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: MICROBIOME ANALYSIS OF PACIFIC WHITE SHRIMP GUT AND REARING WATER FROM MALAYSIA AND VIETNAM: IMPLICATIONS FOR AQUACULTURE RESEARCH AND MANAGEMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is clear and well organized. The revisions have improved the manuscript.
Experimental design: This study uses appropriate analyses to address the question of how aquacultured shrimp microbiomes differ across geographic scales and from their water environment.
Validity of the findings: The authors have addressed my concerns regarding overstating some of their results and I believe that their findings are well supported.
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: MICROBIOME ANALYSIS OF PACIFIC WHITE SHRIMP GUT AND REARING WATER FROM MALAYSIA AND VIETNAM: IMPLICATIONS FOR AQUACULTURE RESEARCH AND MANAGEMENT
Review round: 1
Reviewer: 3

Output:
Basic reporting: Much improved writing from the first submission.  No additional comments.
Experimental design: Experimental design is mostly the same but its description is much improved; details are better defined.
No additional comments
Validity of the findings: no comment
Additional comments: The authors have addressed my concerns from the prior submission.  This is now a much better paper and will be more useful to the field.

I understand from the text why Supplementary Figure 1 is presented, but it really is an odd figure and does not add to the data at all.  I think it should be removed.  It is unusual to have a figure that just shows the proportion of sequences at each taxonomic level, without any additional details.  There is also no legend for that figure so unless the reader has seen the text, the figure makes no sense.

From the tracked change document
line 628> misspelled Mississippi
line 755 maybe > may be
line 876, change "in contradiction" to "in contrast"
line 891, change "math" to "match"
line 894, I would change "severe" to something else like "critical" or "important"


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: MICROBIOME ANALYSIS OF PACIFIC WHITE SHRIMP GUT AND REARING WATER FROM MALAYSIA AND VIETNAM: IMPLICATIONS FOR AQUACULTURE RESEARCH AND MANAGEMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: N/A
Experimental design: Authors adequately addressed most of my questions. However, the are several new results that should be clarified before the acceptance of the manuscript; see REVIEWER RESPONSE 2 in the following sections:

REVIEWER:
Lines 350-355 and 385-387.
How many reads is the difference in the assignation to Vibrio species among using OTUs Vs ASVs?

AUTHORS:
59,645 reads mapped to Vibrio ASVs while 58,8443 reads mapped to Vibrio OTUs. Note that the % similarity used for read mapping in Vsearch is 97% so the difference in mapping statistics will likely be minimal despite the vast difference in the number of ASV vs OTU as read with mismatches will still map. We have included an excel file counts data for only Vibrio ASVs and OTUs in a new supplemental Table 5 which was used to construct Figure 6.

REVIEWER RESPONSE 2:
There is a great difference in the reads mapped to Vibrio OTUS 588,443, while only 59,645 reads mapped to ASVs. This enormous difference among methods should be clarified and discussed in the manuscript.

REVIEWER:
Please include the age and weight (g) of shrimps collected in methods, and discusses the potential effects of the shrimp age in defining the difference of the microbiota among Malaysian and Vietnamese shrimps (Figure 3).

AUTHORS:
We were able to request the age (but not weight as that was not measured during sampling) of shrimps collected for two farms and have included this information in Table 1 and have discussed briefly its effects on microbiota difference. Given that lack of detailed information from the farmers such as the shrimp diet, probiotic application etc, we are conservative in our discussion of the effects of shrimp age on microbiota difference.

REVIEWER RESPONSE 2:
Please discuss the potential effects of the size differences of shrimp (days post-hatching in Table 1) between the shrimp farms (97 days Vs 115 days Vs 65 days). It is well known that shrimp size/age have a strong impact on the microbiota structure, and I think that the clustering observed in Fig 3 is due to shrimp size and not by shrimp farm origin. What is the basis for choosing and comparing these three samples with a large size difference? This could potentially have a confounding effect on the results. Add some discussion points on this.

REVIEWER:
For a better comprehension of the shared and unique microbiomes between intestines and water from the same pond and between farms, the construction of Venn diagrams of shared ASVs at family and/or genus level will be very useful.

AUTHORS:
A Venn diagrams showing shared ad unique microbiomes at the family level has been constructed based on microbial families that are detected in > 90% of samples in any given group (Figure 4A). Additional information used to construct the Venn diagram is provided in Supplementary Data 5.

REVIEWER RESPONSE 2:
Please discuss the biological roles of families found shared and unique of the different Venn comparisons.
Validity of the findings: N/A
Additional comments: N/A


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

Input:
Title: MICROBIOME ANALYSIS OF PACIFIC WHITE SHRIMP GUT AND REARING WATER FROM MALAYSIA AND VIETNAM: IMPLICATIONS FOR AQUACULTURE RESEARCH AND MANAGEMENT
Review round: 2
Reviewer: 2

Output:
Basic reporting: Well done; much better inclusion of references.
Experimental design: Well defined and rigorous.
Validity of the findings: Findings are well stated and presented.
Additional comments: The authors have addressed all my concerns and it would appear, the concerns of the other reviewers as well.


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: MICROBIOME ANALYSIS OF PACIFIC WHITE SHRIMP GUT AND REARING WATER FROM MALAYSIA AND VIETNAM: IMPLICATIONS FOR AQUACULTURE RESEARCH AND MANAGEMENT
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors performed the changes suggested. I strongly recommend it to be published in the journal.
Experimental design: The authors performed the changes suggested. I strongly recommend it to be published in the journal.
Validity of the findings: The authors performed the changes suggested. I strongly recommend it to be published in the journal.
Additional comments: The authors performed the changes suggested. I strongly recommend it to be published in the journal.


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: ADIPOSE TISSUES OF MPC1± MICE DISPLAY ALTERED LIPID METABOLISM-RELATED ENZYME EXPRESSION LEVELS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript by Zou and colleagues reports on the phenotype of mouse heterozygote for the MPC1 subunit of the mitochondrial pyruvate carrier and analyzes the transcriptome of epididymal fat (eWAT) and interscapular brown adipose tissue (iBAT) by RNA-seq. Following observation of reduced eWAT size, the transcriptome analysis revealed that genes coding for fatty acid metabolism were deregulated in MPC1 +/- adipose tissue. Changes in specific genes were confirmed by qPCR and western blotting to show that proteins involved in lipogenesis were down regulated and proteins essential for lipolysis and lipid oxidation were up regulated in MPC1+/- eWAT and iBAT. Overall, the manuscript is clear despite a few specific points that need to be addressed, as described bellow.
- Abstract: line 21, Use of "Despite" is confusing as the phenotype of smaller adipocyte is consistent with decreased eWAT and body weight.
- Line 36: The original paper showing that MPC1 is essential for embryonic development (Vanderperre et al. 2016) should be cited here rather than later in the introduction.
- Line 40: typo. "pregnant does" should be "pregnant dams"
- Line 59 eWAT is the largest visceral "WAT" not the largest visceral eWAT as stated.
- Line 178: The sentence about eWAT activation is confusing and does not relate to anything analyzed in this study. Consider removing the sentence.
Experimental design: The experimental design is appropriate. The validation by qPCR and western blotting of changes identified by RNA-seq reinforces the validity of the findings.
However, there are several issues with figures that need be fixed and in some cases, better explained, as listed bellow.

- Figure 4, the legends should be more detailed. For example by stating that mRNA levels were analyzed by qPCR. In addition, panels A and B are not identified in the figure.
- Figure 5, panels are also not identified in the figure and the names of protein are misaligned with blots.
- Figure 5, pannel A: the actin blot is the same in WT and MPC1+/- samples. Based on the uncropped images provided as supplement, it appears that this is an honest mistake by the authors as they do have an actin blot that contains 6 lanes as the other blots.
- Figure 5: the uncropped images show 6 lanes for each western blot, but the author chose to show only 4 samples. While there may be a good reason for this, it raises questions about why the author pick only some samples to show. More disturbing is the fact that it appears that they do not show the same samples for the different antibodies. The ACC blot appears to be samples number 1,2,5 and 6 while the ATGL blot shown samples 2,3,4 and 5. Can the author explain what these 6 samples are? In addition, an n of 3 for mice samples should be the minimum to analyze levels and provide statistics. I suggest the authors generate an n=3 for the western analysis and show all 6 samples in a contiguous image (rather than separated boxes for WT and MPC1+/- as currently shown) and repeat the level analysis on 3 samples for each genotype.
- Finally, the method used for quantifying the western blot signal should be described.
Validity of the findings: The mouse model used in this study was generated using CRISPR/Cas9-mediated gene knockout. Based on the reference provided, it appears that the gRNA sequence used to delete the MPC1 allele has several predicted off-target effect with 0 or 1 mismatch, which makes it more likely that these sites could also be mutated in these mice. The authors should provide targeted sequencing data for the most likely predicted off-target sites to show that these sites were not mutated in the founder animal.

