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: LARGE-SCALE MOVEMENTS OF COMMON BOTTLENOSE DOLPHINS IN THE ATLANTIC: DOLPHINS WITH AN INTERNATIONAL COURTYARD
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript provides useful information on the actual and potential connectivity among local populations of bottlenose dolphins in various Macaronesian island systems, and the Portuguese mainland. It is a nice example of collaboration among research groups, and nicely shows the mobility of these animals. In this regard, it is certainly a useful study worth to be published. I commend the authors on undertaking this important work.

However, I do have some concerns.

There are some unclear aspects and inconsistencies. Moreover, the authors place quite a bit of emphasis on the distances travelled in the Discussion, but they do not actually provide these distances in the Results (or elsewhere).

The treatment of existing literature is somewhat incomplete, as some relevant literature sources are missing from the manuscript.

In addition, the manuscript would benefit from proof-reading and revision for language.

I provide some specific comments below and encourage authors to carefully consider them when revising their manuscript.
Experimental design: Description of methods is rather vague and details are missing to get a good idea of what was done and why. There may be some important caveats (photographic quality), which may have significant influence on the results, but that do not appear to be addressed at all. Moreover, certain terminology is used either incorrectly or is at least a bit misleading – for example the “social analysis” is not truly a social analysis, and should therefore be framed a bit differently. This is not to say that the exercise of “mapping” known connections among individuals is not useful – it is – but it should be clearly identified as such, and not confused with social analysis, because that gives the impression of this being a social structure or a social network study.
These issues should be resolved before the manuscript is appropriate for publication.
Validity of the findings: As stated above, this manuscript provides useful information on the actual and potential connectivity among local populations of bottlenose dolphins in various Macaronesian island systems, and the Portuguese mainland. The results themselves are interesting and important enough.

However, in some places the authors infer or speculate beyond what the data show. I am not necessarily discouraging the authors to explore various implications, but they should "tone down" the implied certainty or relevance. More details in the specific comments.

As mentioned above, there are some methodological consideration which may affect results. These should either be incorporated in the study better, or at least clearly and carefully addressed in the discussion.
Additional comments: SPECIFIC COMMENTS

TITLE: My suggestion would be to drop the “novel insights” and just keep “Large-scale movements of common bottlenose dolphins in the NE Atlantic: dolphins with an international courtyard”.
This way the title is both shorter (and “cooler” in my view), without trying to force the “novel insights” into it. With “novel insights”, it sounds as if you are merely providing some new insights into an already studied thing. But instead, as far as I’m aware, this large-scale study is brand new – so remove the “novel insights” accordingly.

ABSTRACT
L39: Replace “photos” with “photographs”. Also, a space is missing between up and 1791.

L43: “as well as among the three archipelagos” – This is confusing. By the three archipelagos I presume you mean Azores, Madeira and Canaries? If so, the way it is written sounds as if no matches were made among those. But you just said a few lines earlier that there were matches between Azores and Madeira and Madeira and Canaries. So something doesn’t add up here.

L46: What are “all known residency patterns”? Please re-word of remove from the abstract. I understand it is probably explained better later on in the paper. However, the abstract should be almost as a “stand-alone” piece explaining the study, so it should be clear on its own.

INTRODUCTION

L60: Range implies overall distribution range of the species. You probably mean “ranging patterns” or “home range” of specific populations? Please re-word accordingly.

L61: Coastal, not costal.

L63: Oudejans et al. 2015 is not in reference list.

MATERIALS AND METHODS

L97: please replace “habitat” with “area”. Also, change “in” to “along” or “off” Iberian peninsula.

L102: Why is there an apostrophe in “cetacean” (cetacean’)...?

L107-108: “representing three different archipelagos, and a small area on the coastal Portuguese mainland” - This has been said already, please avoid unnecessary repetitions.

L112: “compared” – I suggest replacing to “used” or “included”, because the way it is written now, it makes it seems that those two catalogues were compared to each other (rather than to other catalogues as well).

L114-115: “A third set of data from this region, containing 201 photos of 42 individuals collected from 2014-2016 was added.”
Wait... does this still refer to Azores? If so, why make it confusing like this? Just say they were 3 catalogues from the beginning, rather than first saying there were two, and then saying another one was added... If there were three, then say it was three from the start.

L117: “from 2014” – I assume this means only in year 2014 (as opposed to from 2014 onwards)? To avoid confusion, remove “from” and just leave the year.

L128: Please explain what you mean by “ambiguous matches”.

L129: But not false negatives...?

L122-129: There is no mention of whether photographic quality was assessed and how. This has major implications on the results and relates back to the ambiguity and false positives (and false negatives). This is a major issue and if not addressed, may seriously bias the reported results. This should be either incorporated into the analysis, or at the very least incorporated into the caveats and interpretation of results in the Discussion. Poor photographic quality may create matches where there are none (false positives) or create absence of matches where there actually should be some (false negatives). Both of these may severely affect the outcomes of matching. This is something the authors need to consider.

L132-133: Please explain a bit why only Madeira animals were used for this. In other words, if the idea was to get an overall sense of connectivity among all putative local populations, then it would make sense to include all. On the other hand, if the idea was to only look at social connectivity among Madeira animals, then why was not the same done for other places such as Azores, etc.? It seems a bit arbitrary.

L137: Some revision of language and spelling needed here (“remain” individuals, “felled”,...).

L137-139: This statement is not logical. Interactions between dyads form part of the social interactions at the level of the community. These terms are not mutually exclusive. Even when looking at interactions at the level of community, the sighting frequency is still important, so the logic of the statement does not hold. However, I do understand what the likely aim was here: to get the basic idea of connectedness among individuals. If so, I suggest this is re-worded, so it accurately reflects that. Also, the sub-heading should be changed – this is not really “social analysis”, it is a crude estimate of connectedness (which is fine and useful). Therefore, please re-word accordingly, otherwise this gives the false impression that the authors carried out an actual social analysis, which is not the case.

L140-142: It is not entirely clear to me what was done or why. More details are needed here, with a better explanation. For example, since the paragraph starts with saying only Madeira animals were included, it is confusing where “with Macaronesian individuals” fit in here. If I am reading this correctly, the authors formed a network of Madeira animals and then added “Macaronesian individuals” (whatever this means – are these all other animals, or only those animals re-sighted between Madeira and other island systems...?), but this is not entirely clear. Also, unclear what “main clusters” refers to. Furthermore, “to highlight their associations” – again not sure what exactly is meant by this, because some of these associations can be misleading if animals seen only once were included (see previous comments about this).
So, more detail and clarity is needed here. It is all a bit vague at the moment.

L142: Please provide more detail about why and how this was included as an attribute in the analysis. I may try to guess this because I know what “attribute” means in the context of social analysis, but it is not immediately apparent, especially not to a general reader. Also, it is unclear to me how this attribute was incorporated into the analysis.
(re-reading this now, I realise you probably meant how this is labelled in Figure 6 – in this case, you can remove this sentence here altogether, because Figure 6 already provides that information)

RESULTS

L148: Some revision of language needed.

L148: Please be specific. What does “most of which” mean? Please say exactly how many matches between Madeira and Canaries, and how many between other places. It is too vague.
Also, be specific in both the number of matches but also number of animals.

L149: “Additionally, three individuals were seen both in the Azores and the Madeira archipelagos”
Additionally to what? To those 26 matches? Or is this part of the 26? It is not clear, because previous sentence says MOST of 26 matches were between Madeira and Canaries (but not all). So, are these three individuals part of those remaining matches among 26, or in addition to that?

L151: But what about Canaries-Sagres and Azores-Sagres...? Why are these not included in the comparisons?

L166: What do you mean by “possibly accompanied by”? Were they seen together or not? If not, do not speculate beyond data. Or if speculation is warranted, leave it for Discussion, not Results.

L173: Please provide movement distances for all animals.

L178: “...including 20 Macaronesian individuals seen in association...” – What does this mean?

L178: Which diagram? You should refer to specific figures.

L180-183: Some sentence re-wording is needed for proper language structure.

L182: I may be missing something, but from Fig 6, it does not show that animals seen in both Azores and Madeira associated with “migrants”, because no lines goes from them to other migrant (yellow) animals.

DISCUSSION

L186-187: This is demonstrably not true, because the authors state in the Results (and Figures) that no animal was seen in all three archipelagos. And yet, now the authors state that 26 dolphins moved between three archipelagos. Please clarify the sentence to accurately reflect which is the case.

L189: Why is “AND” capitalised?

L193-195: This is a rather incomplete list. See some more examples below, and incorporate them into the manuscript:
- Gnone, G., Bellingeri, M., Dhermain, F., Dupraz, F., Nuti, S., Bedocchi, D., ... & Azzellino, A. (2011). Distribution, abundance, and movements of the bottlenose dolphin (Tursiops truncatus) in the Pelagos Sanctuary MPA (north‐west Mediterranean Sea). Aquatic Conservation: Marine and Freshwater Ecosystems, 21(4), 372-388.
- Hwang, A., Defran, R. H., Bearzi, M., Maldini, D., Saylan, C. A., Lang, A. R., ... & Weller, D. W. (2014). Coastal range and movements of common bottlenose dolphins off California and Baja California, Mexico. Bulletin, Southern California Academy of Sciences, 113(1), 1-13.
- Defran, R. H., Weller, D. W., Kelly, D. L., & Espinosa, M. A. (1999). Range characteristics of Pacific coast bottlenose dolphins (Tursiops truncatus) in the Southern California Bight. Marine Mammal Science, 15(2), 381-393.

