misuse in between urban and rural areas would benefit the article. Keyes K et al 2014 discuss possible reasons why these differences may occur.
•	Comment on how illegal opioid market, not accounted for in the database, can also affect the dynamic changes in prescription opioids. According to the 2018 National Drug Threat Assessment, Fentanyl and other synthetic opioids are primarily sourced from China and Mexico. According to figure 2B, Fentanyl decreased in the period researched. Could it be a reflection of availability from other sources?
•	You may want to include in the discussion comments on the recent Law approved in Texas restricting the opioid length of supply for acute pain and how do you think this fit with the data presented.
•	Reference 24: add access date
Experimental design: This article presents an original primary research within the Scope of the Journal. The aim of this research is clear and meaningful due to the opioid crisis in the US. It is crucial to research States where the opioid crisis isn’t as bad to support policies and interventions on the more affected States.
Validity of the findings: The database used is comprehensive although limitations exist, and they were raised in the discussion session. If the data base is publicly accessible, a reference to it with a link should be added to the reference list.
Aims were met and succinctly mentioned in the concluding session. However, the first part of the conclusion is not the conclusion of this study, therefore it needs to be rewritten to clarify the conclusion of this study compared to statements based on previous studies or assumptions.
Additional comments: I commend the authors for this work. The manuscript is well written and mostly easy to understand. The main weakness is at some points it feels like the information was not joined together.


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: ENVIRONMENTAL DNA DETECTION AND QUANTIFICATION OF INVASIVE RED-EARED SLIDERS, TRACHEMY SCRIPTA ELEGANS, IN PONDS AND THE INFLUENCE OF WATER QUALITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, I think that the authors are direct to their points and the introduction is well-written. I have suggested some corrections and made more specific comments below.

L44-47: I might suggest that you flip those sentences, so you start by explaining what eDNA is and then transition into how it can successfully be used to detect a variety of aquatic organisms. Additionally, as Peer J is an open-source journal, some readers may not know exactly what eDNA is or the capabilities of eDNA.

L45-46: You use the reference style of “Goldburg and Strickler, 2015”, but at L50: you use the style of “Thomsen & Willerslev 2015". Please double-check citation style throughout.

 L47: You do not always need to filter litres of water to detect an organisms’ eDNA. I would suggest that you revise to say something about how you can filter eDNA from water and detect organisms without necessarily needing to directly observe them. As this is one of the most compelling part of eDNA, that it can often detect low-abundance organisms.

L55: Uncertain if this should be “trematode” or “trematodes”?

L56-57: I would argue that we cannot always find relationships between the amount of eDNA and the biomass or abundance. Some studies can, some cannot. In my opinion, the fact that here you claim to find that relationship is one of the key findings of this paper (although see below for additional comments on this claim).

L64: Please add a reference here.

L66-68: I think a reference is necessary here. I could not seem to find this reference cited in your “References” section.

L68-71: I think a reference or references are necessary here.

L87-88: This sentence is worded awkwardly and I believe there are other inhibitors other than humic acid. Perhaps, you could revise to read something like, “For example, humic acids are known to inhibit DNA polymerase in PCR and therefore could directly impact eDNA detection.”

L100: I would suggest to delete the “ ,” after (qPCR) and say something like “paired with visual observations of turtles.”
Experimental design: L101: 100 ponds! That’s quite a robust sample size! But then later the number of detections is concerning. If red-eared sliders are a common invasive species in Japan, please add in your discussion more about why you did not detect more turtles.

L118-119: Uncertain exactly what “From a point within each pond, …” means. Please explain how water sampling locations were selected more. Randomly? As water/conditions allowed?

Why did you chose to limit your sample size to only 1, 500mL bottle per pond? That seems very low replication.

L153-155: Please provide the ascension number from GenBank or some additionally information on what you used as your positive turtle standard. This will be important for other seeking to use this assay in the future.

L164: 55 cycles is a long time. Please provide additional information on why the qPCR methodology is so long. It’s not necessary unheard of, but without information about detection levels (see comment below), I don’t know why 55 vs. 40?

L:166-167: Please provide more information about your limit of detection (LOD) and limit of quantification (LOQ). It is not stated here how low your assay was capable of detecting red-eared slider DNA. This limits the ability of future eDNA researchers to use this assay.
Additionally, your R2 values and %efficiency are low and I’m concerned about your ability to accurately measure eDNA concentration. Please provide additional information or references to support this. For example, see Hinlo et al. 2018 (Performance of eDNA assays to detect and quantify an elusive benthic fish in upland streams. Biological Invasions 20:3079-3093.) examined quantified eDNA, but reported R2 values of 0.999 and %efficiency of 96.908.

L174: Unclear why a PCR inhibition removal kit (e.g., Zymo Research, OneStep PCR Inhibitor Removal Kit) was not used on your same samples to examine if you directly had inhibition in your turtle eDNA that could influence your results. Please explain.

L218-220: Please explain why you used linear models and not linear mixed-effect modeling with site a random variable? Then use AICc model selection on only the variables that are not co-linear?

L232: Did you sequence confirm that the successful amplifications were in fact red-eared slider? Even just one or two eDNA samples to make sure that your assay works (you tested on cyt b, but what about your positive eDNA detections?). Sometimes and even with the specificity of a probe, you can still amplify non-target organisms. Please clarify how you are certain that positive detections are not "false-negatives" from primer-dimer.

Do the authors conclude that 1 amplification by Ct 55 is an amplification and the take the mean of that? Please clarify.
Validity of the findings: The authors did an excellent job in the transparency of their data-a lot of information in contained in the Tables and Figures.