Line 193, their data do not suggest that TG have been metabolized by peripheral tissue as opposed to decreased TG synthesis. This sentence need be better identified as speculation.
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: ADIPOSE TISSUES OF MPC1± MICE DISPLAY ALTERED LIPID METABOLISM-RELATED ENZYME EXPRESSION LEVELS
Review round: 1
Reviewer: 2

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: In the manuscript "MPC1 deficiency enhances the metabolism of triacylglycerol in MPC1+/- mice", Zou et al show that heterozygous deletion of the gene for mitochondrial pyruvate carrier protein 1 in mice increases the expression of genes involved in the catabolism of triglycerides in epididymal white and intrascapular brown adipose tissue. The morphology and large-scale gene expression pattern were analyzed in these two tissues of MPC1+/- mice and compared to wild-type mice. Although the manuscript contains some interesting results there are vital control experiments which are lacking (see comments below). Therefore, the manuscript in its present state is not recommended for publication in PeerJ.

Major comments
The title of the manuscript should be changed to reflect directly the results obtained, such as for example: "Adipose tissues of MPC1+/- mice display altered expression levels of enzymes involved in lipid metabolism"

Fig. 2. A mitochondrial marker should be used to detect differences in the number or volume of mitochondria in eWAT of MPC1+/- mice with respect to WT.

Fig. 5. As the whole manuscript relies on the assumed lower expression level of MPC1 protein in eWAT and iBAT of MPC1+/- mice with respect to WT, this should be demonstrated by immunoblotting using material from these tissues.

A supplementary Table listing all the genes of Fig. 3 should be included for data transparency and to show in which background the genes of interest were selected.

Minor comments
Line 17 says "the liver", but no analysis on liver is described in the manuscript.

Line 19. The meaning of "an MPC1 protein part deficiency" should be clarified.

Lines 20 and 170. The meaning of "heterozygous mice exhibited low energy metabolism" should be clarified.

Line 21, says "Despite". Maybe "In concomitance with" is more appropriate.

Fig. 4. Probably the abbreviations "AGTL" in the graphs should be "ATGL".

Fig. 5. The use of "relative expression level" in the graphs should be explained (relative to what?).

The paragraph on the connection between MPC1 and cancer (lines 313-326) should be rewritten and made more clear.

All the figure legends should be revised to provide the complete information required for the reader to understand the figures.

The manuscript needs to be improved by using English language help for overall clarity and removal of mistakes.


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: ADIPOSE TISSUES OF MPC1± MICE DISPLAY ALTERED LIPID METABOLISM-RELATED ENZYME EXPRESSION LEVELS
Review round: 1
Reviewer: 3

Output:
Basic reporting: See general comments
Experimental design: See general comments
Validity of the findings: See general comments
Additional comments: This is a straightforward and simple study that examines the effect of whole-body MPC1 knockout on adipose tissue metabolism. The authors show that MPC1 +/- mice are smaller and have reduced adipose tissue mass, and changes in the expression of adipose tissue metabolism genes. The paper is adequately written, clearly laid out and the results are presented clearly. However, the data are extremely preliminary and very limited in their scope, most likely because the authors have very recently published a related manuscript containing some of the basic phenotypic data that should be included here (Zou et al., 2018). I have a few additional specific comments:

1)	Clearly the major weakness of the paper is that the authors did not analyze the phenotype of adipose tissue-specific MPC1 knockout mice. This is important as it is known that whole-body MPC1 knockout mice are embryonically lethal. Moreover, the MPC proteins have important roles in liver metabolism and are critical for brain (and probably adipocyte) development. Together this makes the interpretation of the MPC1 +/- mouse data very difficult, if not impossible. If the authors are intent on pursuing studies in this whole-body model, the authors need to address the following points regarding the very basic analyses of the MPC1 +/- mice phenotype:

i)	Where is the quantification of MPC1 and MPC2 protein levels in the MPC1 +/- mice? This is essential to perform. Do the authors have these data? Knockout of MPC1 usually leads to the degradation of MPC2 protein, and vice versa. I only see mRNA data in figure 4, but this is not very convincing.
ii)	Were there changes in liver, muscle, heart and pancreas mass? Did the authors perform whole-body qMRI analysis?
iii)	What about whole body energy balance, including food intake and energy expenditure?
iv)	Did the mice have changes in glucose metabolism? The authors could examine fasting plasma glucose levels (at a minimum) very easily. Similarly it would be important to quantify circulating triglycerides and/or FFA levels. This can also be done easily and cheaply.
v)	I note that some, but not all, of these data are presented in a related manuscript by the same group (Zou et al., 2018). It would have been more impactful to combine these data into one larger, more comprehensive analysis. A brief look at the data in this manuscript shows the expected abnormalities in whole body RER, hepatic glucose metabolism etc. This reinforces my concerns about focusing on a specific tissue in the context of a whole-body knockout.

2)	In figure 1, only 6 mice were examined in each group. This is quite a small number of mice for these studies. Photos of adipose tissue should be shown from more than one mice in my opinion. In the previous paper (Zou et al., 2018), 6 mice were also used for all studies. Are these the same six mice shown here?
3)	In figure 2, the authors should improve their presentation of the adipocyte size distribution. The axes should be swapped and the number of adipocyte diameter bins should be expanded so that the overall distribution of adipocyte size can be better appreciated.
4)	In figure 5, only two mice were used for quantification and statistical analysis. This is inappropriate and more samples should be analyzed to enhance the rigor of the studies. The provided “uncropped” Western blot images look like cut film to me. Do the authors have whole-films with molecular weight markers included?
5)	The discussion does not address the weaknesses of the study adequately. Specifically the issues relating to point 1 above are not introduced. This would be important to do in a revised 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: ADIPOSE TISSUES OF MPC1± MICE DISPLAY ALTERED LIPID METABOLISM-RELATED ENZYME EXPRESSION LEVELS
Review round: 2
Reviewer: 1

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: The authors have addressed all my comments. With the exception of a few text edit required as described below, the manuscript in sound and should be accepted for publications.
Minor edits:
Line 21-22: Should use "consistent with" rather than "In consistent"
line 221: should say 1625 genes "with" a significant difference to WT


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: ADIPOSE TISSUES OF MPC1± MICE DISPLAY ALTERED LIPID METABOLISM-RELATED ENZYME EXPRESSION LEVELS
Review round: 2
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: The revised version of the manuscript now entitled "Adipose tissues of MPC1+/- mice display altered expression levels of enzymes involved in lipid metabolism" (26773-v1) by Zou et al. has been improved and my previous comments have been dealt with properly. However, it still needs English language editing to improve clarity and remove errors. Furthermore, new errors have been introduced in the revised version.


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

Input:
Title: ADIPOSE TISSUES OF MPC1± MICE DISPLAY ALTERED LIPID METABOLISM-RELATED ENZYME EXPRESSION LEVELS
Review round: 2
Reviewer: 3

Output:
Basic reporting: See general comments
Experimental design: See general comments
Validity of the findings: See general comments
Additional comments: My concerns about the interpretation of the adipose tissue findings in relation to the whole-body knockout phenotype have not been addressed in this revised article. Many of the findings in the adipose tissue could be due to primary effects elsewhere (liver, muscle, pancreas), and potentially on the development of the adipose tissue (and other tissues). I think presenting the adipose tissue metabolic data in the absence of additional whole-body data is misleading.


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: FROM THE CAGE TO THE WILD: INTRODUCTIONS OF PSITTACIFORMES TO PUERTO RICO
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is poorly written and is much too long for the research findings.  It is receptive in places, and extremely "wordy" and contains extraneous information.  It could be reduced by half without loss of important content.  Suggested improvements are contained in review sent to the authors.
Experimental design: Use of Max Ent model is appropriate for predicting suitable habitat for the exotic parrots and the census methods used for counting roosting parakeets is appropriate.  Remainder of ms. base on natural history observations and anecdotes.
Validity of the findings: Results of the niche model seem reasonable, but authors do not provide adequate discussion of model results especially where observations do not fit model predictions.  Conclusions includes some stuff unrelated to their research findings.
Additional comments: This manuscript makes a valuable contribution to our understanding of introduced psittacines on the island of Puerto Rico, by summarizing current status of the introduced species and by using niche modeling to indicate which species have occupied their potential available niches.  This is of value for understanding how introduced species adapt to their new environments.  The niche modeling predicts that some species have colonized much of the potential available habitat on the island or for some species have much more habitat available for colonization.  In the case of the monk parakeet, some sight records have been recorded it in habitat, which was predicted to be unsuitable by the model results.  The expansion of the range of the white-winged parakeet recorded in the study is also supported by increased counts of the parakeets at roosts, which would contribute to the range expansion.  The niche modeling and population counts are new contribution to our undersanding of exotic species on Puerto Rico.  However, the manuscript is poorly written – overly “wordy”, includes extraneous details (not clear how the parasitic mite information is relevant to range expansion), and very repetitive (especially in the Discussion section).  The authors use common and scientific names interchangeably in an inconsistent manner throughout the ms. The diet information on the white-winged parakeet is poorly integrated in the discussion of the parakeets population growth and range expansion on the island.  Furthermore, the Discussion section includes numerous anecdotes and much speculation, which is unwarranted. The Conclusion section includes discussion of issues, which were not related to their research findings.  In addition, considerable space is used to review issues and recommendations related to management and control of exotics (from previous studies and not from their own research findings), rather than focusing on the research findings.  The discussion section could be shortened by eliminating the anecdotal material on the various exotic psittacines and by providing an appendix with species accounts summarizing the current status of various exotic psittacines.  Overall, the results of this study contribute to our understanding of exotic species and their status on Puerto Rico, but of less interest to those concerned with general topics related to naturalization of non-native species.  The manuscript could be shortened by half without loss of critical content.  The manuscript requires substantial revision and elimination of extraneous material before it is publishable.  My specific comments and suggestions follow below:

Line 88 – “many species have been imported to be sold as pets in PR” – Change to: “many species have been imported for sale as pets…..”

