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

Input:
Title: A LATE SURVIVING PLIOCENE SEAL FROM HIGH LATITUDES OF THE NORTH ATLANTIC REALM: THE LATEST MONACHINE SEAL ON THE SOUTHERN MARGIN OF THE NORTH SEA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript report an alleged “late” Surviving Monachinae seal in the southern margin of the North Sea. The material is restricted to a single, partial, humerus, lacking the distal end.

I see some issues with this manuscript that I will point following here and additional notes are inserted in the manuscript pdf.

Age.
The age, in general terms, was adequately established, but from reading the text, it seems that an age between 3.71 and 2.72My, thus, late Early to Late Pliocene is more accurate than just Late Pliocene.

Identification of specimen.
All the comparisons were done with Monachinae species, in the assumption that there is no doubt that it belong to this subfamily. As themselves noted, some features are not common to most (if not all) Monachinae, and even have one feature ( “a moderately well-developed fossa for the origin of the triceps brachii muscles” p.16, l.251) is also present in a species considered now as a Phocinae.

As the authors stated, in essence, the identification relies on the morphology of the deltopectoral crest (“The shape of the deltopectoral crest in the humerus IRSNB M2308 allows to identify the specimen as a monachine seal” p.16,l.264-265) which indeed, shows a condition most common in living and extinct Monachinae.

However, as the same authors noted in a previous publication:
“... many extinct Phocinae, such as C. maeotica, K. benegasorum, Phoca vitulinoides, and S. sintsovi (Cozzuol, 2001; Koretsky, 2001; Leonard Dewaele, 2016, personal observation) show varying conditions that are intermediate between extant non-phocid pinnipeds (Odobenus Brisson, 1762 and Otariidae Gray, 1825), monachines and extant phocines (Fig. 3). From what is present of the distal portion of the humerus, the deltopectoral crest of Prophoca rousseaui (and Leptophoca proxima) appears to terminate near the distal end of the diaphysis, as in O. rosmarus, otariids, and monachines (except L. weddellii and L. carcinophaga) and a number of extinct phocines (e.g., C. maeotica, K. benegasorum, Phoca vitulinoides, and S. sintsovi).” (Dewalele et al 2017).

Consequently, the condition of the deltopectoral crest in this specimen is not enough to assign this specimen to the Monachinae. Also, the presence of a prominent supinator crest is also unusual between Monachinae and the specimen described here had this condition, that may be considered an apomorphy of Phocinae. Unfortunately, the absence of the distal end of the humerus makes impossible to check the presence of a entepicondylar foramen, another derived feature of Phocinae.

All those makes necessary a better comparison and I think essential to add Phocinae, especially those that exhibit this (plesiomorphic) condition to it.

Monachinae geography.
One of the relevance of this finding is the late surviving of the subfamily in north coast of Europe. In the map of Figure 4 the authors plot the present and past distribution of Monachinae in the North Hemisphere. In it they show that Monachus monachus was restricted to the south and east of Mediterranean and to the Atlantic coast of North Africa. However, as was shown by Gonzales (2015), M. monachus was present in pre-historic times at least up to the Cantabrian coast, northern Spain, based on paleo-archeological material. Despite this no invalidate the relevance of the finding presented by the authors here, it shows that Monachinae were present until quite recent in localities much closer to the one form which the new specimen comes than they identified.
Experimental design: Not applicable
Validity of the findings: Since the feature used to determine the systematic position of the specimen is questionable, the central argument of the manuscript may be challenged and it needs, at least, be better justified.
Additional comments: Note on taxonomy.
Despite the division of the family Phocidae in two subfamilies (Monachinae end Phocinae) is widely accepted, in the last years several papers by Koretsky and co-authors proposed a different view, with four subfamilies (namely Monachinae, Phocinae, Cystophorinae end Devinophocinae, Koretsky & Rahmat, 2013, 2015, between others). As clearly the authors do not adhere to this view, I will like to see at least a mention of the reason for not.

Minor changes suggestions.
I suggest adding a small location map for the finding.
The reason for restricting the age to Late Pliocene contrary to late Early to late Pliocene should be better elaborate.