However, I have serious concern over the authors’ abilities to correctly determine eDNA concentrations (see above about R2 and % efficiencies). Further, I would suggest that this discussion needs to be revised to discuss the authors’ shortcomings. I don't agree that not-finding a significant result or no trend means that this paper is unworthy of publication. Instead, I suggest that others are more straight-forward in not making large conclusions from small data and that they should discuss what future research could be done.

L235: 100 lakes sampled and being able to only determine red-eared sliders in 11 ponds, I am uncertain how your N=20. If 10 ponds detected with visual, 9 with eDNA, I’m confused how that is 20.

L231-235: I would be hesitant to infer much from detection in only 10 ponds.

L257-259: I disagree that you can make that conclusion based on only being able to detect eDNA at 9 ponds. Your results actually indicate that you’re able to detect the turtles better with visual observation.

Please revise this to discuss more about the short-comings of your paper and how future research can add to your study.

Additionally, you discuss in the introduction about how visual surveys are not the best method to detect these turtles. Maybe eDNA sampling at one point vs. visual surveys is not the comparison that is needed and in the future, maybe more work should be done to compare other samplings methods vs. eDNA (at with more than n=1 at each of your ponds).

L295: I believe this should be “Barnes et al. 2014”?

L292-308: The relationship between Chl a and eDNA is interesting, however, I would suggest that you again state that eDNA was only detected in 9 ponds.

Tables/Figures:

Table S1: Please explain why some values are missing. Were these not measured? Or were they removed from analyses?

Figure 2: The eDNA concentration is not log-transformed here, but is in Figure 3? Please explain that, as I do not see a trend here that you are concluding. Instead this relationship seems to be largely driven by the point of 10,000 red-eared turtles. Further, were you able to count 10,000 turtles?

Figure 3: I would suggest that you add regression curve + 95% CI to all graphs. I understand that only Chl. a had a significant effect on eDNA copy number, but it looks odd to not show the other trendlines.

Figures 2 and 3: Suggest to add r2 values and p-values to figures?
Additional comments: It is very exciting to see a turtle eDNA paper, as reptile eDNA is understudied! Further, more studies that directly examine the relationship between number and eDNA are necessary, as there do appear to conflicting results dependent on organism and system studied.

However, I have serious issue with the overstatement of conclusions that the authors report here, largely due to the small number of ponds where turtles were actually collected. I am also hesitation to conclude much give the low R2 values and wide-range in % efficiency. I would suggest that the authors specifically address those concerns, as this assay and study does present interesting findings that other researchers would be very interested in reading!


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: ENVIRONMENTAL DNA DETECTION AND QUANTIFICATION OF INVASIVE RED-EARED SLIDERS, TRACHEMY SCRIPTA ELEGANS, IN PONDS AND THE INFLUENCE OF WATER QUALITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Literature references, article structure, figures, tables, and data sharing are all sufficient. The English needs clarification or correction in some cases (many of which I have pointed out in the General Comments). Hypotheses are not explicitly stated, but could easily be added to the last paragraph of the Introduction.
Experimental design: Methods are generally well-described, but a few details need to be added as noted in the General Comments section. One statistical analysis, the testing of eDNA concentration against water quality metrics, is confounded by varying slider turtle abundances. I have provided suggestions for overcoming this issue in the General Comments section.
Validity of the findings: Conclusions about the effect of water quality metrics on eDNA concentrations are based on a confounded analysis as mentioned above and in the General Comments. This major issue needs to be corrected before any conclusions can be made. The other conclusions in the manuscript are fine.
Additional comments: The manuscript "Detection of environmental DNA of the invasive red-eared slider in ponds for evaluating their distribution with comparison to water quality" by Kakuda et al. presents the results of a survey of turtle presence/abundance and water quality in 100 ponds in Japan. The authors compare visual versus environmental DNA (eDNA) survey results, test eDNA samples for PCR inhibition, and test the relationship between eDNA concentration and various water quality parameters. The authors find evidence for slide turtles in 30 out of 100 ponds, with 10 ponds having just visual detections, 9 ponds having just eDNA detections, and 11 ponds having both visual and eDNA detections. Only one pond had evidence for PCR inhibition, which was surprising to me. Finally, the only significant relationship between eDNA concentration and water quality was a negative relationship with Chlorophyll a. The authors conclude that eDNA is as good as visual observation for slider surveys, and that Chl. a may increase DNA degradation rates in the field, but more research is needed for the latter.

This is a very interesting study and provides valuable data the increases our understanding of the field “ecology” of eDNA. The introduction includes relevant literature citations. The methods, while sometimes needing more details (see Minor Comments below), are sound. My two major issues with the manuscript are 1) the writing, which is sometimes unclear (see Minor Comments below for some suggestions), and 2), more importantly, the eDNA concentration/water quality analysis. For the latter, the authors use linear regression to test relationships between eDNA concentration and various water quality parameters. However, they neglect to include slider turtle abundance into this analysis, even though they clearly show that slide turtle abundance as measured visually is strongly correlated with eDNA concentration (Lines 233-235, R2=0.48, p<0.001). It could be that the negative relationship that they find between eDNA concentration and Chl. a is entirely due to a negative relationship between slider turtles and Chl. a. To deal with this confounding issue, the authors could include visually-estimated slider turtle abundance data as a covaraiate, but this would limit them to just 11 data points, which I'm not sure provides enough statistical power. They may be able to add 9 more data points by assuming low abundance in the eDNA detection-only sites. But a new analysis that considers slider turtle abundance is clearly needed in order to properly evaluate the Chl. a result.