Line 94 – “having gone extinct” Change to: “are now extinct”.

Line   97 -  “managing agncies” – Change to:  managers want

Line  102 – “alerted” Change to: warned

Line 103 – “there is yet no evidence”.  Change to: evidence of crop damage yet.

Line 109 – Change to:.... negative effects that the parrot might have on native biota and agricultural sector if the parakeet kept expanding its range.

Line 111 – Change to: In this study we review the introduction and persistence of Psitta…. by evaluating their historic and present distribution.  In addition, we assess the invasive ecology of the white-winged parakeet in more detail, including aspects of its natural history, estimated population size and growth rates and predicted distribution based on niche modeling techniques.

Line 115 – Change to: “Finally, we identify possible factors that may have contributed to the successful establishment of Psitt in PR…..”

Line 122 to 126. Eliminate this section.

Line 131—Change to: We also recorded species occurrences based on observations made during 2013-17.

Line 133 – Change to: “which contain records from amateurs and professional ornithologists. Online searches were also conducted for photographic records from local groups including: “

Line 145-145 – Modify to ….”were used to assess the “invasiveness” of various parrot species in non-native locales (outside of Puerto Rico) using the invasive categorization scheme of Blackburn et al. (2011, Table 1).  The latter scheme was also used to classify the invasive status of introduced psittacines in Puerto Rico.

Line 160 – “…. with at least 20 records were included in the subsequent analyses.” Eliminate “in this paper”.

Line 162 – Change to: “We assessed the current distribution of the introduced psittacines by surveying Ebird for geo-referenced records….”

Line 166 – Change to: “using methods of Falcón, el al. (2012) and…”

Line 167 – 170 – Change to: “Species distribution models were constructed with the Maximum Entropy Method (MaxEnt ver 3.4.1;…), which is a learning machine method that uses presence only data in combination with predictive variables to model a species’ geographic distribution”.

Lines 202-208 – Eliminate this introduction paragraph – too general.

Line 220 – Change to: “Many psittacines, including B. versicolurus roost….”

Line 233 – “observations on natural history information….”  Change to “observations on food and nest resources (plant species).”

Lines 256 257 – Change to: “mark-recapture (Casagrande & Beissinger, 1997).  These four methods produced 95% confidence intervals, which overlapped each other.”

Line 258 – “To test the effectiveness of this method…”  Change to [?]: “To test the effectiveness of the mark-recapture method with the…”

Line 259 – Change to: “…, we conducted three counts spaced a week apart from September to October…”

Line 262 – Change to: “For estimates of population size and growth rates, we assumed that…”

Line 267 – Remove: “(these cannot be separated with this method)”

Line 299 – Change “On the other hand,…” to: At least 46 psittacid species are now present on the island (Fig 1, Table 1), of which 24% are only found in the pet trade, 48% have been observed in the wild (present), but not known to be breeding (established), and 28% are established (naturalized) and are known to have bred or are currently breeding.

Line 313 – “Of the species with population increase,…” Change increase to increases,..

Line 321 – “The green-checked amazon (….)…”  Change to green-cheeked amazon.

Line 337 – Eliminate Puerto Rico from end of sentence (paper is on psittacid in PR, so save space).

Line 341 – change “were” to “where”

Line 342 – “Furthermore, the only other historic record are…”   Change word “record” to records

Line 345 – “especially to coastal and heavily (human) populated zones,…”  change to: especially in coastal and urban/suburban areas, but they….

Line 347 – Change to:  To predict potential distribution of psittacine species whose populations are increasing, we obtained a total of 106,493 occurrence records from their native…

Line 357 - B. versicolurus – I suggest be consistent in use of scientific and common names.  I suggest give scientific name with common name together when first used in the manuscript, then use common names afterwards. – In this paragraph you start with white-winged parakeets and then move to the scientific name  and change back and forth – this gets confusing.

Line 367 – “Ecology of Brotogeris versicolurus in PR” – change to common name and use common name hereafter.

Line 385 – Eliminate – information on feather mites – not relevant to your manuscript.

Line 399 – “was estimated in 604 for San Germán …”  Change to: “was estimated to be 604 for San Germán…”

Lines 405 – 410 –Repetitive, reduce length.

Lines 414- 434 – Repetitive – authors have repeatedly made the point that exotic psittacines on the island are a result of the pet trade.  No need to repeat it.  This paragraph should be eliminated – examples here are anecdotal – masked lovebird example could go in a table.

Lines 435 – 465 -  This paragraph as the previous paragraphs is just a collection of anecdotes on the status of introductions – these anecdotes could perhaps go into an appendix, which would summarize, species by species the history and status of each introduced species.  Discussion should summarize and synthesize patterns related to success or failure of exotic establishments.

Lines 466 – 468 – First paragraph repeats earlier information that exotics most common along coastal plan and in urban areas.

473 – 474 – Repetitive statement that white-winged and monk parakeets are most sighted and widespread species on island.

Line 478 – “…., and species more commonly found in captivity.” – change “more” to “most”.

Line 489 -   This paragraph summarizes potential negative effects of introduced psittacines as found elsewhere, but is there any evidence for these negative effects in PR?

Line 505 – “before native birds have access to them (because native species only eat them when they are ripe – I am skeptical of this statement – many native species will take unripe fruit including bullfinches and spindalis and other tanagers and finches.

Line 506 – Correct this citation: Snyder, Wieley & Kepler 2007…..” Change “Wieley” to Wiley (correct on line 509 also).

Line 517 – Paragraph – “Another aspect to consider is that the three species of endemic parrots in Puerto Rico were once much more common and used to occupy many of the areas and habitats now occupied by exotic species”  - The current habitats occupied by the exotic species is likely very different from the habitat occupied by the endemic psittacines – the exotics are using habitats with many exotic plant species (e.g., “novel” communities) including second growth forests/woodlands and suburban and urban areas, which were not occupied by the endemics.  Thus as with the exotic finches (see Raffaele, A.H. 1989. The ecology of native and introduced granivorous birds in Puerto Rico. In Biogeography in the West Indies: past, present, and future: 541–566. Woods, C.A. (Ed.). Gainesville, FL: Sandhill Crane Press), the exotic psittacines are occupying novel habitats which were not available to the extinct endemics – in other words the exotic psittacines are occupying vacant niches, which may not have been available for occupancy by the endemics.

Lin 520 – Change “damages” to word “damage”.

Line 525 – “Parrots are known to incur in such functions…”  Change to:  Some parrots are known to be both seed predators and dispersers…”   Nothing or little is known about the PR parrakeet’s and parrot’s role as seed predators or dispersers, so this seems highly speculative.

Line 531 – “and they often predate on them in the wild”  Change to:  and they often destroy seeds in the wild.

Line 533 – “But they also predate seeds of the pink trumpet…”  Change to: “But they also depredate seeds of native species such as pink trumpet…..”

Line 535 – Change “predate” to word “depredate.”

Line 547 – “However, rarely the number of individuals trapped was reported”.  Change to: However, the number of individuals trapped was rarely reported.”

Line” 553 – 560 – Paragraph here is about recommendations for sale and reporting of psittacine sales – is not relevant to this paper.  Eliminate

Lines 561- 568 -  “If control and management is necessary and/desireable…”  Paragraph focus is not relevant to paper.  Eliminate – recommendations are found elsewhere in literature and not unique to PR.

Line 570 – Ecology of Brotogeris versicolurus in PR – Use common name in title, to be consistent.

 Line 577 –583 --Eliminate the mite discussion – perhaps publish in a separate note, but not relevant here.  Eliminate this paragraph as natural history notes on mites are irrelevant to your study, or you do not make a connection to the study.  Publish as a separate note.

Line 589 – This paragraph is highly speculative – can you be certain you’ve found all of the roost sites?

Line 602-603 – Change to:  “Additionally, as the size of a species’ population increases, so does the geographic distribution (Veit 1997), so we can expect the population result in spread….”

Line 604- “(although this is not always the case,”). Shorten to “(but not always, …”).

Line 605-607 – Eliminate sentence about Yellow-headeded Blackbird details – represent unnecessary details – you can cite Veit (1997) in earlier sentence as suggested.