References.
Dewaele, L., Lambert, O. & Louwye, S. 2017. On Prophoca and Leptophoca (Pinnipedia, Phocidae) from the Miocene of the North Atlantic realm: Redescription, phylogenetic affinities and paleobiogeographic implications. PeerJ 5(5)

González, L.M. 2015. Prehistoric and historic distributions of the critically endangered Mediterranean monk seal (Monachus monachus)in the eastern Atlantic. Marine Mammal Science, 31(3): 1168–1192

Koretsky, I. & Rahmant, S. 2015. A new species of the Subfamily Devinophocinae (Carnivora, Phocidae) from the Central Paratethys. Rivista Italiana di Palentologia e Stratigrafia, 121(1):31-47

Koretsky, I. & Rahmant, S. 2013. First record of fossil Cystophorinae (Carnivora, Phocidae): Middle Miocene Seals from the Northern Paratethys. Rivista Italiana di Palentologia e Stratigrafia, 119(3):325-350


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

Input:
Title: A LATE SURVIVING PLIOCENE SEAL FROM HIGH LATITUDES OF THE NORTH ATLANTIC REALM: THE LATEST MONACHINE SEAL ON THE SOUTHERN MARGIN OF THE NORTH SEA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is understandable and professional. The structure of the manuscript follows the standard of the Journal. The different sections of the manuscript are adequately developed, allowing a fluid reading and understanding of the text. However, some points of the study need to be clarified (see below).
Experimental design: The main research subject is clear and relevant, improving the general knowledge of the pinniped fauna from the Pliocene of the North Atlantic Ocean.
Validity of the findings: See my comments below.
Additional comments: The most important issue is the fact that the authors do not explore the morphological differences between the humeri of Monachinae and Phocinae and neither perform comparisons of IRSNB M2308 with extinct members of Phocinae. This is relevant because it has been described that some fossil Phocinae exhibit “Monachinae” features (and also the other way around, with some species of fossil monachine with phocine features). See some examples in Figure 3 in Ray (1976), Cozzuol (2001), Wyss (1988). How would these observations affect the main conclusion of this study? Which other traits of the humerus are useful for differential diagnosis between the subfamilies? I suggest performing an extensive qualitative and quantitative comparison including species of Monachinae and Phocinae, with the aim to clarify this issue. Similarly, Fig. 3 should be improved by adding additional comparisons with relevant fossil Phocinae and Monachinae. Finally, please provide a table with comparative measurements of the humeri of the species mentioned in the text (e.g. Pliophoca etrusca, Callophoca obscura, Homiphoca, etc).

I also question about how the authors rejected the possibility that the differences between the humerus of Callophoca obscura and IRSNB M2308 (i.e. the overall size of the bone, the height of the lesser and the greater tubercle relative to the head, development of the pectoral crest) are not because of sexual dimorphism. I suggest, to include some type of analysis addressing this matter. How morphological variation because sexual dimorphism could affect some of your conclusions?

The discussion develops adequately the major implications of the finding. For clarity, I suggest to organize it in subsections (identity, biogeographic implications or so). Also, see my comments above. Finally, I suggest performing a deeper discussion about the biogeographic and diversity implications of this finding. For instance, is it consistent with classic reviews such as Ray (1976), Deméré et al. (2003)?

Other minor comments:
-	Line 57-59, you state that "The elephant seals of the genus Mirounga Gray, 1827 live in sub-Antarctic waters and along the western shores of South America, but also in the Northeast Pacific, from California to Alaska”. However, permanent colonies of SES are found in Puerto Madryn, Argentina, which is located on the southeastern coast of South America.
-	In the line 78 are mentioned Mediterranean, Caribbean and Hawaiian monk seals belonging together to the genus Monachus. However, a recent study (Scheel et al., 2014) proposed that Hawaiian and the extinct tropical monk seal belong to the new genus Neomonachus. Correct the assignation or justify why you do not.
-	Line 178 says (Fig. 3A-D, versus Fig. 3E), but I should say Fig. 3F.
-	Line 179 mentions the Fig. 4F, but it should be 3F.
-	In Figure 3, be consistent with the anatomical views and information presented in the text. Further, I suggest improving contrast/light levels of IRSNB M2308, especially in anterior and lateral view.
-	I suggest modify Fig. 4 and include the ages of the taxa next to the name of their corresponding species to make easier the visualization of the biogeographic and temporal consequences of this new finding.