Below I note some minor comments that I hope will improve the readability f the manuscript and replicability of the study. I have not made suggestions for the Discussion section as I think that needs to be completely re-written after a new eDNA concentration ~ water quality analysis is done.

Minor Comments

Title
This is a confusing and long title. I suggest something like “Environmental DNA detection and quantification of invasive red-eared sliders, Trachemy scripta elegans, in ponds and the influence of water quality”. The scientific name for red-eared sliders needs to be included in the title.

Abstract
L26. Environmental effects on eDNA don't necessarily prevent accurate estimates, but they do complicated estimates. I would change “prevent” to “complicate”.
L28: Change “Also” to “Further”
L29-32: Suggest rewrite “Here we examined the relationship between the visually-observed number of red-eared sliders (Trachemys scripta elegans) and eDNA concentrations across 100 ponds. Additionally, we evaluated the effect of water quality on slider eDNA concentration in these ponds.”

Introduction
L46: Change to “Environmental DNA consists of DNA fragments...”
L47: Some eDNA surveys use only 250ml, so change to “a few liters or less...”
L49: Change short to “shorter”.
L56-57: Delete sentence “We can detect the eDNA of various taxa...” because it's redundant with 1st sentence.
L60: Change “detect” to “determine”.
L67: The statements “which was 64% of all turtle abundance” is confusing – were red-eared sliders 64% of all invidual turtles observed?
L76: Change “the previous eDNA studies” to simply “previous eDNA studies” (delete “the”)
L87: Rewrite “In fact..” sentence because it is awkward. I suggest “Inhibition of eDNA detection can be caused by compounds such as humic acids that inihibit the DNA polymerase used in PCR.
L90-94: Mention that both the Strickler et al. and Barnes et al. studies are mesoscosms earlier. I suggest: “Mesocosm studies have found that eDNA degradation decreases with low water temperatres, low UV-B levels, and high PH (Strickler et al. 2014) and with increasing biochemical oxygen demand, Chlorophyll a concentration, and total eDNA concentration (Barnes et al., 2014).”
L95: Change “conclude on” to “understand”
Line 100: Change “Additionally, we aimed to” to “Specifically, we evaluated”.
*Also, you should include a hypothesis/es in the last paragraph of the Introduction

Methods
L109: How/why were these ponds chosen? Also, you talk about city, rural, and mountain areas but do not consider these in the analysis or the discussion. Were there any interesting patterns amond these three area types?
L118: What time of day were visual surveys done? Was weather considered as a cofactor, since turtle bask more on sunny days?
L128: Would be helpful to specify again that 500mL was filtered for eDNA collection.
L132: Suggest rewrite sentence to “For eDNA samples, the 'cooler blank' and a 'filter blank' consisting of DNA-free distilled water were filtered in the same was as the samples.”
L137-138: The statement “in the 100 samples without six data is confusing”. I suggest changing to “data missing from six of the 100 sites”. Also, from the table is looks like more than six sites are missing water quality data, so please check this number.
L166: Please describe how you estimated your standard curves.
L181-182: Suggest rewrite “To test for PCR inhibition in the DNA samples, 1μl of plasmid including the cytochrome b gene from Trachurus japonicus..”. Also, what did you mean by “1.5 X 104 copies”, why not say 156 copies?
L226: What R package was used for the VIF analysis? Need to include that.

Results
L232: Delete “Of the 11 ponds...” because this suggests that the 10 visual-only and 9 eDNA-only ponds were included in the 11 visual + eDNA detection ponds. Rewrite these first two sentences to something like “Of 100 samples ponds, we detected red-eared sliders in 11 ponds with visual and eDNA surveys, in 10 ponds with just the visual survey, and in 9 ponds with just the eDNA survey (Table 1).”


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: ENVIRONMENTAL DNA DETECTION AND QUANTIFICATION OF INVASIVE RED-EARED SLIDERS, TRACHEMY SCRIPTA ELEGANS, IN PONDS AND THE INFLUENCE OF WATER QUALITY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Some minor issues with English grammar that can easily be addressed by the authors.
Experimental design: No comment-
Issues with sampling design (one sample/pond) are stated as downfalls in the discussion section.
Validity of the findings: No comment-
Findings confirm previous research and present novel results on the relationship between Chl a and eDNA.
Additional comments: It seems that the authors have taken appropriate steps in revising the manuscript to address the concerns of the reviewers. Overall, the paper is generally well-written and would likely be of interest to the eDNA community.

I suggest minor corrections to the English language to clarify grammar and assist with sentence flow.
L39: I suggest "inhibition" not "inhibitions"

L42: Suggest stating, "...the ponds, as microbial activity is thought to decrease eDNA persistence." It was not immediately clear what was being stated here.

L95: Suggest revision to read something like, "Mesocosm studies have found that a number of factors are important to decreasing the degradation of eDNA, such as cooler temperatures, higher BOD, increased Chl a, (Barnes et al., 2014; Strickler et al., 2014)."

L99: Suggest revision to say, "Because the effect of Chl a was different in laboratory vs. in situ experiments, more evidence is needed to understand the effects of water quality on eDNA degradation."

L 101-103: Can delete? This is repetitive to sentence before.

L196: One copy/µL?

L240: Revise to "...were measured at 630..."

L305-308: Suggest revision for clarity to be something like, "In the nine ponds where only eDNA detected turtles, we suspect that turtles were too rare to be observed by visual observation. In addition, eDNA methods have often been cited to detect rare and cryptic species (Barnes & Turner, 2016)." Then delete the next sentence as it is repetitive (Lines 307-308).