Line 608 – “….have a proportional increase of long dispersers…:  Change to: “…have a proportional increase in long dispersing individuals (vagrants)….”

Line 608 – Change word “incrementing” to “increasing”.

Line 618 -  “populations suggested that they were they only existing…”  Change to: “populations suggested that they were the only existing roosting…”

Line 622 – Change “variations” to word “variation”.  (singular not plural).

Line 633 – “Puerto Rico and parks…” Change to:  “highly abundant in Puerto Rican forests and parks.”

Line 638 – Change word “exacerbating releases” to:  increasing the parakeet numbers in the wild.

Line 645 – Eliminate word “indeed”.

Line 655 – Eliminate paragraph on PR parrot as your research findings do not address this issue.

Line 661 – This paragraph on hurricane impacts does not belong in the conclusions as your research findings are unrelated to hurricanes – Conclusions should focus on your research findings, and hurricane discussion here is irrelevant.  Eliminate entirely or move elsewhere.

Line 676 – This paragraph on studying endangered exotics does not belong in the conclusions section – perhaps move to Discussion section.

Line 698 – Change “Wieley” to “Wiley”.


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: FROM THE CAGE TO THE WILD: INTRODUCTIONS OF PSITTACIFORMES TO PUERTO RICO
Review round: 1
Reviewer: 2

Output:
Basic reporting: A few small issues with the English language.

For main comments see below.
Experimental design: For main comments see below.

The information on the roost counts was not presented in sufficient detail.
Validity of the findings: See comments below
Additional comments: In the paper “From the cage to the wild: Introductions of Psittaciformes to Puerto Rico with emphasis on the invasive ecology of the white-winged parakeet” the authors have made a useful compilation of information on the introduced parrots of Puerto Rico. They have also gone more in depth in to the status of the White-winged Parakeet. The manuscript is overall relatively easy to follow, but it has some rather major issues that will need to be addressed.

Starting with the title, it is clear that the paper has two main themes. Introduced parrots in general and White-winged Parakeets in specific. Unfortunately, the paper reads like two papers that have been put together in the same manuscript. I think that the authors need to separate out the two topics in to different papers and submit each individually to either the same or different journals. By restructuring in this way, the authors will be able to devote more attention to each manuscript and make each stronger on its own.

The authors use a Maxent analysis using a combination of locations from the native range and introduced range to predict potential distribution on the island. Most observations of the introduced birds in Puerto Rico fall within the predicted Maxent ranges, but not all. It is unclear to me why the authors chose to do a single Maxent map using a mixture of locations from native and introduced ranges. The authors should provide better justification for this. Or ideally, do two Maxent maps one using locations from the native range and another using locations from Puerto Rico to determine how they differ. If the overview of introduced species is separated off in to a separate paper, it will provide enough space to include an analysis like this and its discussion.

However, there is a single much larger issue with the spatial / habitat analysis in this paper. The authors have not addressed the issue of urban versus native habitat use by the introduced parrots. One classic hallmark of introduced parrots is that they often use preferentially (or sometimes exclusively) anthropogenic habitats. From looking at the maps generated, many of the clusters of points signifying high use by introduced parrots overlap with the population centers on the island. This is an issue that must be addressed in the spatial analyses through the use of some sort of layer that indicates urbanization or the like.

However, there is another reality that could be adding to this finding. The eBird locations may be preferentially clustered in areas with more people. Are there well known parks with high numbers of eBird checklists that do not report introduced parrots? The authors need to discuss both the quantitative and anecdotal/qualitative information about urban versus natural habitat use by the introduced parrots.

The authors also need to discuss the use of eBird data in general. Sometimes eBirders use county or city locations when entering their data. As a result, the actual point location is not very accurate. How did the authors filter or check their data for this? While eBird is a wonderful resource, the authors need to discuss points from the literature about possible strong and weak points of these data and how that may have impacted their findings.

With regards to the focus on the White-winged Parakeets, the authors should go in to more detail about the censuses they did. In general this part of the paper seemed “rushed.” There was an overall lack of detail, development, and discussion. Again, if this is broken in to its own paper, the authors will have the opportunity to develop this information fully.


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: FROM THE CAGE TO THE WILD: INTRODUCTIONS OF PSITTACIFORMES TO PUERTO RICO
Review round: 2
Reviewer: 1

Output:
Basic reporting: Although I am not a native English speaker, I find that it there are many errors of language and syntax, making the readability of the manuscript not fluent. A severe language polishing is recommended.
Experimental design: OK
Validity of the findings: OK
Additional comments: Line 42: delete “where they are introduced”
Line 68: change “Among the species commonly sold as exotics are Psittaciformes” with “Psittaciformes are among the exotics species most commonly sold as pets”
Line 76. This is true, but you have to say that most “unsuccessful” introductions have not been documented. See, for instance: Mori E., Monaco A., Sposimo P., Genovesi P. (2014). Low establishment success of alien non-passerine birds in a Central Italy wetland. Italian Journal of Zoology, 81: 593-598.
Lines 110-114: I suggest you to take a look at this: https://www.inaturalist.org/projects/alien-parrots-observatory
Line 212. Delete the dot after “Psittacara”
Line 272. Change “sought to assess” with “assessed”.
The Discussion chapter is too long and unbalanced with respect to the rest of the MS. I suggest you to summarise it and to reduce it at least by 40%.

Emiliano Mori


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: CONFIDENCE IN EATING DISORDER KNOWLEDGE DOES NOT PREDICT ACTUAL KNOWLEDGE IN COLLEGIATE FEMALE ATHLETES
Review round: 1
Reviewer: 1

Output:
Basic reporting: There is an interesting are of research and the study meets ethical requirements.

However, there are some important problems that must be solved before the paper will be found suitable for publication in the scientific journal.

From the Introduction it is not clear what kind of eating disorders are commonly seen in female athletes: how often we can see ED and how often we can see improper nutrition which can significantly effect their health.  The Authors should have on mind that not all readers are familiar with this problem in athletes and therefore, it necessary to shortly but precisely introduce it.
Experimental design: There is also not clear if there is an obligatory training for coaches and students on nutrition, nutritional requirements for athletes, relation between nutrition and health, or it is expected that they have some general knowledge as other members of the population should have. Without specific knowledge it can be hard to distinguish between eating habits and eating disorder, which significantly influence the way of treatment. It is an important problem, which significantly influence the questions used in the questionnaire and the answers. The Authors assess the knowledge on etiology, symptoms, treatment etc. It indicated that study participants are expected to possess more that “general knowledge”. It would be important also to present data on subject characteristics related to their university activities (eg. faculty, area of their studies, etc.). The sources of student knowledge on nutrition and eating disorders should be also assessed and presented.

It seems that the questionnaire was used before and how it was validated.
Validity of the findings: It is not clear why the Authors pay attention only o anorexia nervosa, bulimia and starvation, and do not pay attention on eating habits and eating behaviors as well on general knowledge on nutrition and health.

Table 1 should contained also participants characteristics not “simply” numbers of participants involved in different sport activities.

  Fig.1 and Fig.2– what “a, b, c “ means???

Fig.3 and Fig.4 – there is no reason to present the figures as no association was found, and the figures do not introduce a specific new information important for the readers

Under Discussion the new data coming form the study should be underlined
Additional comments: There is  a lack of refection on the data obtained and what should come out based on the results of this 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: CONFIDENCE IN EATING DISORDER KNOWLEDGE DOES NOT PREDICT ACTUAL KNOWLEDGE IN COLLEGIATE FEMALE ATHLETES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors used appropriate English throughout the manuscript. Within the attached review, authors will see additional references to support the manuscript. Sufficient background was provided for most of the aims; however there was additional analysis conducted, but no justification/background to support the analysis (examination of the relationship between age and knowledge). Rationale/background for examining female athletes, needs to consistent throughout the document. Some sections state it may be the responsibility of the athlete to identify signs and symptoms of EDs in other athletes, whereas others it states it should be the dietitian or healthcare provider. With there only being one institution involved in the study, it is unknown what the current healthcare coverage is, so making this assumptions is risky if they indeed have adequate healthcare coverage for student athletes.  Does this institution even have access to a dietitian? If not, then what would be the next step? This all needs clarity for such a small sample size, it is almost impossible to generalize to other institutions.
Experimental design: The format of the current methodology section needs organization and clarity, the attached review provides in-depth information on how this section could flow better.
Validity of the findings: Findings are novel however aims need to be clarified and additional background need to be provided for the last aims on examination of the relationship with age and knowledge. If the institution does indeed have adequate medical coverage, this conclusion may be suggestive to focus on prevention and intervention strategies for student athletes for awareness of EDs not necessarily "responsibility" to identify signs and symptoms. This will take the "athletes as the untrained healthcare" provider out of the justification.
Additional comments: NAIA has 250 institutions and 65,000 students athletes; therefore, a sample size of 51 is low. Without a power analysis to determine the effect size and power it is difficult for the reviewer to determine if this is adequate or not. Rationale for the study is conflicting, in the introduction it is clearly stated “instead of team dietitians or athletic training identifying disordered eating behaviors; the responsibility of identifying disordered eating behaviors may fall on the coaches and teammates.” It is recommended the authors be careful with this statement as the institution’s name is in the methods section and may not be reflective of the healthcare providers the student athletes have access too. Due to having only one institution participating in the study, it is important for the authors to be clear on the clinical coverage currently provided for those student athletes or the types of current resources these student athletes have or may not have compared to other institutions in the NAIA. Due to legal issues, administration would never leave the responsibility up to the athletes, so it is recommended authors soften the language for the justification and make sure they have clear evidence prior to making assumptions on the care for the student athletes and who is responsible for the care (needs to be consistent throughout the document).