Also, albeit shorter distances, but also possibly useful to consider in the context of bottlenose dolphin photo-ID and movements, may be:
- Bearzi, G., Bonizzoni, S., & Gonzalvo, J. (2011). Mid-distance movements of common bottlenose dolphins in the coastal waters of Greece. Journal of Ethology, 29(2), 369-374.
- Genov, T., Angelini, V., Hace, A., Palmisano, G., Petelin, B., Malacic, V., ... & Mazzariol, S. (2016). Mid-distance re-sighting of a common bottlenose dolphin in the northern Adriatic Sea: insight into regional movement patterns. Marine Biological Association of the United Kingdom. Journal of the Marine Biological Association of the United Kingdom, 96(4), 909.

L196-197: “The distances reported here for the individuals that moved between Madeira and the Canary Islands are comparable...” – The authors have not provided any distances in the Results, and one cannot readily infer that from the maps. Please provide actual movement distances for all individuals seen in different archipelagos.

L198-199: Impossible to assess this statement, because there are no distances reported.

L207-210: This statement is a bit vague and not very informative. What was the aim here, what is the point the authors are making?

L207-208: You may want to consider the following (more recent) relevant publication on this topic: Bräger and Bräger 2019: Movement Patterns of Odontocetes Through Space and Time. In: B. Würsig (ed.), Ethology and Behavioral Ecology of Odontocetes, Ethology and Behavioral Ecology of Marine Mammals, https://doi.org/10.1007/978-3-030-16663-2_6.

L214: comparably

L221: Please add the species name here, because Shark Bay dolphins are not the same species.

L222-223: “will be improved”, “will decrease” – How can you be so sure? “Will” implies certainty. Consider replacing with “may” or “could”.

L224: What is “(11)”? This should be a citation, I presume.

L242-243: Also see the following in relation to this, showing marked within-population variability, and making the point of variability among populations:
Genov, T., Centrih, T., Kotnjek, P., & Hace, A. (2019). Behavioural and temporal partitioning of dolphin social groups in the northern Adriatic Sea. Marine biology, 166(1), 11.

L247-250: These results do not show that. I’m afraid the authors are making inferences beyond the available data. Yes, the authors can provide this speculation as one way of interpreting results, but please “tone down” the certainty related to this. You may certainly refer to it as a possibility, but please do not say that results show this, because they do not.

L262: Some revision of English is needed (find, found).

L269-270: This is very vague – please provide more rationale.

CONCLUSIONS

L273: Does it? I thought so too, but the authors themselves report the study of Tobeña, so apparently this study does not provide first evidence of this. Please remove “first”. It is still relevant enough without it.

L275-276: Please provide references.

L282-285: Some references would be good here.

L297: What do you mean “shall be effective”? Of course they should be effective, but not clear what this statement aims to say.

L307-308: Please provide the justification why this would be the most effective way. I am not saying a sanctuary is not a good way forward, but I am not convinced it is necessarily the most effective way. For example, you may have no MPA at all, but the regulation of issues/threats (fisheries, for example, or dolphin-watching) may actually provide greater benefit to the animals – which does not need to be in the form of an MPA necessarily.
This statement is too generic and not supported. So either provide a better justification (and some references), or remove it.

FIGURES: Please add movement distances into Figures 3-5.

FIGURE 6: The additional levels of symbols, and the description and legend of location is not needed, because it location is already depicted by squares and the names of the locations, so the extra legend is a bit obsolete.


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: LARGE-SCALE MOVEMENTS OF COMMON BOTTLENOSE DOLPHINS IN THE ATLANTIC: DOLPHINS WITH AN INTERNATIONAL COURTYARD
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: It’s not stated but did the researcher review each photo/fin from these separate catalogues for quality and distinctiveness?  I understand that each institution/researcher will have its own way of procedures including grading the photographs for quality (focus, contrast etc.) and for dolphin dorsal fin distinctiveness but it is well known that various researchers don’t see quality and distinctiveness the same (Urian et al. 2015) which can add to false positives/negatives.  To avoid the ambiguity of grading between different researchers/intuitions and different catalogues I suggest the researcher reviewing the photos for the greater Macronesia catalogue should separately grade for distinctiveness and quality for each photo/dolphin when reviewing them for that catalogue.  It then could be reported on how many photos/individuals were removed due to low quality/distinctiveness if indeed any were.

It’s reported that matches were confirmed by a second researcher, but I am more concerned with false negatives (missed match – recording one animal as two).  A second experienced research should review the entire catalogue for false negatives.  Even better would be to use a newer computer assisted matching program.  I am not familiar with Darwin, (and it seems that only one catalogue was used with Darwin) but comparing 1250 individual dolphins by naked eye is a huge undertaking which assumes some risk of human error.  Using a program such as FinFindR (https://github.com/haimeh/finFindR) or Flukebook would help a great deal with matching in a minimal amount of time (Thompson et. al 2019, Levenson et al. 2015).  I’d hate to see that they have missed a few matches that could add to the robustness of this manuscript due to human error.

Levenson, J., S. Gero, J. Van Oast, and J. Holmberg. 2015. Flukebook: a cloud-based photo-identification analysis tools for marine mammal research. Accessible at: https://www.flukebook.org

Thompson, J.W., Zero, V.H., Schwacke, L.H., Speakman, T.R., Quigley, B.M., Morey, J.S. and McDonald, T.L., 2019. finFindR: Computer-assisted Recognition and Identification of Bottlenose Dolphin Photos in R. bioRxiv, p.825661.

Urian, K., Gorgone, A., Read, A., Balmer, B., Wells, R.S., Berggren, P., Durban, J., Eguchi, T., Rayment, W. and Hammond, P.S., 2015. Recommendations for photo‐identification methods used in capture‐recapture models with cetaceans. Marine Mammal Science, 31(1), pp.298-321.
Validity of the findings: no comment
Additional comments: This manuscript presents new movement information on the common bottlenose dolphin in this area.  I commend the authors for this important contribution which would add to the understanding of common bottlenose dolphins in the northeast Atlantic. However, there are a few issues stated in the "Experimental Design" section and in line specific comments that could be addressed that would strengthen the manuscript.  I hope you find them useful.

Line specific comments:

TITILE: NE should be written out.

ABSTRACT:
L30: insert “common” before bottlenose.

L31: replace NE with “North East (NE)”

L33: the K in “km” is not capitalized and further along in the manuscript it is. Be consistent.

L57: change “threats” to “anthropogenic disturbances, such as…”

L102-119: Would the numbers from the catalogues be more appropriate in the results rather than the methods?  I go back and forth on that.

L105: Please define the acronyms the first time in use. Replace “OOM” with “Oceanic Observatory of Madeira (OOM)”

L110: define “MONICET”

L115: define “SECAC”

L117: define “BIOECOMAC” and “MEER”

L121-141:Photo-identification Procedures – While I appreciate the changes in the methods, I would like to see a bit more clarification. Is the description of the photo-identification procedures from the individual catalogues? Or is it describing the methods that were used to create the greater Macronesia catalogue? It wasn’t very apparent at first. A bit of editing would improve the manuscript and give more clarification.

L138: Darwin 2.0 needs a reference.

L144: For clarity after “was compiled” insert “from the individual catalogues”

L148: “two or more locations were included…” Does this mean resighted two or more times or seen in two different archipelago or two island areas within each archipelago?

L148: “100% certainty” Were the photos also confirmed with a second experienced researcher?

L151-162 Associations.  I understand why the authors have included this, but I am not sure how much this adds to the manuscript without the use of more rigorous analysis. There isn’t much detail on how they manipulated the data for use in Netdraw.  SOCPROG could be used to help with this.  But there could be an issue on how the data was collected (from various researchers and different platforms) that the analysis could not be done in the first place.

L175: remove “.” after “Islands”

L229-230: Does the Azores archipelago not share these biogeographic and oceanographic characteristic? Please explain rather than just give the reference.  Shorter distance between the two archipelagos seems to be the reason for the matches.  Flip the sentences and expand on your reasoning.

L278: remove the parentheses around the word “much”

Figures 3-5: The exact sighting locations would be helpful.  Instead of a generic dolphin photo, perhaps photos the dorsal fins of the animals represented would be better.


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: EFFECTS OF FUNCTIONAL CORRECTION TRAINING ON INJURY RISK OF ATHLETES: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 	In some phrases some supporting references are missing. This problem is repeatedly seen in whole of the manuscript. Examples: lines 89 and 94
	Please explain the importance of research. Lines 130-138
	Explain more about FMS before the second paragraph and previous studies. Line 95
	In the discussion section, please use more theoretical foundations to better interpret the results.
Experimental design: Well done
Validity of the findings: Well done
Additional comments: Title: Effects of functional correction training on injury risk of athletes - a systematic review and meta-analysis.

This study investigated the Effects of functional correction training on injury risk of athletes in a systematic review and meta-analysis study.  Results indicated that the functional correction training can reduce the injury risk. Furthermore the functional correction training can improve the FMS™ scores. Even though the subject is very relevant and current, I have some concerns that I would like to be addressed before considering this paper 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: EFFECTS OF FUNCTIONAL CORRECTION TRAINING ON INJURY RISK OF ATHLETES: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: English proofread is absolutely necessary before the paper can be recommended for publication. This include the figures as well.