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

Input:
Title: A LATE SURVIVING PLIOCENE SEAL FROM HIGH LATITUDES OF THE NORTH ATLANTIC REALM: THE LATEST MONACHINE SEAL ON THE SOUTHERN MARGIN OF THE NORTH SEA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well-written in clear English, short/concise, and has an appropriate reference list.
Experimental design: No issues are evident with the methods. I do wonder about the statement regarding the suitability of naming a new genus and species from a holotype humerus - the authors are quite cautious here, yet in another recent paper, Dewaele and others (Royal Society Open Science) erect two new genera and species with humeri as holotypes. This contrasts starkly with the attitude expressed in the current paper, and I wonder if this bears more explanation.
Validity of the findings: The identifications in the paper are sound.
Additional comments: I reviewed an earlier draft of this paper, which was in already excellent shape. The authors have implemented all of my earlier suggestions, and therefore I recommend acceptance practically as-is. I note one very minor issue: It should be written as "analog caliper" in the table 1 caption. (is 0.1 mm really possible with non-digital calipers?).

Kind regards,

Robert W. Boessenecker, Ph.D.
College of Charleston
Charleston, SC


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

Input:
Title: A LATE SURVIVING PLIOCENE SEAL FROM HIGH LATITUDES OF THE NORTH ATLANTIC REALM: THE LATEST MONACHINE SEAL ON THE SOUTHERN MARGIN OF THE NORTH SEA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The reply for authors are adequate enough despite I am still not completely convinced of the subfamiliar assignation of the specimen.
Despite this, I have no regrets in accept authors opinion and recommend the acceptance of the manuscript.
Experimental design: None
Validity of the findings: Acceptable.
Additional comments: None


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

Input:
Title: A LATE SURVIVING PLIOCENE SEAL FROM HIGH LATITUDES OF THE NORTH ATLANTIC REALM: THE LATEST MONACHINE SEAL ON THE SOUTHERN MARGIN OF THE NORTH SEA
Review round: 2
Reviewer: 2

Output:
Basic reporting: This paper is a straight forward study highlighting the latest known record of the Monachinae in the North Atlantic, providing further information on the biogeographic history of the group. Geological background and comparisons seem appropriate, and I have no reason to doubt the authors conclusions. References are mostly up to date, although I note that Figure 6 does not include references to Pliocene monachine records from Spain/Montepellier region of France, referenced in Berta et al. 2015 paper on Pliophoca. Figures seem good and English is also good, although I have a few minor grammatical suggestions I will list later.
Experimental design: Methods seem sound and the paper overall seems to fit the scope of the journal.

My only suggestion (which is likely outside the scope of these revisions, but something I strongly encourage the authors to pursue in the future), is that I would have like to seem some sort of quantification of the shape differences highlighted in Figure 5.For instance, generating an area measurement for the region under the curve representing the deltopectoral crest would allow better characterization of this variation and allow more rigorous comparison between taxa. Again, I only include this in here as something for the authors to pursue in the future, not something I expect them to perform for this revision.
Validity of the findings: Overall everything is sound, and most importantly I believe the referral of the specimen as far as taxonomic identity and age is correct.

I do think, in lines 359-363, the authors makes some pretty big assumptions that are a bit to speculative.I don’t think we can necessary assume it that sea temperature changes were the most important factor driving monachine extinction in the high latitude North Atlantic region, especially when the fossil record for the Late Pliocene and Early Pleistocene isnt that great for the region. Their extinction might be related to change in sea temperature, but given the success of the Monachinae in Antarctica, this seem to be a pretty big assumption, and other factors such as sea level changes may have played as large or larger a role, and maybe should be referenced.
Additional comments: The following are minor revisions, largely related to grammar or other small details:

Abstract, Results: “Confronting the information” = “Combining the information” Also in the abstract you mention the deltopectoral crest and other features as indicating monachine affinities. What are the other features?

Line 47: remove “commonly”

Line 52. Perhaps rephrase selected morphological features. After all, almost all morphologic classification is based on some set of features. Perhaps it would be more useful to use “few morphologic characters” instead.

Line 60: It would be useful to list what morphologic features clearly separate the group, rather than just state they are clearly separated.

Line 92: I thought the referral of fossils in the North Atlantic to Homiphoca has largely been refuted?

Line 187: “allowing to assume” = “allowing us to assume”

Line 219: “banded seal” = “harp seal”. I have never seen banded seal used as a common name in English.