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

Input:
Title: CONFIDENCE IN EATING DISORDER KNOWLEDGE DOES NOT PREDICT ACTUAL KNOWLEDGE IN COLLEGIATE FEMALE ATHLETES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript was improved according to previous comments. No additional comment.
Experimental design: The description was improved according to previous comments.
No additional comments.
Validity of the findings: The Results and Discussion was significantly improved, however, it is still necessary to explain in each figure legend  what "a, b, c" mean. Maybe it is somewhere at the end of the manuscript but it is hard to find for the reviewer and readers will also have this problem.

It is not necessary to present all data, especially that the study group is small and the results should be called preliminary and additional study in larger group are needed. It is necessary to underline that you found no relationships and figures did not add additional information important for data interpretation.
Additional comments: The manuscript was found to be significantly improved according to previous reviewers' comments, however, still some revision is needed as indicated.
Under Discussion still some reflection is needed as the reviewer is not surprised that  older subjects have more knowledge than younger (time is an important factor needed to improve our knowledge in different areas) as well as that "low knowledge" can be accompanied by "quite high" confidence as people who get some knowledge often do not realize the complexity of the problem and the need for cooperation with health professionals.


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: CONFIDENCE IN EATING DISORDER KNOWLEDGE DOES NOT PREDICT ACTUAL KNOWLEDGE IN COLLEGIATE FEMALE ATHLETES
Review round: 3
Reviewer: 1

Output:
Basic reporting: The manuscript was improved . No additional comment
Experimental design: The description was improved .
No additional comments.
Validity of the findings: Contrary to the opinion of the authors, the reviewer strongly postulates to clearly indicate under each figure legend differences between which groups are described by letters a, b, c. It is commonly accepted that figure legend contains all information needed to understand the information presented on this figure.

If the authors want to present all data they collected, even data found not significant, such data should be presented in supplementary materials not in the manuscript. Figure 3 should be send to supplementary materials.
Additional comments: The manuscript was found significantly improved. However, the aim of the review is to draw the authors attention to the elements that should be corrected Unfortunately, the authors of this work stubbornly stick to their choices and do not respond to the requests of the reviewer.

Contrary to the opinion of the authors, the reviewer strongly postulates to clearly indicate under each figure legend differences between which groups are described by letters a, b, c.

If the authors want to present all data they collected, even data found not significant, such data should be presented in supplementary materials not in the manuscript. Figure 3 should be send to supplementary materials.


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: CONFIDENCE IN EATING DISORDER KNOWLEDGE DOES NOT PREDICT ACTUAL KNOWLEDGE IN COLLEGIATE FEMALE ATHLETES
Review round: 4
Reviewer: 1

Output:
Basic reporting: No additional comments. The manuscript was found suitable for publication.
Experimental design: No additional comments. The manuscript was found suitable for publication
Validity of the findings: No additional comments. The manuscript was found suitable for publication
Additional comments: No additional comments. The manuscript was found suitable for publication


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

Input:
Title: DON’T WORRY, BE ACTIVE: HOW TO FACILITATE THE DETECTION OF ERRORS IN IMMERSIVE VIRTUAL ENVIRONMENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article is original, well written and easy to follow. I only suggest reviewing the points listed below in order to improve the text.

A.	Please define the acronyms listed below:
1.	Line 226-227: Please define the acronyms into brackets “(A vs P)”;
2.	Line 497: Please define the acronyms “DE3” and “DE4”;

B.	Please check out the reference to the figures in the text. Specifically at lines 211-213, Figure 1 is wrongly referenced. I believe Fig.1B and 1C might be Fig.1A and 1B instead.

C.	A few typos to correct:
1.	Line 47: Please change “Mavpor” with “Mavor”;
2.	Line 109: Please change “Arns” with “Arms”;
3.	Line 137: Please change  “Bulthoff” with “Bülthoff”
4.	Line 154: Please change “ideothetic” with “idiothetic”;
5.	Line 263: Please change “active.” with “active”;
6.	Line 418: Please change “exemplar” with “example”;
7.	Line 531: Please change “Undergraduates'” with “Undergraduate”;
8.	Line 548: Please change “reduced” with “reduction”;
9.	Line 551: Please change “allow” with “allows”;
10.	Line 722: Please change “thorough” with “through”.

D.	I suggest improving the description of the participants taking part to the study. I have a few questions regarding this point. Did you make any preliminary study on the tested population? For example, did you check if the participants had comparable seek and hike errors recognition ability or did you test the participants for color sensitivity? Did you consider these factors in the depiction of the experimental setting?
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: DON’T WORRY, BE ACTIVE: HOW TO FACILITATE THE DETECTION OF ERRORS IN IMMERSIVE VIRTUAL ENVIRONMENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This work cannot be published in its present form. Its appears that the paper has been translated from its original language into English. There are few spelling mistakes however the grammar is entirely unacceptable and the use of words in almost every paragraph make interpretation of the experiment reported extremely difficult to understand.
Experimental design: The 'Active' versus 'Passive' paradigm, as the authors report, has been used in numerous previous experiments. From most of these experiments it seems to me that active exploration produces better performance than passive and there are various reasons for this.
The experimental design is perhaps reasonable although the fact that the sessions occurred during visits by students etc. on a haphazard basics leads me to be believe that the conditions were not highly controlled.
Validity of the findings: There is little novelty here apart from the fact that a comparison is made between affordance and perceptual error in passive versus active viewing.
The data is reasonably well stated given the obvious problems with the use of the English language.
I cannot see that the conclusion adds anything to our general knowledge.
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: DON’T WORRY, BE ACTIVE: HOW TO FACILITATE THE DETECTION OF ERRORS IN IMMERSIVE VIRTUAL ENVIRONMENTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is written in English and is generally clear, although there are numerous instances where there are grammatical or stylistic errors that detract from the work. It is recommended to have the manuscript reviewed again in detail to catch these. Note that the results are clear so issues with language do not affect the ability to review the work.

The abstract and introduction could be more direct and focus with respect to the aim of the paper. While the authors provide a reasonably comprehensive review of relevant previous work, I found it difficult to keep in mind what the purpose of the experiment was.
Experimental design: The experimental design was clear. As noted above, the research question could be better defined prior to the experimental design.
Validity of the findings: The statistical approach appears sound, although as it is not a standard approach it is good to guide the reader through the approach used, which the authors mostly do. While impact and novelty are not of concern, and negative/inconclusive results are permitted, I will require all results to report measures of effect size and power analyses. This is both important to understand just how strong the reported effects are (and their likelihood for being reproducible) as well as to assess those effects that were not found to be significant. Reporting of effects sizes and statistical power are particularly important as there were some unexpected results found be the researchers.
Additional comments: I thought that this is a useful paper on what appears to be a novel research design. If the authors make more of an effort to streamline the paper and make it more approachable it will help readers tremendously to best understand the work. The addition of measures of effect size and power analyses will significantly strengthen the results section and temper the interpretation of the results 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: DON’T WORRY, BE ACTIVE: HOW TO FACILITATE THE DETECTION OF ERRORS IN IMMERSIVE VIRTUAL ENVIRONMENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: the authors reviewed the paper and improved the English form of it. The manuscript is overall improved and more readable.
Experimental design: The authors improved the description of both their aim and of the method used. I really appreciated the details added about the participants' selection and characteristics.
Validity of the findings: The authors improved the results section through an improved statistical analysis.
Additional comments: The authors improved overall the manuscript that to my opinion should be accepted for publication.


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

Input:
Title: DON’T WORRY, BE ACTIVE: HOW TO FACILITATE THE DETECTION OF ERRORS IN IMMERSIVE VIRTUAL ENVIRONMENTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The language used in the manuscript is now clearer. I will leave this to the editor to decide if the quality of the language is appropriate for this journal.
Experimental design: While the vestibular system is certainly involved during self-motion here, it is not acting alone. Consequently the authors should temper their use of "vestibular" as there is no attempt to isolate the vestibular system from other systems that are active during self-motion compared to passive motion.
Validity of the findings: It is very helpful that the authors have included measures of effect size throughout the manuscript. I have remaining issues with the reporting of statistics and the interpretation of the results:

L444, L449: report the full p value (don't round). If the p value is actually 0.05 exactly then there is no effect, but perhaps a trend. Given that the sample size is well powered, but the effect size is very small, this is most likely not an effect and should be interpreted this way with caution

L621: if reporting that all of these are trends and below 0.09, the full range should be reported and the language should not be "marginally significant" but rather trended towards reaching significance. In small underpowered designs trends can be important, but in well powered designs these really should be reported as not being significant.