Otherwise, the reporting is more or less clear and concise. References are sufficient. A hypothesis should be included in the Introduction section.

Figures are well prepared (apart from grammatical mistakes).
Experimental design: Research question is well defined and relevant. The results of the literature review are important for practitioners and researchers. The search strategy was well designed.

The only major comment I have regarding the experimental design is related to inclusion/exclusion criteria: It is unclear what was the requirement for the control group. From what is written, I assume that the control group athletes continued with their regular training? Does this mean that your comparisons analyze regular training + corrective training versus regular training only? Or regular training vs. corrective training? I would also strongly suggest that the authors structure these criteria according to the PICOS tool for better clarity.
Validity of the findings: No comments.
Additional comments: Some specific suggestions for improvements:

ABSTRACT
I would omit the ‘’non-randomized’’ in the abstract background section. This could imply that you purposefully include only ‘’non-randomized’’ trials. You nicely state later that both randomized and non-randomized trials were considered. Maybe, instead of the background, you could start the results section with ‘’12 non-randomized trials were included into the meta-analysis’’.
Line 58. It is not clear what 1.7165 stand for. Is it the mean difference?
Line 62. Insert a full stop after ‘’athletes’’
Line 62-63. Either insert a full stop after ‘’effect’’, or write ‘’therefore’’ in small caps.

INTRODUCTION
Line 95. What do you mean by function-based exercise? Please explain more clearly in the manuscript.
Lines 130-138. What was your hypothesis after analyzing the available literature?

METHODS
Line 186. Bilateral instead of left and right.
Line 190-192. Please revise this sentence. It is unclear what you mean.
Line 225. You need to elaborate more on the sensitivity analysis. What exactly was done?

RESULTS

Line 252. Session, not lesson.
Line 328. Random, not randomized
Lines 342-347. This section should be integrated into the Discussion, not Results.

DISCUSSION
Line 377-378. Your review does not show WHY the training was effective. Therefore, you can only speculate about the reasons, and thereby, sentences such as this one are not appropriate.


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: EFFECTS OF FUNCTIONAL CORRECTION TRAINING ON INJURY RISK OF ATHLETES: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Tables and figures are clear for me, and looks attractive for readers. The references should be corrected. Because some of the citations cannot be found in reference list.
Authors should also consider about proper structure of this article, because some of the pages are double.
Experimental design: In my opinion some of sentences (highlighted in the text) should be explained more, for better understanding of international community. Methodoly are clearly descibed with the help of figures and tables.
Validity of the findings: No comment.
Additional comments: I believe that the article provides a good overview and summary of the FMS.
Readers are able to see problems occurring making researches, and to plan their own with better rigour.
This paper should be good base for preparing potential research with the use of FMS.
I hope if authors make cosmetic changes, this paper would be valuable for scientific world.


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: EFFECTS OF FUNCTIONAL CORRECTION TRAINING ON INJURY RISK OF ATHLETES: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: acceptance
Experimental design: OK
Validity of the findings: OK
Additional comments: OK


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: EFFECTS OF FUNCTIONAL CORRECTION TRAINING ON INJURY RISK OF ATHLETES: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comments left.
Experimental design: No comments left.
Validity of the findings: No comments left.
Additional comments: Regarding the contents of the paper, I am satisfied with the modifications that the authors have made. Well done, congratulations. I still think the paper could be somewhat improved in terms of grammar and style. For instance, there are way too many decimal places at several points in results.


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: EFFECTS OF FUNCTIONAL CORRECTION TRAINING ON INJURY RISK OF ATHLETES: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 3
Reviewer: 1

Output:
Basic reporting: OK
Experimental design: OK
Validity of the findings: OK
Additional comments: All comments have been addressed, well done.


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: WARMING RESISTANT CORALS FROM THE GULF OF AQABA LIVE CLOSE TO THEIR COLD-WATER BLEACHING THRESHOLD
Review round: 1
Reviewer: 1

Output:
Basic reporting: The language of the manuscript is clear and professional throughout. The introduction covers the background well, and the discussion aids in interpreting the results in relation to the knowledge gap outlined in the intro. The discussion around contrasts in physiological responses between the symbiont community and host was particularly interesting.
Experimental design: This manuscript presents original research within the scope of the journal. The research questions and resulting hypotheses are clearly stated and flow logically from the background information presented in the introduction.

The experimental approach is appropriate to address the research questions. However, when the authors outline their two experimental phases (lines 148-167), it is clear that less than 3 months occurred between the “winter” and “summer” phase. What was the justification for this period of time between phases? Is this based on the average duration between minimum and maximum temperatures in the region, or was there some other consideration made? I fully support the authors in the longer hold at ambient between phases but I am wondering if 3 months was appropriate or if it should have been longer? Although I expect that a longer hold would not have significantly changed the outcomes of the summer phase.
Validity of the findings: The results are presented, interpreted and then discussed in an appropriate manner. The conclusions are well-supported by the data.

However, I was surprised that in some cases, there was no effect of genotype, based on some models producing equal marginal and conditional R2 values. The corals were collected from a nursery (lines 105-106), so I am wondering if they have been established previously as genetic individuals or whether it could be possible that some of the genotypes were actually clones? It is also interesting that in some of the cases where there is a difference between the marginal and conditional R2 values, genotype effects account for as much as 30% of the variation in the data (specifically, A. eurystoma, summer heat shock, photosynthesis).
Additional comments: Overall, the authors are presenting a very interesting study, highlighting a little-studied issue in which corals that are high-heat adapted may or may not be cold-adapted, too. This is an important issue to consider given the projected increase in severe cold-weather events under future climate change.


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: WARMING RESISTANT CORALS FROM THE GULF OF AQABA LIVE CLOSE TO THEIR COLD-WATER BLEACHING THRESHOLD
Review round: 1
Reviewer: 2

Output:
Basic reporting: Well-written and succinct.
Experimental design: This is a simple, elegant experiment to assess the lower physiological thermal bounds of corals in the Gulf of Aqaba and any potential links between low- and high-temperature resilience. It builds on previous work and is a nice contribution to the literature.
Validity of the findings: The author's evaluation of their data is appropriate, see general comments.
Additional comments: The authors have conducted a small but well-conceived experiment on cold-water stress on coral physiology. Overall I found the experiment conducted and analysed well and a good contribution to the rather limited literature of low-temperature coral stress.

My primary criticism is the description of the summer high-temperature exposure as "acute thermal shock". The link between cold stress and subsequent high-temperature stress seems flawed in that the summer samples do not appear to be stressed; the quantum yields are high and cell densities seem largely unchanged except for the 18.6C treatment in the summer phase in Acropora. The other parameters seem to be what would be expected of corals in the summer. Is +3 degrees above the monthly mean the same as +3 over annual mean (i.e. is June the warmest month)? Based on Fig. 4 the corals are still several degrees from the summer maximum. In short: are the authors convinced that their 30C treatment was a stressful event? Was the temperature high enough and sustained for enough time? If not, then the link between low- and high-temperature treatment need to be re-evaluated. It's certainly true that the cold treatment did not make them especially sensitive, however. In any case I don’t think “acute thermal shock” is the correct term given these physiological data.

Line 56: I disagree that the mechanisms of warm and cold thermal stress are the same. High-temperature stress alters protein folding processes, while low-temperature stress can slow/inhibit multiple (or all) cellular processes. Membrane fluidity is likely a factor in both.

The paper would also benefit from a very brief hypothesis in the discussion, clearly marked as speculation, as to what cellular mechanisms might link low and high temperature responses. I understand that this is an ecophysiology paper, but a sentence or two on what processes are shared between the two (even if it has to be drawn from non-coral literature, e.g. plant) would strengthen the manuscript.

Figure 3 B & D: The increase in R at low temperatures is odd. Is this due to highly variable measurements?

Figure 4: Are these temperatures experimental or from the field, that is, is 34C experienced by corals in GoA? If not, some vertical lines in the figure to denote the typical thermal range of the corals used in these experiments would make the figure more clear. Different shapes of experimental vs. field points might also improve the figure.

282 Host biomass (as measured by protein concentration) was maintained.
356 tested


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: WARMING RESISTANT CORALS FROM THE GULF OF AQABA LIVE CLOSE TO THEIR COLD-WATER BLEACHING THRESHOLD
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript addresses cold-stress on symbiotic corals living in the Gulf of Aqaba. The researchers found that the symbiont density, chlorophyll content, and photosynthetic rate were significantly lower at temperatures that were reduced 1, 3 oC compared to ambient wintertime temperatures for both S. pistillata and A. eurystoma, implying that these corals are living close to their cold-water bleaching limits. The Fv/Fm, ETR data show that the algae is compromised at 1oC, and definitely at 3oC lower than ambient in the winter.

That said, there are a few things that seem a bit odd in this study:

1.	The symbionts densities seem a bit low. I remember them being about 1 x 106 /cm2 for S. pistillata (e.g. Muscatine 1980’s). Perhaps because the corals were collected from “a coral nursery” adjacent to the Eilat lab? Perhaps because they were collected from deeper water?