Line 335: delete “the it should be noted that” as unnecessary

Line 376: replace “pagophilic evolution” with “pagophilic traits associated with ice-breeding”


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: ARTIFICIAL LIGHT AT NIGHT ALTERS LIFE HISTORY IN A NOCTURNAL ORB-WEB SPIDER
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is an interesting and thorough study, well described and performed. The manuscript is very well written throughout.
Literature coverage is appropriate.
Experimental design: Using a split-brood design, offspring from 18 females of an orb-web spider were subjected to two treatments that differed in the amount of light provided during 12hours of dark. Spiders either received 0 or 20 lux during the night and were raised from egg to adult. Growth and development of 440 individuals were carefully monitored and the number of moulting events was documented. Prey availability was standardised and increased twice throughout development to accommodate increasing demands.
The experiment was designed to test the hypothesis that artificial light at night (ALAN) would affect survival and life-history as well as female fecundity.

The experimental design varies a single variable (light at night) on two levels. A more complex design might have been valuable, as for example two levels of prey supply. Perhaps this could be planned as a follow-up?
Validity of the findings: Results show that the main effect of the treatment (light during the night) was an accelerated development with costs in adult body size and female fecundity. How well this result can be interpolated to a natural situation is unclear and I wonder whether the amount of prey provided was relatively lower than the average in nature. Perhaps that was intentional since an ad libitum provision of food might have masked the effects of ALAN? An explanation of the reasoning behind the applied feeding regime should be provided.
Additional comments: Despite a thorough description of experimental conditions, I have a few minor questions, none of which are serious concerns:
1.	Eriophora is a nocturnal species. How did that affect web building and hunting behaviour? Did they perhaps take down webs during the day? Or did you observe any other differences during night and day, perhaps related to activity in some way? Any light-dependent activity could have affected prey capture directly, I think.
2.	The light regime may have also influenced behaviour of the flying insect prey and possible differences in the flies’ activity may affect the probability of getting entangled into the web. Perhaps the authors have collected information about whether the provided prey items were actually eaten or not?
3.	During each phase, a single prey species was provided, first Drosophila, then Musca and finally crickets. I am curious whether the provision was adequate to cover all needs for normal development.  Could prey supply be part of an explanation for the high mortality rate? This is not a serious concern, as conditions were the same in both treatments but it might be relevant to briefly discuss whether the prey regime might have been rather poor or not despite the constant prey supply. There is no discussion about the appropriateness of prey quantity to simulate natural conditions and growth. Obviously, prey numbers were high enough to allow growth and moulting but did the duration of development and the size at maturation reflect natural conditions? Growth of almost a year seems a long time for a spider. Perhaps there are data on sizes and fecundity of wild females? If so, I would find a comparison interesting.
4.	Adults received crickets matched to their size. Does that mean that smaller females received smaller crickets but the same number? If so, why was that done? Could they have eaten more if you would have given them more?


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: ARTIFICIAL LIGHT AT NIGHT ALTERS LIFE HISTORY IN A NOCTURNAL ORB-WEB SPIDER
Review round: 1
Reviewer: 2

Output:
Basic reporting: I think the article is very well written, clear and structured in an easy way that allows comparisons between the different sections of the manuscript, especially between methods, results and discussion. Two small points:

1. I think while the structure of methods, results and discussion is very similar and helped me assessing each component of your study (maturation time and survival, body mass, reproductive success), the same is not true for the statistical analyses section. I would sugest you to keep a clear distinction also in this section, clearly stating what were the models used for assessing changes in mat. time and survival, body mass and reproductive success. At present this section is a bit messy and it is not always clear which model was used for which analysis,

2. While I appreciate the long list of literature you cite and refer to, I think you have missed a key recent study in this same journal (Yuen and Bonebrake 2017 - Artificial night light alters nocturnal prey interception outcomes for morphologically variable spiders - Peer J). Here the authors showed something counterintuitive, spiderwebs in lit areas were less successfull in intercepting moths than webs in unlit areas, which is contrary to your expectations about potential benefits of artificial light at night for spiders' foraging success. I think that reporting the findings of this study in your own article would make it a much more unbiased and comprehensive discussion of your results.
Experimental design: The design is sound and well explained. Few minor questions/comments:

1. L126-141. Did all spiders mate? If not, was there any difference in the probability of mating between control and LaN spiders?