L310: Add "s" to suggest

L313: Delete this empty space and bring the last sentence of the paragraph up to line 313.

L316-317: Suggest revision to read something like, ", as eDNA produced false, non-detections when red-eared sliders were present."
L319-321: Revise to, "Others, such as Takahara et al., (2012) have found positive correlations between eDNA concentrations and the biomass using both aquaria and outdoor experiments."

L322: Delete "ed" from supported to read "support"

L323-324: Suggest rearranging sentence to read, "Although few studies examining the effects of biomass or density on eDNA have used reptiles, Lacoursière-Roussel et al. (2016) found eDNA was highly correlated to the relative abundance of wood turtles. Then I suggest deleting the next sentence (L325-327) as this information has already been stated in the above sentences.

L327: Suggest replacing "for estimating" to "to estimate"

L334: Inconsistency in how pond No. 33 is labeled, see Line 332. Revise to be same format.

Line 335 and paragraphs on Chl a. Do you think that the dark tannin colored water is just less influenced by UV-B rays and temperature was cooler (unfortunately there is no measure of temperature on this) and thus, eDNA persists longer? Not necessarily a factor of microbial activity?

L346-349: The authors did a nice job of pointing out that differences between laboratory vs. in situ eDNA studies often find contradictory results.

L367-369: I disagree with this sentence, as you state on Line 360-362 that eDNA was  only detected in 11 ponds more replicate water samples would be useful. In addition, you report at L254 and L260 that visual surveys detected turtles in 10 of the ponds and eDNA was only successful in detecting turtles in 9.

L372-374: Suggest delete sentence on "For eDNA surveys,...", as it has already been stated above.

L 445, 463: Trachemys scripta should be italicized. L496: italicize Cyprinus carpio

Table: Suggest that Table 2 B be a standalone table.


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: ENVIRONMENTAL DNA DETECTION AND QUANTIFICATION OF INVASIVE RED-EARED SLIDERS, TRACHEMY SCRIPTA ELEGANS, IN PONDS AND THE INFLUENCE OF WATER QUALITY
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: The only criticism I have is the statement on Line 367-368 ("This result contrasts with other studies... which suggested that multiple sampling points would increase the ability to evaluate distribution and abundance..."). These results do not contrast with these studies, since you did not compare multiple versus single samples per site. I would guess that multiple samples per site would increase your ability to detect T.s. elegans, but this was not tested in this study. I would reword this sentence accordingly.
Additional comments: no comment


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

Input:
Title: IN VITRO HISTOMORPHOMETRIC COMPARISON OF DENTAL PULP TISSUE IN DIFFERENT TEETH
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This study investigated the histomorphometric properties of dental pulp tissues obtained from different tooth types. The effect of age and sex was also reported. The results showed differences in the weight and also cell density of the dental pulps among the groups. The authors suggest using these parameters to predict the availability and quality of dental pulp stem cell isolation. There are some concerns regarding the aim and methodology of the present study.

There has been a lack of standardization in tissue samples in the present study. It is hard to standardize the tissue volume when determining the cell number due to the differences in pulpal volume in different tooth types and different donors. In addition, the authors did not include the scale bar to the histological slides and the image resolutions are too low for the evaluation. Regarding the isolation of dental pulp stem cells, undoubtedly younger patients are a more suitable candidate due to the decreased pulp volume and cellular contents by aging. Although there is a decreased cellular intensity by aging shown by the previous and present results, the percentage of DPSCs might not be changed. In this point, the isolation of DPSCs and also the evaluation of their biological properties such as proliferation and differentiation properties are important. Previously, Bressan et al (Plos One, 2012) reported that DPSCs isolated from 16-66-year-old patients could retain the proliferation and differentiation potential. In the present study, the authors examined the dental pulp tissues derived from young donors up to 25 years, which any difference regarding the percentage and properties of DPSCs would not be expected.

Regarding the correlation between the histomorphometric properties of the dental pulp tissues and the isolation of cells would be further investigated with the determination of biological properties of the isolated DPSCs. In this point, the statement cannot be made by the results of the current study.


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

Input:
Title: IN VITRO HISTOMORPHOMETRIC COMPARISON OF DENTAL PULP TISSUE IN DIFFERENT TEETH
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled "In vitro histomorphometric comparison of dental pulp tissue in diﬀerent teeth" reports a novel methods that demonstrated a signiﬁcant positive correlation noted between length and width of the dental pulp with age in both males and females. I believe the manuscript still requires some minor revision before further consideration for publication.
Experimental design: The authors has described the experimental design very well. However some minor changes that has been mentioned in the general comments are needed to be completed.
Validity of the findings: The current manuscript demonstrates that the mean dental pulp weight of supernumerary and third molar teeth was greater than deciduous and premolar teeth and finally, the histological analysis revealed that the apical portions of dental pulp in supernumerary and premolar teeth have the highest cell density and provide a greater number of cells for in vitro culture. Overall, the current finding has important applications in the dental pulp stem cells isolation.
Additional comments: 1.	The author should change the abbreviation “DP” in the results section of the abstract to the full word and use the word first time in full and then only in abbreviation.

2.	At line 55, the author has used the cells name in full words as well as in abbreviation “Dental pulp stem cells (DPSCs)” and the same format has repeated at line 141. The same mistakes have repeated throughout the manuscript. Please follow the proper format.

3.	Materials and Methods: Tissue removal and processing section, “Tooth samples were longitudinally cut using a micromotor”. The authors need to confirm whether the pulp has been little bit damaged or remain intact while using the microcutter? Please mention the diameter of the cutter.
4.	Figure 2A, the DP weight of only one male is more than 100mg whereas all the other DPs weights are showing less than 30mg. Why there is a huge difference in weight in DP weight of male? Explain them precisely.