2.	Line 117: the authors say that the symbiont was Symbiodinium and reference a 2013 paper. Todd LaJuenesse has recently reorganized the Symbiodiniaceae (Current Biology 28:2570, 2018), with Symbioinium microadriaticum probably inhabiting S. pistillata, and Cladocopium sp. living with the Acropora. Unless the authors have more recent news, I would use this.

3.	Line 365: I do not see the “protein concentration, chlorophyll concentration, and net photosynthetic rate all appear reduced in the summer for S. pistillata”. Do you mean compared to wintertime data?

4.	Line 377: “corals live relatively close to the lower bleaching temperature”. I think this is true for most northerly coral populations (out of the tropics). Many of the corals on the Florida Reef Tract might actually die before they actually bleach. See third graph next page!

5.	Line 390-3: There is more variation around the mean temperatures during global warming. This means that temperatures potentially go higher in summer, lower in winter. Let’s see…could go either way?

This manuscript seems a bit pedestrian, but certainly publishable!
Experimental design: This manuscript addresses cold-stress on symbiotic corals living in the Gulf of Aqaba. The researchers found that the symbiont density, chlorophyll content, and photosynthetic rate were significantly lower at temperatures that were reduced 1, 3 oC compared to ambient wintertime temperatures for both S. pistillata and A. eurystoma, implying that these corals are living close to their cold-water bleaching limits. The Fv/Fm, ETR data show that the algae is compromised at 1oC, and definitely at 3oC lower than ambient in the winter.

That said, there are a few things that seem a bit odd in this study:

1.	The symbionts densities seem a bit low. I remember them being about 1 x 106 /cm2 for S. pistillata (e.g. Muscatine 1980’s). Perhaps because the corals were collected from “a coral nursery” adjacent to the Eilat lab? Perhaps because they were collected from deeper water?

2.	Line 117: the authors say that the symbiont was Symbiodinium and reference a 2013 paper. Todd LaJuenesse has recently reorganized the Symbiodiniaceae (Current Biology 28:2570, 2018), with Symbioinium microadriaticum probably inhabiting S. pistillata, and Cladocopium sp. living with the Acropora. Unless the authors have more recent news, I would use this.

3.	Line 365: I do not see the “protein concentration, chlorophyll concentration, and net photosynthetic rate all appear reduced in the summer for S. pistillata”. Do you mean compared to wintertime data?

4.	Line 377: “corals live relatively close to the lower bleaching temperature”. I think this is true for most northerly coral populations (out of the tropics). Many of the corals on the Florida Reef Tract might actually die before they actually bleach. See third graph next page!

5.	Line 390-3: There is more variation around the mean temperatures during global warming. This means that temperatures potentially go higher in summer, lower in winter. Let’s see…could go either way?

This manuscript seems a bit pedestrian, but certainly publishable!
Validity of the findings: This manuscript addresses cold-stress on symbiotic corals living in the Gulf of Aqaba. The researchers found that the symbiont density, chlorophyll content, and photosynthetic rate were significantly lower at temperatures that were reduced 1, 3 oC compared to ambient wintertime temperatures for both S. pistillata and A. eurystoma, implying that these corals are living close to their cold-water bleaching limits. The Fv/Fm, ETR data show that the algae is compromised at 1oC, and definitely at 3oC lower than ambient in the winter.

That said, there are a few things that seem a bit odd in this study:

1.	The symbionts densities seem a bit low. I remember them being about 1 x 106 /cm2 for S. pistillata (e.g. Muscatine 1980’s). Perhaps because the corals were collected from “a coral nursery” adjacent to the Eilat lab? Perhaps because they were collected from deeper water?

2.	Line 117: the authors say that the symbiont was Symbiodinium and reference a 2013 paper. Todd LaJuenesse has recently reorganized the Symbiodiniaceae (Current Biology 28:2570, 2018), with Symbioinium microadriaticum probably inhabiting S. pistillata, and Cladocopium sp. living with the Acropora. Unless the authors have more recent news, I would use this.

3.	Line 365: I do not see the “protein concentration, chlorophyll concentration, and net photosynthetic rate all appear reduced in the summer for S. pistillata”. Do you mean compared to wintertime data?

4.	Line 377: “corals live relatively close to the lower bleaching temperature”. I think this is true for most northerly coral populations (out of the tropics). Many of the corals on the Florida Reef Tract might actually die before they actually bleach. See third graph next page!

5.	Line 390-3: There is more variation around the mean temperatures during global warming. This means that temperatures potentially go higher in summer, lower in winter. Let’s see…could go either way?

This manuscript seems a bit pedestrian, but certainly publishable!
Additional comments: This manuscript addresses cold-stress on symbiotic corals living in the Gulf of Aqaba. The researchers found that the symbiont density, chlorophyll content, and photosynthetic rate were significantly lower at temperatures that were reduced 1, 3 oC compared to ambient wintertime temperatures for both S. pistillata and A. eurystoma, implying that these corals are living close to their cold-water bleaching limits. The Fv/Fm, ETR data show that the algae is compromised at 1oC, and definitely at 3oC lower than ambient in the winter.

That said, there are a few things that seem a bit odd in this study:

1.	The symbionts densities seem a bit low. I remember them being about 1 x 106 /cm2 for S. pistillata (e.g. Muscatine 1980’s). Perhaps because the corals were collected from “a coral nursery” adjacent to the Eilat lab? Perhaps because they were collected from deeper water?

2.	Line 117: the authors say that the symbiont was Symbiodinium and reference a 2013 paper. Todd LaJuenesse has recently reorganized the Symbiodiniaceae (Current Biology 28:2570, 2018), with Symbioinium microadriaticum probably inhabiting S. pistillata, and Cladocopium sp. living with the Acropora. Unless the authors have more recent news, I would use this.

3.	Line 365: I do not see the “protein concentration, chlorophyll concentration, and net photosynthetic rate all appear reduced in the summer for S. pistillata”. Do you mean compared to wintertime data?

4.	Line 377: “corals live relatively close to the lower bleaching temperature”. I think this is true for most northerly coral populations (out of the tropics). Many of the corals on the Florida Reef Tract might actually die before they actually bleach. See third graph next page!

5.	Line 390-3: There is more variation around the mean temperatures during global warming. This means that temperatures potentially go higher in summer, lower in winter. Let’s see…could go either way?

This manuscript seems a bit pedestrian, but certainly publishable!


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: WARMING RESISTANT CORALS FROM THE GULF OF AQABA LIVE CLOSE TO THEIR COLD-WATER BLEACHING THRESHOLD
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have adequately addressed concerns raised by all three reviewers.
Experimental design: Appropriate
Validity of the findings: Appropriate
Additional comments: Minor edit on line 275.


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: WARMING RESISTANT CORALS FROM THE GULF OF AQABA LIVE CLOSE TO THEIR COLD-WATER BLEACHING THRESHOLD
Review round: 2
Reviewer: 2

Output:
Basic reporting: Appropriate.
Experimental design: Appropriate.
Validity of the findings: Appropriate interpretations.
Additional comments: The authors have made the requested changes and I'm satisfied with the manuscript.


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

Input:
Title: WARMING RESISTANT CORALS FROM THE GULF OF AQABA LIVE CLOSE TO THEIR COLD-WATER BLEACHING THRESHOLD
Review round: 2
Reviewer: 3

Output:
Basic reporting: I have read the rebuttal letter, with the 3 reviewer comments/authors comments.
Experimental design: I have read the tracked changes manuscript. Minor changes made by authors.
Validity of the findings: It is my opinion that the authors addressed most of the comments.
Additional comments: Nothing more to add!


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

Input:
Title: NEEDS OF FAMILY MEMBERS OF PATIENTS ADMITTED TO A UNIVERSITY HOSPITAL CRITICAL CARE UNIT, IZMIR TURKEY: COMPARISON OF NURSE AND FAMILY PERCEPTIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The authors include in their paper Table 3 that is not referenced throughout the text. This table provides summary information on the differences in means in each dimension of the questionnaire, and it may be useful to refer to it in the corresponding section of the text.

2. The bibliography used in the manuscript is adequate, but it is necessary to review the format in which it is reflected, both throughout the text and in the reference section.
Throughout the main text, the authors include a full stop between the end of the sentence and the reference number, to add another full stop after it. Authors should consider removing the first of these points. For example, in line 77, 98, 102, 105, and more.
In the reference section, there are several errors of style that can be amended to improve the manuscript. Considering that the authors are using the Vancouver referencing style, they should amend some errors such as avoiding underlining in the references (lines 308, 319, 341, and more), separating the names with commas and not points (lines 315 and 333), and avoiding using a colon after the authors' names (line 326). Also, in line 329 "MS" appears in the reference, and the authors should check if the surname is missing or those initials refer to other author. In line 343, the full name is used in one of the authors, and initials are used in the others. I would recommend that authors review the entire bibliography to ensure its correct formatting.
Experimental design: 1. The authors use the Critical Care Family Needs Inventory to conduct their study. This questionnaire was validated in family members of people admitted to the ICU by Leske JS (Heart Lung 1991; 20(3):236-244) and its Turkish version was validated by Büyükçoban S et al (Peer J 2015;3:e1208. https://doi.org/10.7717/peerj.1208) also in relatives of persons admitted to ICU. However, the authors use this questionnaire in nurses, without pointing out any reference that this questionnaire is validated in that population. I would recommend the authors to include that reference if it exists, and if not, explain the relevance of using this questionnaire in that population.