What I would like to see in the results, discussion and abstract then is more effort in being skeptical of the results. For those results that are clearly significant and with large effect sizes then treat them as such. For the remaining trends and very low effect sized results these really should be reported as providing no or very little evidence.
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: ASSOCIATION OF URBAN FOREST LANDSCAPE CHARACTERISTICS WITH BIOMASS AND SOIL CARBON STOCKS IN HARBIN CITY, NORTHEASTERN CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: I stopped reading this manuscript at the methods section.  This is a paper supposedly about soil carbon and tree carbon stocks in the urban forest of Harbin City.
Whilst I think this paper look potentially interesting - there is insufficient detail in the methodology to come to any conclusions about the work.  There is no detail about how or even whether they soil sampled in order to estimate carbon stocks.  There is no information at all about how they converted any tree information they collected in their field plots to carbon storage.  Without this information I am not willing to go through the rest of the manuscript.
Experimental design: I stopped reading this manuscript at the methods section.  This is a paper supposedly about soil carbon and tree carbon stocks in the urban forest of Harbin City.
Whilst I think this paper look potentially interesting - there is insufficient detail in the methodology to come to any conclusions about the work.  There is no detail about how or even whether they soil sampled in order to estimate carbon stocks.  There is no information at all about how they converted any tree information they collected in their field plots to carbon storage.  Without this information I am not willing to go through the rest of the manuscript.
Other points - I do not understand why the authors think that administrative boundary will effect carbon storage - trees and soils do not conform to administrative boundaries.
Validity of the findings: Cannot assess this as method were insufficient.
Additional comments: I think potentially this could be worthy of publication BUT the methods section is too incomplete to assess this.  See above comments.  Remember the purpose of a methods section is to allow other to reproduce your methods.  This would be impossible for your manuscript as it is.  Whilst you describe the GIS approach used the field studies information is completely inadequate.
How did you sample your soils for carbon?
How did you analyse your soils?
What tree parameters did you measure in your field plots?
Did you identify tree species?
Did you use allometric equations to convert tree information to biomass and then carbon?


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: ASSOCIATION OF URBAN FOREST LANDSCAPE CHARACTERISTICS WITH BIOMASS AND SOIL CARBON STOCKS IN HARBIN CITY, NORTHEASTERN CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: I would recommend the authors to refine the English word usage and style. There are many  typos and grammar errors in the manuscript.

Some literature needs to be added to enhance the background and discussion of the manuscript.

Figures and tables need further improvement.

See attachment PDF for revision.
Experimental design: I believe the research fits the scope of PeerJ.

Research question defines well and fills an unknown knowledge gap.

Methods are robust, but some parts (especially the linear regression part) need further clarification.
Validity of the findings: Data is robust and findings are significant.
Additional comments: Please see the annotated pdf for revision.


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

Input:
Title: ASSOCIATION OF URBAN FOREST LANDSCAPE CHARACTERISTICS WITH BIOMASS AND SOIL CARBON STOCKS IN HARBIN CITY, NORTHEASTERN CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The English need improving in this manuscript as it is difficult to follow and incorrect words are used in places. e.g. L74 coped - this is not the correct word - do you mean coupled? This occurs throughout manuscript - in another place the authors say mean average - they mean the same thing.
The relevant references are there it is just that the clarity of the writing needs improving - the manuscript could certainly be shortened.
Although in general the figures and tables are well presented and described there are issues.  see below.
Experimental design: I think the aims of this research are interesting but they are not well defined and I think there are problems with the independence of the way they have set up the scaling up work.
Description of the sampling in the methods is now much better.
Validity of the findings: Fig. 5 - where are the error bars on this figure? Presumably you used an average in each class?  Were the statistics run on the site specific data or the averages?
Table 2 - need to provide area figs for each of the different classes.  Also it is unclear why you get different storage figures when you estimate in different ways here.  If you think that Forest type, Administrative district, ring road and history all effect carbon storage then they should be not be treated independently or used to calculate 4 different storage estimates.  For example, presumably LF occurred in all administrative districts, and across the urban gradient. In addition in relation to this and other averages presented there are no errors associated with the averages provided.
I do not think that the discussion is well written and does not concisely bring together the results and is also in some places lacking references - e.g. see l292-304.
Table 4 - what is area mean? if you are using an area mean there is only one point and so how have you run a correlation - this in part would be solved if you had presented the d.f. or sample size in the analyses. Presumably the area mn is based on some kind of classification of the different patch area mean in different groups (e.g. LF)?
Additional comments: L123 - do you mean million not billion
Please try to report the data using the same number of decimal places.
L219 - presenting total storage in different districts is pretty meaningless to a global audience - there are no data provided on the area of trees in each district.
L395 - comparing the topsoil and aboveground carbon storage only beneath trees doesn't give any indication of the importance across an urban area of soil carbon storage relative to aboveground storage.  Grassland covers the majority of the greenspace in an urban area and the carbon storage there is considerable greater than that aboveground.
l415-416 - there is no practical mention of how and where this afforestation might occur - it is not going to really be feasible in existing greyspaces and so generally it will occur in the existing grassland in the city.


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: ASSOCIATION OF URBAN FOREST LANDSCAPE CHARACTERISTICS WITH BIOMASS AND SOIL CARBON STOCKS IN HARBIN CITY, NORTHEASTERN CHINA
Review round: 2
Reviewer: 2

Output:
Basic reporting: English and literature cited are fine. Figs and tables need some improvement (see attachment).
Experimental design: Research fits with PeerJ scope. Research question defines well, and methods are robust.
Validity of the findings: Findings provide insights about urban forest fragmentation at Harbin.
Additional comments: After the major revision, the manuscript improves significantly. However, several minor revisions are needed to make this manuscript more completed (see attachment). The only thing I am concerned now is the accuracy assessment of image processing. Please follow my advice and improve it.


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: ASSOCIATION OF URBAN FOREST LANDSCAPE CHARACTERISTICS WITH BIOMASS AND SOIL CARBON STOCKS IN HARBIN CITY, NORTHEASTERN CHINA
Review round: 3
Reviewer: 1

Output:
Basic reporting: Reporting int he manusucript is fine.
Experimental design: This is adequate.
Validity of the findings: Findings are valid.
Additional comments: L330 doesn't make sense.
Abstract results - no mention of soil just forest cover.
L384 - you have no evidence that tree planting in greenspace will increase soil carbon storage.  You have not examined whether there are any differences in soil carbon between tree covered soils and soils in greenspaces covere with other vegetation.