5.	Figure 2 legends: “Statistical signiﬁcance was deﬁned as p ≤ 0.05”. However there is no symbol representing statistical signiﬁcance in the figure. Add the symbols.

6.	Figure 3 legends: “Values were calculated from three slices of each of the 10 teeth in each group and are expressed as the mean ± standard deviation. *P ≤0.05”. Add the symbol * in the figures?

7.	In “Figure caption section” more detail description are needed to add for making clear easily follow.
8.	There are grammar errors in the manuscript and the tenses are confused. Therefore, the manuscript should be thoroughly edited.
9.	In “Table caption section” more detail description are needed to add for making clear easily follow.
10.	I advise the authors to correct the grammatical errors because there are a large number of grammatical mistakes or odd turns of phrase, and too many for a reviewer to list.


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: IN VITRO HISTOMORPHOMETRIC COMPARISON OF DENTAL PULP TISSUE IN DIFFERENT TEETH
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank the authors for their revision according to the previous comments, but still, some revisions are required.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Page 7, line 61, please remove the sentence “This fact motivates our research and concomitantly ensures the novelty of the results”.

Page 8, line 77, please add PBS brand.

In the results section, please add the P values (p<0.05 or p>0.05) after each sentence.

For the histological images, please label dentin and pulpal side of the samples. The images are still not well enough for the resolution.

In Table 2, the statistical comparisons should be also performed among coronal, cervical, middle and apical part of each tooth type. According to that, the ideal cell location might not be limited to the apical region of supernumerary and premolar teeth.


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: IN VITRO HISTOMORPHOMETRIC COMPARISON OF DENTAL PULP TISSUE IN DIFFERENT TEETH
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript entitled "In vitro histomorphometric comparison of dental pulp tissue in diﬀerent teeth" reports a novel methods that demonstrated a signiﬁcant positive correlation noted between length and width of the dental pulp with age in both males and females. I believe that the manuscript in the current format have adequate significance to be of interest to the readers of the journal.
Experimental design: The paper is well presented and the experimental design used for the study is acceptable.
Validity of the findings: The current manuscript demonstrates that the mean dental pulp weight of supernumerary and third molar teeth was greater than deciduous and premolar teeth and finally, the histological analysis revealed that the apical portions of dental pulp in supernumerary and premolar teeth have the highest cell density and provide a greater number of cells for in vitro culture. Overall, the current finding has important applications in the dental pulp stem cells isolation.
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: VCF2POPTREE: A CLIENT-SIDE SOFTWARE TO CONSTRUCT POPULATION PHYLOGENY FROM GENOME-WIDE SNPS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript requires some careful proofreading at the very least. Among other typos:
- people from Colombia are Colombians, not Columbians
- in the abstract, we can read gnome, instead of genome
- the "Simons Genome Project" should read as the "Simons Genome Diversity Project".
- etc

A few references are missing, even when programs are cited, including for bwa, MUSCLE and samtools.

Some sentences are simply not true, such as "these programs are not suited for
large-scale whole genome data" (second sentence in the abstract). It is plenty of tools for inferring NJ trees, most notably including ngsDist or plink (to calculate pairwise distances from NGS data), and fastme to infer a tree from a matrix of pairwise distances.

Some aspects are not totally clear from the manuscript, regarding the calculation of pairwise distances. I expose them in the "general comments to the author"
Experimental design: The main motivation of the paper is to present another user-friendly tool for phylogenetic inference from a VCF file. Although not novel, my opinion is that user-friendly tools are always welcome, especially if they can be easily run as javascript client applications. I would suggest to add some extra functionalities, such as reading compressed VCF files (users do not want to uncompress huge files in their personal computers), calculating genetic and drift distances, the possibility of re-rooting the tree, or to display the branch lengths on top of the corresponding branches.
Validity of the findings: The authors claim that their implementation is faster than others. However, I find some issues a bit unclear:

- the running time depends on two independent variables (i) the number of individuals, and  (ii) the number of sites. The computational time is expected to grow quadratically with the number of individuals, because it involves calculating all pairwise distances. I suggest the authors explore their running times as a function of these two variables, separately. For a reader with little expertise in bioinformatic analyses, the file size (in Gb) is a bit cryptic.

- In fig2, the running time seems to grow exponentially for vcf2poptree, while only linearly for the solutions implemented in Galaxy. I suspect then that at some point Galaxy will perform better than vcf2poptree. It is interesting to know when.
Additional comments: Subramanian and colleagues present a user-friendly tool, vcf2poptree, which runs as a client application in personal computers. Although many scientist are nowadays comfortable with existing tools, even if they require some basic bioinformatic skills, I truly appreciate the effort of adapting analytical tools to wider audiences. However, I have serious issues with the clarity of the manuscript, as is in its present form:

1) I think some statements are simply not true, or at least not correctly phrased, such as "these programs are not suited for
large-scale whole genome data" (second sentence in the abstract).
It is plenty of tools for inferring UPGMA and NJ trees, most notably including ngsDist or plink (to calculate pairwise distances from NGS data), and fastme to infer a tree from a matrix of pairwise distances. There are also wonderful and more sophisticated models for calculating trees with potential admixture, such as TreeMix. All these tools scale perfectly well to NGS data. Please, cite all relevant literature.