2. The authors should consider explaining why they have taken into account whether the nurses had a history of ICU admissions. This aspect, a priori, may be an interesting factor because it may condition the nurse's perception of the needs of the family members, and a brief explanation of that importance in the paper would be appreciated.
Validity of the findings: Authors should consider including some Conclusions after the Discussion section, and these Conclusions should be according to the aims of the study.
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: NEEDS OF FAMILY MEMBERS OF PATIENTS ADMITTED TO A UNIVERSITY HOSPITAL CRITICAL CARE UNIT, IZMIR TURKEY: COMPARISON OF NURSE AND FAMILY PERCEPTIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study of the biopsychosocial needs of ICU patients and their families is widely studied. Old references should not be used. I suggest updating the conceptual framework with current scientific literature.
It is strange that the introduction emphasizes the need for healthcare professionals to consider patients and relatives needs, but the study focuses on nurses. This needs to be justified as many of the aspects explored in the questionnaire are interdependent interventions.
Experimental design: The manuscript describes that the characteristics of the patients are quite heterogeneous. An elective postsurgical patient is not the same as a septic patient. The expectations and needs of the people screened can vary significantly depending on the circumstances of admission. The inclusion and exclusion criteria should address these issues, or at least explain how this has been controlled.
It is also necessary to evaluate not only the minimum time of admission to the ICU, but the accumulated time (the emotional exhaustion of a family that has been in the ICU for 20 days is not the same for a family admited for 2 days). Aspects such as the type of shifts that nurses work should also be valued. The type of family connection also needs to be better explained since the regular visitor is not the same as the sporadic.
Regarding the design, the time at which the study was carried out has not been indicated, which is very relevant although it is suspected that it was before the pandemic.
The size of both samples is very different and homogeneity tests would be necessary to verify that they are comparable.
Although the methodology used is widely explained in the methodology, it is necessary to make it clear throughout the text (including the abstract) that it is the Turkish version of the CCFNI.
Validity of the findings: I find the explanation of the results redundant since they are reflected in the tables. However, more discussion of such striking aspects is missed, such as the fact that the second important question for families is the twelfth for nurses. It is striking that honest information is valued more by nurses than by families.
It would be enriching if the relationship between the clinical variables of the patients and the needs of their families were analyzed. The current analysis without taking into account the context of the patients is of little use.
I believe that although the study responds to the initial objective, the manuscript is uninteresting without more complete accurate analyzes.

In the discussion again the problem referred to health workers is taken up, however the study has only been carried out on nurses. On the other hand, certain quite bold claims are made based on very local literature and with a limited level of evidence.

One of the most striking aspects are the recommendations made on the information. I do not believe it is fair to ask that nurses be involved in the process when it is a two-way process and the scientific literature has shown serious deficiencies in health teams, not only in nurses. Contextualization is necessary about why nurses do not consider giving information a priority. Is it their role in Turkey? Do they have the information requested? Is the information accessible?
Additional comments: In general, the study can provide the necessary knowledge to guide work and functions of ICU nurses in order to provide a better biopsychosocial care. The final paragraph of the manuscript is very important, in which the real situation of nurses is contextualized and I believe that this point should be present from the beginning (these circumstances may have an uncontrolled influence on the results of the 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: NEEDS OF FAMILY MEMBERS OF PATIENTS ADMITTED TO A UNIVERSITY HOSPITAL CRITICAL CARE UNIT, IZMIR TURKEY: COMPARISON OF NURSE AND FAMILY PERCEPTIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have made substantial and sufficient improvements
Experimental design: The authors have made substantial improvements and have satisfactorily answered my questions. Thank you for clarifying the methodology used.
Validity of the findings: Although the study has limitations, I consider that the current version of the manuscript adequately collects the results and that they are valid.
Additional comments: Thank you very much for the improvements made and congratulations on the study. I would recommend that you continue with this line of research. It would be interesting to take a qualitative approach and involve more nurses in the research group.


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: GENETIC SIGNATURES OF POPULATION BOTTLENECKS, RELATEDNESS, AND INBREEDING HIGHLIGHT RECENT AND NOVEL CONSERVATION CONCERNS IN THE EGYPTIAN VULTURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The submitted manuscript presents fresh data from a long-term monitoring study of a Spanish population of Egyptian vultures over a period of thirteen years. A comprehensive population genetic analysis is complemented with a screening of several common avian pathogens. Because the largest part of the species‘ European breeding range is on the Iberian Peninsula, these data from actually declining Spanish populations are of a certain importance for conservation management.
The manuscript is well written and structured. However, the discussion is a single long paragraph that could benefit from some substructuring by some subheadings.
To my impression reference to previous work could be a little more substantial. In l. 83 it is mentioned that „information on genetic diversity of populations and species is still limited“. Though the papers by Agudo and collaegues on the Egyptian vulture are mentioned later in the manuscript, there are many more population genetic studies on other vulture species, such as Davidovic et al. (2020) Sci Rep 10: 20394; Poulakakis et al. 2008 Biol J Linn Soc 95, 859–872; Çakmak et al. 2018: Ibis 161:793-805; Godoy et al. 2004: Mol Ecol 13: 371-390. This should be taken into account.
Experimental design: My major concern relates to the use of microsatellites for kinship analyses. Microsatellites have been in use for population genetic analyses for a long time, however, recently there is a growing general criticism on the use microsatellite data (single tandem repeats, STR) for specific research questions for a number of reasons. Indeed, I believe that micosatellites are useful tools for population genetic analyses and in my research group we have used this approach for a number of hybrid zone studies – which has been severly criticized during review processes with the argument that STR data would not be appropriate for the study of hybridization patterns between species – which I think is a false argument. In that study we did not even intend at identifying hybrid classes (F1, F2, backcrosses etc.) with our microsatellite data set, and I would admit that this would have been much more problematic. For example, when we compared our microsatellite data set from one hybrid zone  with SNP data set for the same sampling we realized that indeed hybrid classes could be more reliably discriminated against each other using genome-wide SNPs. So I generally believe that microsatellites are useful for analyses of particular population genetic questions whereas they might have limitations with respect to others (e.g. indentification of hybrid classes).
There is a similar criticism on the application of microsatellites for kinship analyses and I feel that this problem needs to be adressed in a revision. There is evidence from several kinship studies that micsats performed less reliably than SNP data sets particularly for groups without parental information, for example: Lemopoulos et al. (2019: Ecol Evol 9:2106–2120.: SNPs „outperformed microsatellites for applications that required individual‐level genotype information, such as quantifying relatedness“); Hauser et al. (2011: Mol Ecol Res 11 (Suppl. 1):150-61); Thrasher et al. (2018: Mol Ecol Res 18: 953-965); Weinmann et al. (2014: Mol Ecol Res 15:502-511); Flanagan & Jones (2019: Mol Ecol 28:544-567). Therefore, the choice of methods needs any kind of justification – in the current version the method is presented straightforward without any critical comment.

Likewise, the QG estimator has been greatly criticized and several alternatives have been suggested (see discussion in Konovalov & Heg (2008: Mol Ecol Notes 8: 256-263). Moreover, this estimator has been shown to become biased when calculation of allele frequencies is based on small sample-sizes (Wang 2017: Herdedity 119:302-313). In the submitted study the pedigree analyses are based on ten individuals (over time, four breeding seasons; 14 individuals in 2018; Fig. 1) … which indeed must be considered small sample sizes. This general criticism should be taken into consideration and a revised methods paragraph should be complemented with a strong justification for the choice of this particular estimator for genetic relatedness.

Demographic events: I agree that bottleneck analysis as done here may be appropriate for local samplings to estimate recent historical events over past decades, however, I doubt whether it makes sense trying to estimate past bottlenecks „over centuries and millennia“ with one local population sampled over a period of 13 years. For this aim, a larger range-wide sampling of a metapopulation would be more appropriate. Therefore, I would indeed recommend to keep the short-term analysis with BOTTLENECK but to discard the M-ratio analysis.

Sample size:
l. 179: „Overall, our study includes the sampling of 42 Egyptian vulture individuals.“ In the text are mentioned: 19 from 2006, 14 from 2018 (l. 173) and further 10 to study relatedness among nestlings (l. 185: eight nestlings and two adults as shown in Fig. 3) – so these sum up to 43 … something must be wrong here.
Validity of the findings: To my impression the validity of the findings depends on a strong justification for the chosen methods to estimate genetic relatedness and an appropriate discussion of potential pitfalls when microsatellites are used for this kind of analyses (see my comments above).
Additional comments: Some minor comments refer to rather formal and editorial issues like typos, correctness of citations etc.

l. 89: „a species‘ range“
l. 99: Once in the main text, the scientific name of the study organism should be mentioned; probably here at first mention (apart from the abstract).
l. 149-151: Have these data on dispersal distance been published? It seems that this information is part of this study’s results (Fig. 1B).
l. 43: „Frankham 2002“ is not in the reference list – is it „Frankham et al. 2002“ or „Frankham 2010“? Please, check the references carefully throughout.