2. Related to the previous point. Did each individual mate only once or multiple times? If the latter, did reproductive success decrease with age, and was this different between light treatments? I am particularly interested in the possibility that spiders under LaN may have had an earlier onset of reproductive senescence.

3. L103-104. It's ok to report the lux range of the collection sites, but what about mean +- SD? I think this information is sorely needed. Based on these values, why did you choose 20 lux as your experimental light intensity? I think you need to specify this.

4. Relatd to the previous point. Any idea whether the light levels at the collection site influence any of your subsequent results? What proportion of the variance was explained by your random effect family in your mixed models?

5. L110. Were these plastic cups transparents? Otherwise how did you ensure that spiders were exposed to LaN?

6. L237-240. This aspect on growth trajectories is really interesting and made me thinking of whether you actually have the data to proper calculate growth curves of spiders in the different treatments, but reading your methods sections it's not clearly to me how many times body mass was measured per individual. Could you calculate growth curves and test for treatment effects in a proper way?
Validity of the findings: I think overall the data is robust and statistically sound, and the results well interpreted. I do have few comments though on the statistical approach and the way that results are reported.

1. L169-170. It's hard for the readers to appreciate the magnitude of the effect of LaN on longevity without clearly stating the effect size of this treatment compared to the control group. You state the effect size for the the reproductive success (L196), and that was really helpful to me to visualize the importance of this result. Pleae try to use the same approach throughout the results section and make use of effect sizes.

2. L177-178. This does not make much sense, as when a significant interaction is present one cannot really interpret the main effects. I suggest you delete the first part of this sentence and report only the result for the interaction term.

3. I question the approach of splitting the datasets into sexes and treatments. This way you are running a lot of independent models, increasing the risk of finding significant effects simply by chance. I would rather use posthoc tests with Bonferroni correction or any other way to correct for multiple testing, or bootstrapping methods that produce reliable confidence intervals upon which the comparison of different treatment groups and/or sexes can be done.
Additional comments: Although I have enjoyed reading your article and I think you present very interesting and strong results, I was also slightly disappointed in that I thought that you had the possibility of a much deeper insight into the effects of light at night on life-histories and fitness, by linking these data to more mechanistic/physiological aspects that might have helped you to explain your results. in the end, these data is very cool and convincing but I miss a clear explanation of how light at night affects maturation time, survival, body mass and reproductive success. Also, how likely is that spiders are exposed to such high levels of LaN in nature? 20 lux is dim, but still in the higher range of what animals can be exposed to in light polluted areas, particularly for mobile animals such as spiders. Also, the explanation for the earlier maturation time is not really comprehensive. What do the authors really think is happening? Do you think that spiders perceive a longer photoperiod, therefore being "tricked" into thinking that it is late in the season and they need to speed up their maturation if they want to be ready at the right time, or perhaps something more fundamental about the circannual and circadian rhythms of these animals is modified? For instance, see Wikelski et al 2008 Phil Trans B for a very interesting take into how different photoperiod might affect energy turnover which in turn might affect the speed of the circannual clock. in general, I think a more fundamental understanding of the mechanisms at play might have made this article much more compelling. But I do like it still!


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: YOUNG, AMERICAN INDIAN OR ALASKAN NATIVE, AND BORN IN THE USA: AT EXCESS RISK OF STARTING EXTRA-MEDICAL PRESCRIPTION PAIN RELIEVER USE?
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, the manuscript reads well, but there are typos, missing words and use of idioms, which take away from the quality of the manuscript, e.g. line 129 ‘Figure 1 shows depicts these…’, line 179 ‘scratch the surface’ and line 189 missing word (selected examples).
Experimental design: See below.
Validity of the findings: No comments.
Additional comments: Thank you for the opportunity to review this interesting manuscript, offering further data and insights into the dire situation in the US of high rates of opioid overdose deaths. I think the focus on sub-populations is very important and perhaps somewhat overlooked in previous work.

Whilst the authors briefly discuss the age of the data (some data waves being more than 15 years old) in lines 170 to 174, reading the Introduction, Results and Discussion, might easily leave the impression that this is new data, e.g. line 147: ‘these new annual incidence rates estimates’ and line 143 to 144: ‘this country’s US-born young people…have such large incidence rates…’ (They had in this dataset but do they today?). I would hope that the authors would make this clearer in the Introduction and Discussion. What’s the value of the dataset? It is historic insight, does it apply to the current situation, and if so, how?