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: SIZE-DEPENDENT FUNCTIONAL RESPONSE OF XENOPUS LAEVIS FEEDING ON MOSQUITO LARVAE
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.A. The article reports on the results of mesocosm experiments to determine the functional response on Xenopus laevis frogs on one size class of larvae of a mosquito species.  The basic presentation is good, although there are a number of small points where wording could be improved or there is lack of clarity; these are listed below.  The main substantive point here is that it is not clearly stated that a single predator individual is used (?) in each mesocosm (this should be in the first paragraph of experimental procedure).  If this is the case, there should be some quantification of between-individual variation in numbers eaten at each of the five prey densities for at least one (and maybe all three) predator size classes.
1.B. Specific corrections
line 70 Holling not Hollings (and maybe also note that he proposed a type-4, which has in fact been observed in many studies (Jeschke review), and predicted in a variety of scenarios (Abrams 1989).
line 79 – Note that in addition, a physical refuge in limited supply can create a type III response.
line 80-82 This sentence is too long and disjointed-rewording needed
line 155 Maybe add some justification for range of prey densities and for the distinct and narrow size class ranges.
line 259 "…data show a treat similar to…" would be better
line 264- 'is' should be 'in'
line 304 Don't you mean 'predator' rather than 'prey'
line 330 'which' should be 'with'??
line 332 'their' should be 'the'
line 335 Delete 'a' before 'higher'
line 339 –Doesn't a lower handling time mean a HIGHER feeding rate?
Experimental design: The design of the experiments is fairly traditional for functional response studies.  However, this design can be misleading, and, as noted above, the presentation did not make it totally clear that there was one predator individual per mesocosm (I am assuming this, as no numbers were given).  There was also no data presented on proportion of prey eaten.  This is important for estimating parameters (using the equation on line 193) to be used in an expression (the functional response) that assumes a fixed density.  The 48 hour starvation period can have different effects on different size classes; certainly it is likely that a large individual would have more energy reserves and would be less motivated to forage at a high rate.  It would have been informative to examine responses over periods longer than the 14 hours used here.
	Theory predicts that variation in foraging time or effort can be a large component of the effective 'handling time' in a type II response (Abrams 1991), and certainly digestion is likely to play a role. Actual physical handling time has been shown to underestimate 'h' relative to the 'h' obtained by fitting a response curve in several studies. There should have been some comparison of the parameter estimates based on actual consumption and those based on the video analysis.
	It is not clear how an 'encounter' was identified.
Validity of the findings: The manuscript is structured around the theme that using a single predator size class to determine the functional response may be misleading in terms of the predator's effect on the prey.  I do not think this is a good message.  In the first place, it is widely known that size affects functional response parameters. The claim that "Often predators of the same species are assumed to be functionally equivalent" (line 28, 43 and later—e.g. line 251) is not backed up with references. People who work populations having significant size variation are unlikely to assume all sizes have the same functional response.  It is true that classical models assume this (line 57), but they also assume that every predator is identical in EVERY respect, and it is not clear that the assumption of identical functional responses is worse that the assumption of identical numerical responses for all individuals.  The reality is that these simple models are not meant to make quantitative predictions about any particular system.  Theoreticians who have worked on size-structured models of predator and prey (e.g. deRoos and Persson book) have not assumed identical functional responses in all size classes. Using this argument is particularly problematic here in that the prey also has size structure, but only one size class is used in the experiments.  If one wanted to build a predictive model, one would want to know susceptibility to predation of all prey sizes as well.  This issue was examined in detail by McCoy et al (2011), an article that is mentioned briefly here and shares an author with this manuscript.  It would be interesting to have some discussion of the combined implications of size structure in both species or speculation about which is more important for modeling.
3B. The two main findings are the type 3 response in the two larger size classes, but not the smallest, and the change in attack and handling parameters with size. There could be a more general discussion of hypotheses that could account for the type-3 response.  The frogs are likely to be balancing costs and benefits of foraging, which is a major mechanism that could produce type-3 responses.
3.C. The effective functional response of a population having individuals with varying parameters is not the same as a response based on all individuals having mean parameter values unless it is linear (Chesson).  It would therefore be of interest to see the range of individual capture rates at each of the densities (a plot with actual consumption rates of all individual's rates given as dots above each of the 5 prey density treatments in fig. 1)
3.D. Various other small issues
line 270 – It does not seem that handling time necessarily increases with larger predator size.
line 283 – Low enough densities of any prey will fail to provide enough reward for foraging.  (Sarnelle et al 2015 CJFAS is an example—several theoretical works predicted this much earlier)  This lower threshold should logically be lower for smaller individuals.
line 237  I did not see any analysis of how parameters based on video analysis differed from those based on data fitting; shouldn't this be present?
line 297 – The claim here seems to assume synchronous development of all frogs; is this the case?  If not, the level of synchrony may be needed to estimate the effect on prey.
l. 299- A more accurate sentence would say 'There are some examples of a unimodal relationship between attack rate and predator size.'
l. 311 – It seems likely that attack behaviors/rates are adapted to the spectrum of prey sizes normally encountered (a broader range and no doubt different mean than in your experiments).
l. 335 and rest of paragraph – Whether a given feature is stabilizing in nature depends on the full response, which includes many other species and size classes—I'd recommend dropping the discussion of stability.
line 355 – A claim about many studies doing something should be backed up by more than one reference or it should be clear that the study referred to is or contains a review of many studies.  I have not read the two Dick et al. studies referred to in the paper, so a wording change may be enough.  However, standard ecological theory implies that the resource requirements and degree of generalization of the predator are much more important than the shape (or even mean value across prey densities) of the functional response in determining the impact of an invasive species.
Additional comments: The authors are right in claiming that few studies examine the predator-size dependence of its functional response, and it would be good to have this example published.  However, the conclusions reached are often stated too broadly, and they should be qualified by noting the absence of other prey types and sizes, lack of examination of predator-dependence and other-species dependence of the functional response (see following paragraph).  There also needs to be some discussion of the possibility that the pre-experiment 48 hour fast affected size classes differently and the possibility that the lowest prey density was not sufficiently low for detection of a type-3 response in the smallest size class.

This article had more references to old literature than most I've reviewed recently—this is good.  However, there were some important ones left out.  For example, there are older studies on size dependence of responses than the ones listed on line 64; the Eveleigh and Chant study in the references is one of these.  The traditional approach to functional responses approaches this relationship as a rate of feeding by an isolated predator on a single prey population.  This has been widely criticized, and there is general acceptance that predator density frequently influences the response (DeAngelis et al 1975; Abrams and Ginzburg 2000; Abrams 2015).  In addition, the presence of higher level predators, other prey, animals that may be confused as prey, etc. all affect predator rates of feeding, and are therefore parts of any functional response that could be used in a predictive model of population dynamics.  There should be some acknowledgement that what is dealt with here could easily be very different from what the relationship would be in the presence of other prey, higher level predators, etc.


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

Input:
Title: SIZE-DEPENDENT FUNCTIONAL RESPONSE OF XENOPUS LAEVIS FEEDING ON MOSQUITO LARVAE
Review round: 1
Reviewer: 2

Output:
Basic reporting: See PDF for notes on clarity.
Experimental design: no comment
Validity of the findings: See PDF for notes on the discussion and conclusion.
Additional comments: Manuscript number: 24758
Full title: Size-dependent functional response of Xenopus laevis on mosquito larvae
Article type: Primary research paper

Reviewer overview:
This manuscript focuses on the dependence of functional responses on predator
body size. Specifically, the authors measure feeding rates of three size classes of
African clawed frogs on different densities of mosquito prey. They found that
functional response type was dependent on predator size. Additionally, both
observational data and parameter estimates found body size dependence of attack
rate and handling time. This work contributes to our understanding of intraspecific
variation in predator feeding rates and its potential impact on prey populations.
Overall, the research is interesting and well designed.

Major concerns: It is not clear why the authors use both observations and model
estimates to determine attack rate and handling time. The manuscript may benefit
by expanding the introduction to include predictions of the observational data and
rearranging the discussion to make clear links between parameter estimates
derived from models and mechanisms from observations of predator behavior.

Minor concerns:
Line 61, 94: effect of prey size on predator-prey interactions is outside the scope of
this manuscript and does not need to be included in the introduction.
Line 63: “these [studies] highlight”.
Line 72: define attack rate and handling time.
Line 74: Type II functional response is destabilizing as predators are unable to
regulate prey populations at densities beyond predator satiation.
Line 82-85: To someone unfamiliar with functional response models, it is unclear
that parameter estimates derived from models fit with data on feeding rates is not
the same as estimating parameters from observational data on behavior i.e. how are
the measurements of attack rate and handling time different between the two
methods? Do they provide different expectations? How does observational data
provide mechanistic understanding of variation in attack rate and handling time?
Line 88: Environmental conditions are outside the scope of this manuscript and do
not need to be included in the introduction.
Line 92: Prey size is not tested in this experiment and should not be in the
introduction as it leads the reader to believe that prey size will be part of the
experimental design.
Line 98: hypotheses for the effect of predator body size on handling times are
presented but there are no hypotheses for the effect of predator body size on attack
rates, unless “smaller predators…limited by” means predator attack rates are
constrained by predator body size.
Line 102: broader range of prey species or prey sizes?
Line 132-134: repetitive – edit for clarity. E.g. “temporary surface-water habitats”
and “colonize newly formed aquatic habitats”.
Line 149: “to quantify the functional response…dependent on consumer body size”.
Line 152-153: are these size classes the same life stage? If not, can you differentiate
the effect of body size vs life stage (Rudolf and Rassmussen 2013)?
Line 195: is there a difference between attack rate and the search coefficient? Table
2 and Figure 2 show search coefficient as used in equation 1, however the research
question posed in line 114 focuses on attack rate.
Line 240: What about the other components of handling time?
Line 244: What is scooping behavior?
Line 260: the phrase “handling efficiency” is confusing since “attack efficiency” is a
variable measured in the experiment and “handling efficiency” is a description of
predator behavior.
Line 287: contradicts line 244. All predators exhibited scooping behavior, but the
smallest predators exhibited sweeping behavior.
Line 288: contradicts findings in table 3.
Line 292-295: remove.
Line 297: Does level of predator ontogeny refer to body size or life stage?
Line 307: Additional size classes on the lower end?
Line 312-318: remove. Move lines 318-324 under earlier paragraph on predator
behavior.
Line 328-329: confusing- “smaller predators being more limited by handling times
than large prey”.
Line 342: high densities of predators can lead to increased intraspecific competition
and subsequent changes to an individual’s functional response, which can also be
size-depedent.
Line 343: Unclear how X. gilli is relevant to this study.
Line 359-362: the conclusion is not the place for new information. Move to
discussion.