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: GENETIC SIGNATURES OF POPULATION BOTTLENECKS, RELATEDNESS, AND INBREEDING HIGHLIGHT RECENT AND NOVEL CONSERVATION CONCERNS IN THE EGYPTIAN VULTURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: I like the way the article is written. In general, I would, however, shorten the introduction on the theory behind the analyses (as the paper focusses on the conservation and genetics of the organism rather than the theory) and move the "Study species and study area" section into the introduction, to have the introduction set the stage for the analyses, results and discussions. The manuscript is written in good english over all, but I have some smaller correction to the english, which I will point out in detail below.
Experimental design: The study is well designed. However, what is not clear to me is the number of individuals included in the "screening of infectious agents". Why only include two individuals if you later state in the paper that you typed many individuals (which you cite as unpublished data). Given that effectively only two individuals were typed the assumptions in the Discussion are too strong. I really enjoyed the inclusion of that test in the study, and would - if possible - suggest to include the data from the other individuals in this manuscript. If that is not possible, I would recommend to remove the ascertainment that there might be an "interaction between inbreeding and malformation due to BFDV" as this is based on too few samples.
Validity of the findings: The findings are important for the conservation of the Egyptian vulture. I enjoyed the comparison between the two sampled years, which is very valuable for conservation management, and in general the sample sizes are adequate (except for the screening for infectious agents). I am not an expert on demographic inference using microsatellite data, but the methods sounded very reasonable and thought through to me.
Additional comments: I really enjoyed reading the manuscript and think that with some adjustments to make the introduction more focused on the biology rather than the theory, this will be a very important contribution to the conservation genetic literature.

Please, find specific minor comments below:

Title and Line 31: Replace "alert on" with "highlight".
Introduction: Please, reduce the discussion of the theory and include information on the species and area here rather than the M&Ms.
Line 105: Replace "to deepen on the extend of population inbreeding" with "to further investigate inbreeding"
Line 122-123: Add reference.
Line 133-134: Add reference.
Line 145-149: Add reference.
Line 185: Do you mean you sampled 10 individual in each of this years? Please, make that more clear.
Line 204: This might be a conversion issue, but the "10 l" and the "5 l" are missing the micro before the l.
Line 233: Change to "9,999"
Line 260: Change to "9,999"
Line 270: Change to "... based on different assumptions. BOTTLENECK is ..."
Line 302: Change to "The inbred"
Line 334: Wouldn't it have been more logical to remove it?
Line 344:Change "nestling" to "nestlings"
Line 349: Change to "when the offspring"
Line 360: Change to "correctly assigned"
Line 365 and 372: Change to "nestlings"
Line 394: Change to "were 100"
Line 423: Please, change to "failed to reproduce in 2017"
Discussion: The discussion would highly benefit from headers for the different sections.
Line 446-447: Add reference.
Line 454: Please, change to "two breeding seasons twelve years apart"
Line 457: Remove "by", it is either "by" or "using" not both.
Line 476: Same, change to "using"
Lien 502: I would add the years to "all and most" - otherwise it reads a bit odd
Line 506, 507 and 508: Change to "related to"
Line 550: Change to "could have been born".
Line 569: Why would the malformations have changed in captivity if they are birth defects? Did the individual die after two years in captivity?
Line 576: "positive for"
Line 580: This is too strong a statement based on a sample size of 1 in each group. Please, rephrase.
Line 582-584: Please, add some data on the BFDV-positive nestlings or remove the sentence. I think that data would really improve the study as this is an important finding. In general, there is way too few data and not enough actual testing to infer an interaction between inbreeding and malformation and BFDV.
Line 585: Change to "includes" - as this is a general statement.
Line 595-597: I would remove the sentence. It doesn't really fit the topic of this section.
Line 602: Change "alert on" to "highlight"

I am really looking forward to see this manuscript published and hope that my suggestions will help to make it even more enjoyable to read. I am thus recommending the paper for "minor revisions".

Good luck with this and future conservation projects!
Stefan Prost, PhD


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: GENETIC SIGNATURES OF POPULATION BOTTLENECKS, RELATEDNESS, AND INBREEDING HIGHLIGHT RECENT AND NOVEL CONSERVATION CONCERNS IN THE EGYPTIAN VULTURE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have submitted a thorough and comprehensive revision of their manuscript under consideration of all recommendations of the reviewers. Further literature was included in the introduction as recommended. Likewise, the reference to previous work was completed. I particularly appreciate the new figures added to the study. This is a great improvement as compared to the first version and provides a comprehensive documentation and presentation of the analyses and results. Well done!
Experimental design: The research topic and basic research questions were already well defined in the first version. The additional discussion on the explanatory power of microsatellite data (despite all limitations) as compared to other sets of molecular markers and further information on methodical details of genetic relatedness and inbreeding measures add further support to the results.
Validity of the findings: The revised manuscript is complemented with an extensive set of supplementary data that substantiate the results. I have no objections against the revised version.
Additional comments: I would like to congratulate the authors to a fine study and a greatly improved manuscript.


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

Input:
Title: GENETIC SIGNATURES OF POPULATION BOTTLENECKS, RELATEDNESS, AND INBREEDING HIGHLIGHT RECENT AND NOVEL CONSERVATION CONCERNS IN THE EGYPTIAN VULTURE
Review round: 2
Reviewer: 2

Output:
Basic reporting: I have read the replies to the comments and the manuscript and I am very happy with the changes.

The only comment I have, is to remove the sentence "This suggests a potential interaction between inbreeding and malformation depending on infection by BFDV, although other non-surveyed pathogens or pollutants could be involved in disease causation, which requires further research." - as a sample number of N=1 is too small to suggest any interaction.

I am looking forward to see the paper published and to follow any upcoming research on this Egyptian vulture population.

All the best,
Stefan Prost, PhD
Experimental design: All good.
Validity of the findings: All good.
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: INTEGRATING MORPHOLOGICAL AND MOLECULAR APPROACHES FOR CHARACTERIZING FOUR SPECIES OF DACTYLOGYRUS (MONOGENEA: DACTYLOGYRIDAE) FROM MOROCCAN CYPRINIDS, WITH COMMENTS ON THEIR HOST SPECIFICITY AND PHYLOGENETIC RELATIONSHIPS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is very interesting and important papers that integrates morphological and molecular approaches for characterizing species of Dactylogyrus from Moroccan Luciobarbus. All experiments are correct, statistical analysis is sound and conclusions are well supported. Literature is well referenced and relevant, general structure conforms to PeerJ standards, figures are relevant, high quality, well labelled and described. The manuscript is clearly written in professional, unambiguous language. Having said that, I would like to note that the manuscript has a number of weaknesses which should be addressed.

The M&M section includes the well-described subsection entitled as “DNA isolation, amplification and sequencing”, but the results of this work is poorly present in the manuscript. I mean that authors should provide the table with information on host species, locality and accession number (or place where it will be inputted if the manuscript will be accepted) for each isolate obtained in the present study. Moreover, the phylogenetic analysis presented in the manuscript completely includes sequences from already published papers or papers in press.

Subsection “Molecular characterization” provided to each species of studied Dactylogyrus contains information with the number of specimens that were sequenced and short information about intraspecific genetic variability, but again I did not see any information and analytic data, e.g. isolates deposited, counted pairwise genetic distances, phylogenetic tree.
L197, 258-260, 346, 421 Numbers for hologenophores deposited in the museum are provided, but information on the number of corresponding isolates deposited in GenBank is absent
Concluding the above authors should:
1)	Deposit sequenced isolates of species in GenBank
2)	Provide additional table with information on such isolates (at least host and locality data, accession numbers).
3)	Provide an accession number of the sequenced isolate to each hologenophore
4)	Provide graphical information (table and/or tree) that would reflect the results of molecular analysis for isolates sequenced in the present study (see also comment below).

The discussion section does not include the conclusion paragraph with a general conclusion and future perspectives of the study.

Minor comments
L23-26 Four species of studied Dactylogyrus spp. parasitizing on Luciobarbus characterized as those, which have “an attachment organ with a cross-shaped (five radial) ventral bar and the male copulatory organ containing an accessory piece with a distal portion directed backwards along the circle of the curved copulatory tube”. But Dactylogyrus marocanus does not fit to the provided description. The abstract should be corrected.

L218-220. Authors stated that “Seven specimens from three host species (C. fritschii, L. ksibi, and P. maroccana) were sequenced, with no intraspecific genetic variability between specimens parasitizing different host species found.” I do not believe that seven specimens of Dactylogyrus marocanus from three host species were identical. Please provide a table with counted pairwise genetic distances between them. You can allocate this table in Supplementary data.