Minor comments:

Lines 39 to 42: Please elaborate as to what you have in mind in regard to ‘targeted prevention initiatives’ and ‘more universal prevention efforts’.

Line 42: The meaning of ‘more serious drug involvement’ is not clear. As the authors convincingly argue, opioid overdose deaths are very serious, and hence this sentence comes across as a self-contradiction. Perhaps the authors are referring to PO misuse transitioning into heroin use or use of fentanyl contaminated fake medicines?

Line 44: Epidemic of what?

Line 47: The meaning of the term ‘hazard-laden’ is not clear. Is it needed considering that the authors provide a good and useable definition of ‘extra-medical’ PPR use? Furthermore, there would be examples of ‘extra-medical’ use that would not be ‘hazard-laden’.

Line 51: IBID. Epidemic curve of what?

Line 54: Are casualties not the same as fatal overdoses?

Line 59: What is meant by ‘have been neglected’? Gap in research and/or treatment?

Line 81: Please provide a brief explanation for the use of this dataset (see general comments above).

Line 89: Which questions were used to obtain data on PPR use?

Line 115: What is the gender distribution in the sample? Distribution according to US regions? Perhaps it would be useful to include a table with descriptive analysis to characterise the sample?

Line 142: What is meant by ‘PPR involvement’?

Line 178: What is meant by ‘drug involvement’?


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: YOUNG, AMERICAN INDIAN OR ALASKAN NATIVE, AND BORN IN THE USA: AT EXCESS RISK OF STARTING EXTRA-MEDICAL PRESCRIPTION PAIN RELIEVER USE?
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an outstanding paper, clearly written, with a enough information and references to provide background support to the authors' decisions.
Experimental design: This is novel research that, in my opinion, makes the best use of the NSDUH data.  Rarely investigators study incidence of drug involvement.  This has many advantages because many threats to validity are avoided.  The use of a probability sample with appropriate statistical methods are also part of the strengths of this study.
Validity of the findings: The study provides novel and strong new insights to support new research, and the authors offer ideas about potential areas for new studies. The only factor that limits excitement is that the analyses are limited to the period of 2002-2009, and it would be useful to have more recent data.  It is not clear why the authors chose this particular period, and the assumptions behind the idea that a replication with a 2010-2017 period should be informative.  Clearly, the overdose crisis of PPR has received much more attention in the past years, hopefully modifying the conditions that governed during the 2002-2009 period.
Additional comments: This is an excellent report. Publication of this article may be useful to support increased resources for programs serving the populations at higher risk identified here.


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

Input:
Title: YOUNG, AMERICAN INDIAN OR ALASKAN NATIVE, AND BORN IN THE USA: AT EXCESS RISK OF STARTING EXTRA-MEDICAL PRESCRIPTION PAIN RELIEVER USE?
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have successfully edited the document to provide more clarity.
Experimental design: The design has not been changed and was already acceptable.
Validity of the findings: Idem.  The authors have done a good job in revising and amending the manuscript for clarity, which allows to realize the strengths and limitations of the research.
Additional comments: While most of the issues have been resolved, the manuscript can be improved by one last editorial revision.  Some of the phrases that were added in the newest version read awkward (e.g., page 8, line 190).


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: THE CCB-ID APPROACH TO TREE SPECIES MAPPING WITH AIRBORNE IMAGING SPECTROSCOPY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is concise, clear, and includes a good background of advances and challenges of identifying tree species from hyperspectral images. The figures and tables are relevant and well-designed. The data used in this study are available from NEON. The author has provided the information necessary to locate and download the data.
Experimental design: The author introduced the CCB-ID approach to identifying trees, based on two machine learning methods (gradient boosting classifier and random forest classifier). Both methods were rigorously applied in terms of fitting parameters, training (model and probability calibration), and testing. The hyperspectral dataset  was refined to remove spectral outliers and to reduce dimensionality (through PCA). The tree dataset was processed to remove class imbalance (some species with hundreds of samples, other species with tens or less samples). The author provided details of each step of data preprocessing and model fitting, training, and testing.
Validity of the findings: The CCB-ID approach proposed in this study produced promising results (high accuracy and low cross-entropy), but also identified limitations (mapping rare species) and components of the workflow that are problematic and need further development. The author summarized well the advantages of CCB-ID and the limitations and improvements needed.
Additional comments: I appreciate the author’s use of taxonomic approach to thinking about classifying trees from hyperspectral data. The methods are explained in detail. I think this study will inspire further work to fine-tune the CCB-ID approach. I enjoyed reading this manuscript. Below is a list of minor editing suggestions.
Abstract: spell out acronyms (CCB-ID, NIST, ECODSE, NEON); replace “obviate” with a more commonly used synonym – here and throughout the manuscript
Introduction: lines 34-35: spell out NIST and ECODSE
Materials and Methods
lines 77-78: mention that the data are freely available through NEON (in case ecodse.org will become inactive in a few years).
line 120: the specific epithet “styraciflua” is misspelled as “styaciflua”; the same epithet was misspelled as “stryaciflua” in Figures and Tables. Double check all scientific names.
line 193: the use of “sample scale” here is confusing; please explain at the beginning of Methods the difference between sample scale and crown-scale
Discussion
line 270: reword “showed different within…”
Conclusions: line 334: reword “wasn’t”