2) Regarding the calculations of pairwise distances:
- What is the distance between two heterozygous individuals? 0.5 (i.e. two out of the four chromosome comparisons are different)? or 0 (i.e. as the allele frequency is the same in both individuals)? This is very important, because the former case will provide "genetic distances", while the latter "drift distances" (changes in the allele frequency). Conceptually different, both are correct. It would be a good idea to implement both.
- What does happen with non-SNP variants, such as InDels, or with SNPs not passing quality filters? Are they filtered out?
- Does it read polymorphic sites with more than two variants?
- Does the program take into account uncertainties in the genotype calls, if present in the VCF file (e.g. GP field)?
- The NJ method implemented corresponds to the classical algorithm, or includes the improvement presented in Gascuel 1997 MBE?
- Why not implementing at least the Jukes and Cantor correction? It is just adding an equation in the code.
- The authors provide a textbox to specify the total genome size. This is understandable, because the VCF only contains polymorphic positions (usually), and genetic distances are expected to be given by site. However, it is very common to filter out some SNPs during NGS quality processing. The positions corresponding to filtered SNPs should be subtracted from the total genome size. They cannot count neither as monomorphic nor polymorphic, because their status is precisely ambiguous. It is the same for positions that, for whatever reason, are not successfully sequenced (depth = 0). They also need to be subtracted from the total genome size.

Please, clarify all these in your manuscript. As users, we need to know what we are calculating to better interpret the results.

3) Running times (fig 2):
- the running time depends on two independent variables (i) the number of individuals, and  (ii) the number of sites. The computational time is expected to grow quadratically with the number of individuals, because it involves calculating all pairwise distances. Although it is implicitly discussed in the main, I suggest the authors regenerate Fig2, evaluating running times as a function of these two variables, separately. For a reader with little expertise in bioinformatic analyses, the file size (in Gb) is not informative, and I would like to see if the tool is appropriated for analysing hundreds or thousands of individuals.

- In fig2, the running time seems to grow exponentially for vcf2poptree, while only linearly for the solutions implemented in Galaxy. I suspect then that at some point Galaxy will perform better than vcf2poptree. It is interesting to know when.

4) I also suggest you carefully proofread your manuscript, as I catched some typos:
- people from Colombia are Colombians, not Columbians
- in the abstract, we can read gnome, instead of genome
- the "Simons Genome Project" should read as the "Simons Genome Diversity Project".
- etc

5) Others:
- A few references are missing, even when programs are cited, including for bwa, MUSCLE and samtools.
- As a potential user, I would like see some extra functionalities, although I understand some require substantial efforts. For example, reading compressed VCF files (users do not want to uncompress huge files in their personal computers), calculating genetic and drift distances, the possibility of re-rooting the tree, or to display the branch lengths on top of the corresponding branches. Also a bar showing the status of the calculations would be nice for large files (progress bar or similar).

Thanks for your effort. I prefer to sign my reviews. My name is Pablo Librado


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: VCF2POPTREE: A CLIENT-SIDE SOFTWARE TO CONSTRUCT POPULATION PHYLOGENY FROM GENOME-WIDE SNPS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Excellent manuscript. It is indeed clear with adequate references and excellent results and comparisons.
Experimental design: Well suited for the software article. Direct comparisons are provided.
Validity of the findings: Meaningful comparison provided. All the data are standard and tools are standard.
Additional comments: An important need is addressed. The new software has the potential to become high impact.


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: VCF2POPTREE: A CLIENT-SIDE SOFTWARE TO CONSTRUCT POPULATION PHYLOGENY FROM GENOME-WIDE SNPS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors provide a JavaScript-based application, VCF2PopTree, to infer phylogenetic trees (UPGMA or Neighbor-Joining) from variation data in VCF format.

Even though the text is well written in general and both the introduction and literature cited are complete and relevant, some suggestions for improvement are:
1.	Some sentences should be re-phrased to improve clarity: lines 50-51, 74-75, 86-87, 166-168.
2.	The text in the introduction should be written in the present tense in general (e.g. lines 44-47).
3.	I think that whenever the authors refer to inferring phylogenies, they should say “infer” and not “deduce” (e.g. lines 12, 36, 166).
4.	Line 54: add “is” in “an adequate level of computer expertise is required […]”
5.	Lines 140-145: this text is unnecessary
6.	Line 165: I would not refer to VCF2PopTree as “ultrafast”.
7.	Figure 2 is not cited in the text and I think it is irrelevant, mostly because the running time of Galaxy will depend on the queue and vary from time to time.
Experimental design: The pros of VCF2PopTree are:
1.	It is easy to run and does not require any installation.
2.	It is independent of the operating system and browser.
3.	Maybe useful for non-bioinformaticians as a first glimpse of phylogenetic relationships on their variation data.

However, there are some important cons:
1.	It only infers trees by UPGMA and Neighbor-Joining.
2.	It does not apply any correction to the genetic distances (e.g. Jukes-Cantor, Kimura). This is basic and very easy to add.
3.	There is no information on how missing data is handled.
4.	The web-based interface is too simplistic.
5.	It does not have a “running” message. Then, and given that results are not displayed instantaneously even for the test data, the user thinks the application is not working, as nothing happens apparently when you upload the input data.
6.	It takes time even with the tiny example file. I am not sure how stable the application would be with large datasets.
7.	It has few graphical options, basically change from a rectangular to a radial phylogenetic tree, and data needs to be reanalyzed to change from one to the other. I think you should upload input data only once and get both the graphical tree and textual results (Newick and pairwise distance matrices) altogether, and then be able to change graphical aspects instantaneously in one click: e.g. change from rectangular to radial tree, plus other options that VCF2PopTree does not consider now: e.g. include/exclude populations, root on a branch, etc.
8.	Instead of entering the genome size, the user may be allowed to choose a genome version (and VCF2PopTree gets the genome size from it).
9.	The documentation of the package is very poor.