L462. In the sentence “Both phylogenetic analyses yielded similar tree topologies” Insert “ML and BI” between “Both” and “phylogenetic”
Check spelling Dactylogyrus marocanus through the text and tables. There is also D. maroccanus. See L 170 and Table 3
Figure 6. Please provide more detailed information about tree, namely used genes, tree length.
Authors should explain while Dactylogyrus marocanus is out of phylogenetic tree.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: INTEGRATING MORPHOLOGICAL AND MOLECULAR APPROACHES FOR CHARACTERIZING FOUR SPECIES OF DACTYLOGYRUS (MONOGENEA: DACTYLOGYRIDAE) FROM MOROCCAN CYPRINIDS, WITH COMMENTS ON THEIR HOST SPECIFICITY AND PHYLOGENETIC RELATIONSHIPS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I am recommending acceptance after minor revision and consideration of the recommendations and remarks which I have placed directly on the manuscript. The English is very good. The following are the most important concerns:
1) higher taxa are not host or parasites, although their member species might be.
2) My major concern is the apparent arbitrary placement of the root in the phylogenetic tree. As it is, there is no evidence that its placement is correct in the tree. By including arbitrary root (which clearly is a result of the authors' bias), the results are misleading and more than likely erroneous. Remove the root from the tree and let your readers deal with the fact that on is not provided, which by the way, can only be done in a phylogenetic analysis by including an outgroup taxon (which you haven't done). You might also want to reconsider what you have said in the discussion re the tree as well.
3) The mixing of classifications (see ms.)
4) Soft non-repeatable measurements (see ms.)
Experimental design: ok
Validity of the findings: ok
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: INTEGRATING MORPHOLOGICAL AND MOLECULAR APPROACHES FOR CHARACTERIZING FOUR SPECIES OF DACTYLOGYRUS (MONOGENEA: DACTYLOGYRIDAE) FROM MOROCCAN CYPRINIDS, WITH COMMENTS ON THEIR HOST SPECIFICITY AND PHYLOGENETIC RELATIONSHIPS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper is well-written. The introduction provides a clear overview of the state-of-the-art, and situates the current research very well in what has already been done. The use of the English language is, as far as I can tell, fine.

l. 52: do you mean recent descriptions of fish or of parasite species?
l. 383: "specimens" + "was": confusion of singular and plural?
l. 435-437: why no elaborate description of the morphology of the MCO in Dactylogyrus draaensis?
l. 525-527: "unless [...] cannot be excluded": something wrong in this sentence structure
L. 564-565: "Our records [...] and increases...": something wrong in this sentence structure
l. 577: "Getahun", not "Getahum"
Fig. 3A: this species was collected from this host species on two different localities; from which locality did the specimen(s) on which this drawing was based, come?
Fig. 6: perhaps it would be nice to guide the reader through the tree by indicating the clades mentioned in the text, or the host species, or by highlighting the Moroccan species...

General question: is it the first time sequences of the species in question are published? Perhaps it can also be clearly indicated in Table 2 which sequences are new in this study.
Experimental design: The methods are well-explained, and correctly used. I only have some questions:

l. 143: why did the authors opt for the use of uncorrected p-distances if they later select a model of molecular evolution?
l. 145-148: please explain why these species were chosen to be included in the phylogenetic analyses. On the basis of which criteria were they selected? For example, does the analyses include all Moroccan representatives of Dactylogyrus for which sequences are available?
l. 156-158: which partition was used for the concatenated dataset, and how were the selected models implemented? Not all of them are available in the phylogenetic packages used.
Validity of the findings: l. 144 suggests that the taxonomic treatments of the species under study are redescriptions. However, if that is the case, it would be useful to stress whether the authors report any new phenotypical aspects, or any differences in comparison to the original species descriptions. (The authors did this nicely for Dactylogyrus borjensis.)
l. 240-242: did the authors check for size differences between the parasites of different hosts?
l. 275-276: isn't this also the case in D. marocanus and D. draaensis? On Figs. 2 and 5, I also see this expansion of the proximal part (although to a lesser extent than in D. ksibii perhaps), but for D. marocanus and D. draaensis this aspect is not mentioned in the text. Perhaps I don't see what the authors intended to show here of course, but in that case the difference should be made clearer in the drawings.
l. 485: A PP of 0.9 is not "well supported".
l. 486: I do not see the bootstrap value supporting the relationship of D. borjensis towards this clade.
l. 587-588: this tree in itself says nothing about independent origins, as the sister lineages of two of the three clades to which Moroccan representatives belong, are not represented.
Hence, perhaps the conclusions based on the tree should be slightly nuanced.
Additional comments: The phylogenetic coverage of this research project in its entirety (also beyond this manuscript: summarised in the introduction) is impressive in general, and the present manuscript provides a nice overview of what is known on the species in question. I recommend its publication, pending some improvements and clarifications as suggested above.


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

Input:
Title: INTEGRATING MORPHOLOGICAL AND MOLECULAR APPROACHES FOR CHARACTERIZING FOUR SPECIES OF DACTYLOGYRUS (MONOGENEA: DACTYLOGYRIDAE) FROM MOROCCAN CYPRINIDS, WITH COMMENTS ON THEIR HOST SPECIFICITY AND PHYLOGENETIC RELATIONSHIPS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I've looked at the authors' responses, and while they have ignored several, I don't see where a new response to their decisions would make any difference. So go ahead with publication and let the authors' be responsible for their decisions.
Experimental design: see above
Validity of the findings: see above
Additional comments: see above


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

Input:
Title: INTEGRATING MORPHOLOGICAL AND MOLECULAR APPROACHES FOR CHARACTERIZING FOUR SPECIES OF DACTYLOGYRUS (MONOGENEA: DACTYLOGYRIDAE) FROM MOROCCAN CYPRINIDS, WITH COMMENTS ON THEIR HOST SPECIFICITY AND PHYLOGENETIC RELATIONSHIPS
Review round: 2
Reviewer: 2

Output:
Basic reporting: L. 577-579: I agree the sentence is grammatically correct, but its meaning is not… I think you mean that it is hard to say whether worms with a similar morphology are also closely related, because it could be that they share a similar morphology because of adaptations to a similar host, rather than because of phylogenetic relationships. Correct? And therefore the only way to be sure that morphology mirrors phylogeny, is to exclude such adaptive phenomena in the morphology. If that is what you mean, the sentence should read “… CAN be excluded”.

Fig. 6: the caption should explain what the scale bar represents.
Experimental design: no comment
Validity of the findings: L. 537-538: in the previous version I had a question about the statistical support for the sister-group relationship between Dactylogyrus borjensis and the clade consisting of D. benhoussai + D. varius + D. ksibii + D. scorpius. The authors’ reply gives me the impression that this node is supported by a Bayesian PP below 70% and a ML bootstrap below 50%. If this is the case, this node should perhaps best be collapsed, but more importantly: it cannot be concluded whether D. borjensis or rather D. falsiphallus are sister to this well-supported clade of D. benhoussai + D. varius + D. ksibii + D. scorpius.
Additional comments: I thank the authors for the constructive discussion. They have nicely implemented the suggestions of the referees, as far as I can see; they have carefully formulated and justified their responses, and I agree with their arguments in almost all cases. There are two relatively minor elements where our opinions seem to differ, and a single small addition to a figure caption that I believe is needed. If these three things can be addressed (the editor can check this as far as I’m concerned), this manuscript is absolutely ready for publication.

Well done, and my best wishes for a safe 2021 for the authors!


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

Input:
Title: LINFLOW: A COMPUTATIONAL PIPELINE THAT COMBINES AN ALIGNMENT-FREE WITH AN ALIGNMENT-BASED METHOD TO ACCELERATE GENERATION OF SIMILARITY MATRICES FOR PROKARYOTIC GENOMES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, LINflow seems like an important contribution for accurate and fast ANI calcuations. The workflow seems well thought out and the comparisons to other software seem appropriate.
Experimental design: The approach of the pipeline appears to be robust. When I tried to install the software, I ran into mysql connection issues using 2 different operating systems. I would encourage the authors to try to install LINflow on other systems and make sure that the documentation is complete for installation by basic users on operating systems where they do not have "sudo" privileges. When I look at the code, I notice that the python version is hard coded in the header line. I would encourage the authors to use "/usr/bin/env python" to be consistent with the version that the user has installed through conda. I notice that in LINdb.py, there is some description of creating login files, but this should be added to the main page with enough information to make this accessible to new users.
Validity of the findings: The comparisons to other software seem appropriate. The methods for how the heatmaps were generated should be added to the text. If the hierarchical clustering was performed outside of LINflow, then these methods should also be added to the text.
Additional comments: Based on my issues described above, I wasn't able to test the software, which I would like to do prior to providing more in depth comments. The authors use the term "prokaryote" throughout, although this term is phylogenetically inconsistent. At one point the authors use "microbial". If this term can replace "prokaryote", I would encourage them to change throughout.

In Table 3, the time under LINflow should be in "m" and not "n"


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: LINFLOW: A COMPUTATIONAL PIPELINE THAT COMBINES AN ALIGNMENT-FREE WITH AN ALIGNMENT-BASED METHOD TO ACCELERATE GENERATION OF SIMILARITY MATRICES FOR PROKARYOTIC GENOMES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Tian et al present here LINflow, an automated pipeline extending on their previous work with LINbase. The manuscript is generally very clear and well written, and presents a good overview of the relevant literature. I only identified a minor typo in table 3: n instead of m in the LINflow time for set A.

I consider this manuscript and the associated Software an important contribution to the community. However, I remain unconvinced by some assessments, principally due to missing information in the manuscript.
Experimental design: Some design decisions in the Software seem unjustified or only implicitly justified (e.g., k-mer selection in sourmash, or sourmash preference over FastANI). However, this doesn't in any way hinder the reproducibility of the results nor does it affect their validity, and therefore I only raise it as a point of consideration and not really an issue.