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: SIZE-DEPENDENT FUNCTIONAL RESPONSE OF XENOPUS LAEVIS FEEDING ON MOSQUITO LARVAE
Review round: 2
Reviewer: 1

Output:
Basic reporting: Basic reporting is good.  A few ambiguities are point out in the comments.
Experimental design: The experimental design is appropriate.
Validity of the findings: The findings are well justified.
Additional comments: line 72-73.  This phrase; 'proportion of prey consumed is not constant', is vague and many interpretations consistent with this wording would be inaccurate.  Would be better to say; 'the rate of prey consumption by a predator divided by prey abundance declines with prey abundance', which is the defining characteristic of the type 2 response.  Although handling time is a common reason for this, it is not the only one (see Abrams 1990 Ecology).  It would be better to say h is a measure of the nonlinearity of the response, and it corresponds to the amount of time required to handle a capture prey when an inability to capture prey while handling is the cause of the nonlinearity.

line 74 – 'due to high consumption rates at low densities' is not correct.  The destabilization is due to the positive feedback on prey population growth caused by decreased predator consumption rates as a prey population increases.

76-77  Type three is defined by an accelerating increase in prey capture with increasing prey density for a range of low prey densities.  (all responses have low capture at low density)

84-85  I'm unclear about what this last sentence contributes.

214-215  This is confusing- is there a difference between 'large' frogs and 'largest size class'?  I think you mean 'medium' rather than 'large'

227 –'often assumed to be equivalent' This would be stronger with some examples.  It is obviously true in simple models, but these assume everything is identical between individuals.

247 "BOTH zooplankton and…"

265  There is also a possibility that the relative fitness gain from small prey items is too small to make it worthwhile for larger foragers to be active.

281 The paragraph beginning here is somewhat long and you might want to split it with potential explanations for why the observed difference in F.R. shape occurred in the second one.  In that respect Abrams 1991 Ecology and Abrams and Rowe 1996 Evolution both suggest that adults should take fewer risks and are more likely to have accelerating responses at low prey densities—basically because survival is more important relative to growth in adults than in young, which must grow quickly to escape a life stage with high unavoidable mortality.  This was the point I was making with the line 283 comment (Sarnelle reference) in my earlier review.

Since there is a good deal of text about the importance of functional responses for applied questions, it might be good for the authors to acknowledge that shape of a response with respect to one prey is known to be affected (in theory, empirically or both) by the presence and abundance of other prey and of predators.  Even Holling's and Murdoch's early work discussed type 3 arising from the presence of alternative prey.  The response letter indicates the authors think that this is too big a topic to deal with; I think the acknowledgement of the limitation of results to the response to a single prey is important in this case of a generalist predator.

line 329-I think I signed the original review-Peter Abrams


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: SIZE-DEPENDENT FUNCTIONAL RESPONSE OF XENOPUS LAEVIS FEEDING ON MOSQUITO LARVAE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors focus on quantifying the size-dependence of the functional response, a growing interest in food web ecology and important for understanding intra-specific functional diversity. The authors present sufficient background that are generally appropriate for the context of this paper. The following lines can be improved for clarity (line number based on word document with track changes by the authors):

ln 68: Still missing definitions of functional response, attack rate, handling time, therefore found this sentence out of place.

ln 68: This sentence may be better suited in the following paragraph (ln 76) since the paragraph following the next one (ln 103) reviews some of the causes of variation in functional forms. This is probably the most important sentence of the intro as it highlights whats missing in functional response lit and how this study seeks to fill that gap.

ln 76: Found this sentence in the rebuttal document - “The functional response is the key relationship linking predator and prey dynamics and describes the relationship between a predators uptake of prey as a function of the prey density.” Did not see this in the PDF or Word document with track changes. This sentence defines the functional responses whereas a previous version of the sentence did not.

ln 110: I’m not sure the connection between this example and your experiment, or with the previous statement if the focus of this experiment is varying predator size while keeping prey size constant.  Is it that a predator can have the same prey over its lifetime, but there’s an optimal predator:prey size that maximizes feeding rates, and therefore preferences on prey size (Brose 2010, but see Uiterwaal et al 2017)? Maybe this point is better suited in the discussion.

ln 172: Not sure if this is correct wording. You’re estimating the form of the functional response; you’re using the functional response to quantify the effect of predator body size on prey consumption

ln 189: remove during?

ln 282-288: the authors state that this reference shows the form of the functional response changes with prey size, but proceed to describe predator size. Is this a typo, or did the reference alter both predator and prey size?

ln 489: Do smaller predators have higher handling times than larger predators or do they have longer handling times on large prey?

ln 502: This sentence is confusing following the previous sentence following prey densities. Also, are you trying to say that having a lot of predators means that they’re going to control prey populations i.e. what about predator dependence of functional response?

ln 520: Invasive predators aren’t mentioned in the intro or in the methods, so this statement was confusing to me.
Experimental design: With the removal of the video component, the aims, methods, and results are more clear in this version. A few comments to clarify the experimental design:

ln 115: predictions on the relationship between body size and handling time are presented, but missing prediction on the relationship between body size and attack rate.

ln 165: Unclear the range of volume used in the experiment. ± what volume?

ln 225: What indicates a type I response?

ln 238: The intro refers to attack rates, the methods refer to search coefficient. How are these related?
Validity of the findings: A few comments to clarify the authors' findings and add to the discussion:

ln 249: more appropriate for methods section, not results

ln 302: change "different from" to "different between"

Figure 1: Discuss possible reasons for the large variation in feeding for large predators at the highest prey density.

ln 363: authors state that increased handling times may be due to prey being too large to ingest via suction, but later state that this is not the case in their study (ln 467). As this paragraph is a discussion about handling time, moving this observation and including a discussion on its implications on the experimental results to this paragraph would be more appropriate.

ln 463: can you discuss possible reasons for the similarity in the search coefficient between medium and large predators?

ln 475: Might be more appropriate to discuss after ln 453 as it mentions predator detection.

ln 477: Does detection ability/success vary between the different size predators?
Additional comments: In the conclusion, the authors state that it is important to measure the functional response using different prey. An interesting question would be how prey traits affect the functional response i.e. is it prey size alone that affects the functional response, meaning different species of the same size are functionally similar, and therefore predator-prey relationships are predictable based on this trait, are other traits important, or do other traits interact with body size? A focus on traits, rather than species itself, may lead to generalities in predator-prey interactions.

Overall, well written, clear aims, and interesting findings.


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

Input:
Title: EVOLUTION OF AFRICAN BARBS FROM THE LAKE VICTORIA DRAINAGE SYSTEM, KENYA
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Overall this paper follows a standard approach to addressing phylogenetic questions and it mostly does a good job.  The writing is mostly clear and easy to follow.  The two limitations to the study are having only two genes, but a 3-5 years ago this would have been quite good!  The second is the sampling is a little bit limited, but it builds on some broader previous work, thus it is able to related their results back to the broader picture of Barbus in that region of Africa.

My one larger question that I feel the paper has not addressed is why did previous studies, especially Schmidt et al find so much introgression, including in samples from your study region, yet you did not?  You don’t really discuss this point much, although you do emphasise that you didn’t find introgression, but Schmidt did.  Are these species often sympatric?  Any study can get some identifications incorrect, but presumably they can’t all be wrong?  (I didn’t look at Schmidt et al, but presume they found discordance between mitochondrial and nuclear DNA results?  If so, suggested identifications are ok if nuclear markers normally were consistent with their identifications).  Presumably the introgression is older, rather than ongoing today?  If so the contrast in your findings is perhaps a bit odd.  I don’t know what the answers are, but you should include some discussion of this incongruence between your studies.

Most of my more minor comments are below in the order they appear in the manuscript.

Introduction

Line 48, approx. how many species of barbs / cyprinids in Africa?  You do mention a number lower down, but I’m left wondering for a little bit.

Line 77 and 81, barb, not barbs (fix in the abstract too)

Line 118, rewrite sentence to improve English

Methods

Why was Catostomidae used as an outgroup initially?  Is that based on some previous phylogenetic work that suggest they are sister groups?

How did you deal with heterozygous positions in growth hormone?  I presume you used standard ambiguity codes?  Should mention this.

You have not mentioned how you calculated your K2P distances anywhere.

Results and Discussion

You should align your main objectives at the end of the introduction (line 123) with the order that you present them in the discussion (or visa versa).  For instance, your first key objective listed is the last one you address in the discussion.

You don’t provide the models chosen by partionfinder or jmodeltest for either gene, you also don’t give any indication of what partition schemes you explored under partition finder, did you try it by codon position, or just a single partition?  I eventually see that you included these details in Table 1, make it clearer in line 184 what is in Table 1, i.e., best models found and partition schemes tested are provided in Table 1.

When you present some of your K2P distances, I think you are off by one decimal place, e.g., line 222, Maximum Cytb divergence within this clade was 0.39% (K2P), this should be 3.9% I think.  The scale bar on the tree is 0.04, which is equivalent to 4% sequence divergence.

Line 257, max. within clade divergence = 0.59% K2P.  I don’t think this statement is correct, there is almost no within clade divergence in ‘B.’ profundus.  I think you mean between clade divergence to it’s nearest sister group perhaps?  Unclear.

Line 278, make it clear at the start which gene you are talking about, you currently just say haplotype.  How can you be sure that the Schmidt specimen is misidentified vs introgressed?

BTW, it should be RAxML, in many cases you have written RaxML which is what word autocorrects it to.

Shouldn’t start a sentence with an abbreviated genus, spell it out in full.