Finally, the largest problem I see is that in order to perform what VCF2PopTree does, I think it would be more convenient to first calculate a distance matrix from a VCF file (e.g. by using the package VCF2Dis), and then open it in MEGA, where you have many more analysis and graphical options.
Validity of the findings: The MS states that the example dataset contains variation data from ten human populations from the Simons Genome Project. Figure 1 shows the results of analyzing the data with VCF2PopTree.

However, by opening the source file you see that variation data is from chromosome 1 only. This should be stated in the manuscript. In addition, in Figure 1 you can see that the Genome size used as input corresponds to the whole human genome size and not the size of chromosome 1, so the genetic distances displayed in the Figure are meaningless. Finally, the input form of the application is displayed three times, one in each part of the figure, and I think this is unnecessary. At most I would include the form once.
Additional comments: No comment


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

Input:
Title: VCF2POPTREE: A CLIENT-SIDE SOFTWARE TO CONSTRUCT POPULATION PHYLOGENY FROM GENOME-WIDE SNPS
Review round: 1
Reviewer: 4

Output:
Basic reporting: The manuscript is well written.

Literature is sufficient.

In my opinion, it is more useful to convert the VCF format into sequence or SNP data format in order to do a number of population structure studies using more sophisticated applications. Conversion of VCF to fasta format is already performed by SAMtools.

The Figure 1 is relevant in order to explain the application. The second Figure is also relevant in the context of having a simple and fast way to make a tree.
Experimental design: no comment
Validity of the findings: A validation of the results using alternative applications should be mentioned in a sentence.
Additional comments: The authors develop a software for making NJ/UPGMA trees from VCF files using a simple user-friendly page. There are many applications focused on constructing phylogenetic trees, whose use a wide number of evolutionary models of divergence correction. Nevertheless, those applications are not using the VCF format as input file but they use sequence or SNP data formats. The main reason to not use VCF format is because in phylogenetic reconstruction studies, the sequences come from different experiments, that is, the different species sequences are not sequenced and mapped together. In this work, the authors only focus on individuals of the same species or very close related species (the distances are not considering evolutionary models for correcting divergence) to identify the (population) structure of the samples. The authors provide a simple application to do this analysis using VCF format.

If the application user only wants to do a phylogenetic tree or a pairwise distance matrix, this application is a fast alternative to other applications. Nevertheless, there are a number of issues that this application should consider:

- the application ask for the length of the complete sequence analysed. This information, that is not provided in the VCF format is key in the estimation of the pairwise distances that is provided by the application. I would recommend to avoid using the total length and simply use the pairwise distance of the total region (without dividing by the total positions). The reason is because this information is hard to obtain and the user can be confused by the total length of the genome/chromosome (which may contain large fragments of not sequenced regions or gaps), giving incorrect distances.

- if the authors want to show an analysis about the relationships  among individuals, it would be also useful to perform a bootstrap analysis in order to give support to the observed structure. Bootstrap may be useful in case using limited SNPs data.

- The application should read compressed VCF files, at least for gzip format. It is quite painful to unzip a big file in a personal computer. There are available libraries that allow to read these kind of files.

- It is not clear how the missing data is used in this application. I assume that a given SNP position will be used only if all individuals are sequenced at this position.

- The application starts to analyse the data once the VCF file is introduced. It implies that the user has to introduce the information in a sequential way (at least the last must be the VCF file). I would add a new button that indicates the start of the analysis, which would allow to modify the previous parameters/outputs.

In summary, the application is very simple, there are alternatives to do the same analysis, but it is a fast way to do a tree, which may be useful for a number of users not interested in sophisticated structure analysis.


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

Input:
Title: VCF2POPTREE: A CLIENT-SIDE SOFTWARE TO CONSTRUCT POPULATION PHYLOGENY FROM GENOME-WIDE SNPS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors addressed most of my comments, in what I think it is a much better version of the software and the manuscript. I suggest an additional implementation, easy but useful. If the authors agree, the paper can be accepted without sending it me back again.

More specifically, the authors state that sites with missing data are filtered out. I personally think this might be a bad strategy. With the number of genomes increasing, it is possible that a potential user wants to compare hundreds of individuals with their tool. The probability of having at least one individual with missing data growths with the number of individuals compared. So, applying complete deletion, as the authors do, would entail trashing too much data.

An alternative is to add an option for pairwise deletion. This is, in each pairwise comparison, we remove a site only if it is missing in one of the two samples being processed. If the site is successfully genotyped in the two samples, but missing in a third one, the program should keep and analyse that site. The total number of positions analysed will then depend on each specific pairwise comparison.

Same for positions with low coverage/quality. Why trashing a full SNP if only one individual has low coverage? Cannot the program set as missing this poorly genotyped SNP, and proceed with the pairwise deletion approach described above? I truly think that if authors do not implement that, the number of users will be clearly reduced.

English could be polished a lot, but I leave the decision to the editor.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors addressed most of my comments, in what I think it is a much better version of the software and the manuscript. I suggest an additional implementation, easy but useful. If the authors agree, the paper can be accepted without sending it me back again.

More specifically, the authors state that sites with missing data are filtered out. I personally think this might be a bad strategy. With the number of genomes increasing, it is possible that a potential user wants to compare hundreds of individuals with their tool. The probability of having at least one individual with missing data growths with the number of individuals compared. So, applying complete deletion, as the authors do, would entail trashing too much data.