More importantly, some key details from the experimental design are not made explicit here. For example, the exact versions of the Software utilized here should be reported (e.g., L196). FastANI recently corrected an important issue in v1.31, and it’d be important to know if the tests were performed before or after this update. Similarly, the method of hierarchical clustering (a central piece on the evaluation of accuracy) is not reported.
Validity of the findings: In Table 3, the authors report a mix of parallelized and linear times, which are not directly comparable. While multi-threaded execution might be the only way to feasibly obtain matrices from pyani, that is not true for FastANI. Why not doing 1 thread for comparison? Note that sub-lineal parallelization as well as computer-specific issues could result in extremely inflated values here. Importantly, I executed FastANI for data set A in a single thread in a laptop and got a result in only 33 minutes (not >11h). While some machine-specific differences are expected, this is a 20-fold difference worthy of reevaluation.

In L200, the authors state: "hierarchical clustering was performed", without specifying the method used or its implementation. The method of hierarchical clustering is a fundamental piece of information, not least due to the fact that none of the dissimilarity metrics used here are metric distances (they all violate the triangle inequality). Note that the only basis of the accuracy evaluation in the manuscript is the cophenetic correlation between dendrograms, not a comparison between distances.

Related to this last point, I (and expectedly other readers) would be curious to know why the authors decided not to evaluate the distances directly. If this were because capturing the "structure" of the distance data is more important than reconstructing actual distances, then a comparison to proper phylogenetic reconstructions (and perhaps different methods of hierarchical clustering) would be warranted. Moreover, direct comparisons of the distances would still be valuable even in this framework. For example, if the actual values are less important than linearity, linear correlations could be evaluated (instead of error). Or, if the monotonicity of distances is more important than linearity, rank correlations could be studied instead. Importantly, the authors indicate there is a good correlation in the ANI range of 70% to 99.8% (L292), but this is never demonstrated.

Finally, the authors present a hypothesis on why the evaluated accuracy of LINflow was low for set B in L283-284. Why not evaluating this hypothesis directly? It seems the authors could simple execute LINflow with the proposed settings and demonstrate the improvement.
Additional comments: I was unsuccessful on deploying the Software presented here. In particular, it appears the installation instructions are incomplete, and indicate a shifting set of prerequisites that has not been fully updated.

The main point of difficulty in the installation was the prerequisites related to MySQL. The manuscript indicates (and justifies) the preference over SQLite in L124, which I personally celebrate. However, the actual code depends upon a "Python MySQL connector" for which no links or installation instructions are provided. It is unclear why is this even necessary (since the databases are reportedly file-based SQLite databases), but if this indeed is necessary additional instructions are required. Note that the advantage of SQLite is that server-based databases are not required. If this is a requirement anyway, there is no advantage on using SQLite. Notably, the conda instructions didn't resolve this dependency either.

Signed: Luis M. Rodriguez-R


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: LINFLOW: A COMPUTATIONAL PIPELINE THAT COMBINES AN ALIGNMENT-FREE WITH AN ALIGNMENT-BASED METHOD TO ACCELERATE GENERATION OF SIMILARITY MATRICES FOR PROKARYOTIC GENOMES
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1) Figure 3-5 can be combined into one figure. Among them, in Figure 4A, some diagonal ANI values are not highest, and I cannot understand why. Possibly, the order of the genomes in rows and columns are different. For whatever reason, this should be explained.
Experimental design: 1) The authors compared LINflow with three other tools, pyani, sourmash, and FastANI, but whereas the purpose of these tools is to calculate ANI between two genomes, the purpose of LINflow is to assign LINs to input genomes. Therefore, direct comparison between these tools is meaningless. The comparison conducted here should be the results of LINflow with other hierarchical classification schemes based on all-against-all ANI calculation by using these programs.
2) In this respect, I was wondering why the authors compared similarity matrices shown in Figures 3-5. How LINflow creates such a similarity matrix while it does not calculate all-against-all ANI? And what is the purpose of this comparison?
3) Although LINflow can calculate the accurate ANI between the query and the most similar genome using pyani, selecting the most similar genome using sourmash may not be so accurate. Therefore, one of the important issues here should be how this inaccuracy affects the final result. However, the experimental tests in this manuscript seem not to address this issue adequately.
Validity of the findings: 1) The authors insist that LINflow is more accurate than FastANI for the dataset A and C in terms of the cophenetic correlations shown in Figure 6 and this is because these datasets contain different species (lines 285-289). However, in these cases, sourmash is also better (more similar to pyani) than FastANI. This seems to contradict the previous observation by the authors of FastANI (Jain et al. Nat Comm. 2018), where the accuracies of FastANI is better than Mash especially when more divergent genomes are compared. The authors should explain this point more clearly.
2) Similarly, I cannot find any clear evidence for the following sentence (lines 291-292): LINflow reveals genomic relationships more accurately than FastANI and sourmash over the range of ANI values from 70% to around 99.8%.
Additional comments: LINflow is a system for assigning a LIN (Life Identification Number) to each genome in a set of genome sequences.  The resulting set of LINs corresponds to a hierarchical classification of the input sequences based on ANI. LINflow avoids calculating all-against-all ANIs to calculate LINs by incremental updating process, and utilizes two existing programs, sourmash and pyani, for rapidly identifying the most similar genome in the set and for rigorously calculating the ANI between the query and the most similar genome, respectively. I think that this scheme is reasonable enough, and possibly useful for maintaining a large-scale comparative genome database.
As I mentioned in the Experimental design section above, I do not think that direct comparison between LINflow and other ANI calculation tool is meaningful. In fact, I think that ANI calculation tools used in LINflow can be replaced with other tools such as FastANI, and possibly it may improve the performance. Therefore, I recommend the authors not to focus on comparison of LINflow with other ANI calculation tools, but more focus on designing  an efficient genomic data classification scheme.


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: LINFLOW: A COMPUTATIONAL PIPELINE THAT COMBINES AN ALIGNMENT-FREE WITH AN ALIGNMENT-BASED METHOD TO ACCELERATE GENERATION OF SIMILARITY MATRICES FOR PROKARYOTIC GENOMES
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors obviously made an attempt to improve both the manuscript and the software package in this resubmission. Unfortunately, I follow the instructions on the github page for conda and am still unable to run the program on centOS, ubuntu, or MacOSX. The problem is still mysql. I could dig into getting this fixed, but seems beyond the scope of this particular review.

My cluster is unable to run docker images, so I wasn't able to test the docker image on centOS. I also tried on ubuntu, but looks like I would need sudo privileges to set up the container, which I'm unable to do.

Unless the software is easy to install, it is unlikely to be widely used. I would encourage the authors to rethink their dependencies or improve the documentation to allow for each installation and usage in the absence of sudo privileges. One way to test this is to have users on different machines work through the instructions and verify that they can install. Additionally, it appears that the authors didn't change the first line to "#/usr/bin/env python", which will cause serious conflicts if a user tries to invoke the script without a preceding "python".


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: LINFLOW: A COMPUTATIONAL PIPELINE THAT COMBINES AN ALIGNMENT-FREE WITH AN ALIGNMENT-BASED METHOD TO ACCELERATE GENERATION OF SIMILARITY MATRICES FOR PROKARYOTIC GENOMES
Review round: 2
Reviewer: 2

Output:
Basic reporting: I don't have any substantive comments left. I congratulate the authors on this significantly improved manuscript, that I'm sure will have an important impact in the field. I'm eager to use LINflow in production myself.

Only one (very) minor comment: the new text introduces the capitalization pyANI for the tool referred elsewhere as pyani. I believe the all-lowercase version is the correct styling.
Experimental design: No additional comments.
Validity of the findings: No additional comments.
Additional comments: No additional comments.

Signed: Luis M. Rodriguez-R


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: LINFLOW: A COMPUTATIONAL PIPELINE THAT COMBINES AN ALIGNMENT-FREE WITH AN ALIGNMENT-BASED METHOD TO ACCELERATE GENERATION OF SIMILARITY MATRICES FOR PROKARYOTIC GENOMES
Review round: 2
Reviewer: 3

Output:
Basic reporting: My second question has not been addressed yet. My question is: if rows and columns of the matrix are in the same order, the diagonal should correspond to the ANIs of the same strains, which should be 100. But this seems not to be true for FastANI (Fig.3B). Is it correct?

In addition, although the legend of Fig.3 says that there is a supllmenentary figure showing the same matrix with strain names (which can be used to confirm the order of the matrix), I cannot find any such supplementary figure.
Experimental design: I still feel that the authors’ assertion that the main purpose of LINflow is to calculate a similarity matrix is somewhat strange. Instead, I think that the main purpose should be classifying genomes (with accurate ANI values) for which a similarity matrix is required. And this is why LINflow can be a useful tool even if it does not calculate all-against-all similarities assuming that genomic data can be classified in a hierarchical manner. Although I do not force to do so, I recommend the authors to mention this point.
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: LINFLOW: A COMPUTATIONAL PIPELINE THAT COMBINES AN ALIGNMENT-FREE WITH AN ALIGNMENT-BASED METHOD TO ACCELERATE GENERATION OF SIMILARITY MATRICES FOR PROKARYOTIC GENOMES
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I'm still having difficulties getting LINflow installed, but perhaps I'm an anomaly and am willing to accept the manuscript and let the community decide on the usability of the software.


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: LINFLOW: A COMPUTATIONAL PIPELINE THAT COMBINES AN ALIGNMENT-FREE WITH AN ALIGNMENT-BASED METHOD TO ACCELERATE GENERATION OF SIMILARITY MATRICES FOR PROKARYOTIC GENOMES
Review round: 3
Reviewer: 2

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