An alternative is to add an option for pairwise deletion. This is, in each pairwise comparison, we remove a site only if it is missing in one of the two samples being processed. If the site is successfully genotyped in the two samples, but missing in a third one, the program should keep and analyse that site. The total number of positions analysed will then depend on each specific pairwise comparison.

Same for positions with low coverage/quality. Why trashing a full SNP if only one individual has low coverage? Cannot the program set as missing this poorly genotyped SNP, and proceed with the pairwise deletion approach described above? I truly think that if authors do not implement that, the number of users will be clearly reduced.

English style could be clearly polished, but I leave the decision to the editor. Thanks to the authors for the effort.


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: VCF2POPTREE: A CLIENT-SIDE SOFTWARE TO CONSTRUCT POPULATION PHYLOGENY FROM GENOME-WIDE SNPS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I think the authors have made a great effort in introducing most of the suggestions raised by the referees and that the tool is now much more complete, efficient and user-friendly.

I would suggest just making the web form a little bit more intuitive by re-phrasing some fields and adding some help:
- The filters "Quality score" and "Coverage depth" are filters; then, I would replace the text by "Filter chromosome positions by: Minimum Quality Score [__]  Minimum Coverage Depth [__]"
- The box to choose selected genomes only applies if the option "Selected" is selected. Then, I would hide the box and display it only when "Selected" is selected. There are easy JavaScript commands to do so.
- Similarly, "Genome size" only applies under certain conditions, so I would do as above. In addition, "Genome size" is vital to get correct results when it is used, to I would add a small note to help the user know which number is expected there according to the VCF provided (whole size of a genome, or a chromosome, ...)
- "Output format" instead of "Data".


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: VCF2POPTREE: A CLIENT-SIDE SOFTWARE TO CONSTRUCT POPULATION PHYLOGENY FROM GENOME-WIDE SNPS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have included most of the comments of the reviewers. Although this software application is not making new or additional analysis (which can be performed using other softwares), it has the advantage of constructing a distance matrix (or a phylogenetic tree) very fast and using a single application from a VCF file.

Presumably (I guess depending on the memory of the computer), this program is able to  deal with a large number of genomes in short time, which makes this application useful for having a general overview of the structure and relationships of the individuals included. On the other hand, this program has the limitation of dealing with samples very closely related. The reason is because (i) mapping methods used to obtain a VCF file are working with very related samples to the reference, and (ii) the correction methods for estimating divergence are almost inexistent in this software application (only Jukes and Cantor correction method).
Experimental design: The filtered options (Quality score and Coverage depth) are confusing in the interface. They do not indicate if the value included is filtering less than, less and equal than, equal..

Once the VCF file is chosen and read, the program is available to show the plot or the matrix. Nevertheless, a user non familiar with this program may modify the filtering options, without reading again the VCF file, and click for a redraw (thinking that the filtering will be performed). In my opinion, if the user modify the filtering options, the button of "Draw" should be inactive until the VCF file is again read.

The VCF example file is very simple and uniform. I would recommend to include additional VCF files with variability in the quality depths, scores and missing data, in order to see the differences in the trees performed with these different options.
Validity of the findings: no comments
Additional comments: No additional comments


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

Input:
Title: CRITICAL MULTI-STRANDED APPROACH FOR DETERMINING THE ECOLOGICAL VALUES OF DIATOMS IN UNIQUE AQUATIC ECOSYSTEMS OF ANTHROPOGENIC ORIGIN
Review round: 1
Reviewer: 1

Output:
Basic reporting: English is competent enough, but requires some polishing by the authors, and maybe a quick look by an external expert with excellent knowledge of the both technical English and diatom ecology. In general paper is written competently, though some of the sentence i marked can be made more succinct, especially huge list of the species (lines 250-263), which should be made into the table.
Literature referencing seems to be sufficient, and raw data are provided, but I would argue that results of the paper are currently irreproducible and this is tied to the soft being used in statistical analysis, OMNIDIA package in particular. This is THE MOST important issue of the manuscript. While I do understand that  "Lecointe C., Coste M. & Prygiel J. 1993. “Omnidia:” a software for taxonomy, calculation of
 diatom indices and inventories management. Hydrobiologia 269/270: 509-513." is a standard reference and software piece for the analysis in the Diatom ecology, reporting standards of the original paper describing the software is substandard for 2019. It is closed code without any data on the implementation of the ecological indices included. I would suggest that authors should do complete "dissection"  of the math and stat assumptions behind the package, maybe as a supplement. If it is impossible to do so, due to the commercial nature of the programm, you should check the validity of the findings using alternative statistical approaches. Most importantly, I looked OMNIDIA up https://omnidia.fr/presentation/ and programm does not seems to provide any sort of the statistical power of the test measure. My suggestion, back up your fundings with RDA analysis to see which abiotic factors influencing the compositions of your communities and GLM type linear models to see which factors where most important for the every species of the diatoms. I would like authors to provide the data about underlying assumptions on the data, model output and indicators of the size of the difference/ magnitude of the effects - preferably effect size, p-values could be used but effect size is preferable.

Figures are fine, but many other stat test are required before amount of illustrative material will be sufficient to support the claims of the paper.
Paper is not self contained until statistical procedures are better explained.
Experimental design: Research is original and within the scope of the journal.
Sampling design is fine, but more details are required on the abiotic factors measurements.
Authors undertook admirable work and processed impressive amount of the data, but results are currently irreproducible, see above.
Validity of the findings: I can not access the validity of the findings until I see more statistical tests and explanation of the methods.
Additional comments: No additional comments