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: THE CCB-ID APPROACH TO TREE SPECIES MAPPING WITH AIRBORNE IMAGING SPECTROSCOPY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript has good voice and flow throughout. However, there are several opportunities to make more solid statements by adding references to them.
Experimental design: The author clearly explains the methods used, the assumptions made,  and decisions to reach to the conclusions. However, there are some details about the research that only come later in the manuscript. The reader will benefit from having important details presented earlier in the manuscript, such as the sample sizes and how these changed throughout the different stages in the methods.
Validity of the findings: The author reports a new method to build on the efforts to classify tree species. However, the decisions made early in the methods could be influencing the reported overall accuracy and also be influencing further analyses. While the author acknowledges this situation, the way that overall findings are presented could be misleading.
I suggest to explicitly account for the uncertainty added by including a low sample size for underrepresented species in the overall accuracy assessment (e.g. report the accuracy of the method to discern a) all species and b) the species for which there are sufficient samples like Quercus and Pinus).
Additional comments: Line 2-14 This paragraph addresses the need for data to map species, and offer the possibility to do so via image spectroscopy. While this is true for vegetation, the text appear to be true for other groups. Later it is acknowledged, but I recommend to do so earlier.
Line 32 What NIST stands for?
Line 52 What are the characteristics of the referred subset? Are there any examples?
Lines 54-59 would benefit from a reference for the statement.
Lines 73 Are there important characteristics from the types of data used (e.g short description for each data set, sample size, etc.)
Line 109 Is there a reference range used elsewhere? E.g. why 100 and not 1000 ?, which is an exaggeration, but not knowing the typical range the reader cannot have an idea of the authors choice.
Line 146 What is F1?
Line 149 Likewise, what is RCF? Please indicate this and other acronyms
Line 211 How does this accuracy score compares to other participants? This will be useful to see the relevance of this metho
Line 259 With a sample size of 1, the result could be like tossing a coin. Please see the comment to Table 1
Line 301 Is there a reference that leads you to this inference?
Line 334 The use of contractions (i.e. Wasn’t ) was not used earlier, so probably avoid it here for consistence.
Table 1. I understand that the confusion matrix is at the sample level, which implies that the numbers in the tables correspond to the numbers of samples evaluated. If that is the case, why there is only 1 testing sample for several species? The author acknowledges this in the discussion but has not fully explain how this low sample size could be influencing the accuracies. These low samples should probably be excluded from the overall accuracy scores from the method used or at its best, provide the overall method accuracy with and without excluding these species samples.
Scaling –up. How is the data representative for other species/locations? This idea is barely touched in line 342 but not fully developed to see how it can scale-up.


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: PATIENT-CENTERED CARE INTERVENTIONS TO REDUCE THE INAPPROPRIATE PRESCRIPTION AND USE OF BENZODIAZEPINES AND Z-DRUGS: A SYSTEMATIC REVIEW
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper is a well structured and quality submission. The authors have taken a lot of time and effort preparing it. There are some aspects, however, that require clarification and addressing.
1.	The non-English background of the authors is obvious with non-current terms used in some sections. E.g in abstract line 33 ‘side effect’ as opposed to ‘adverse effect’. In the introduction line 58 the word ‘tranquilizers’ is not in common use. May I suggest the use of the term ‘anxiolytic’ or ‘anxiolytic/hypnotics’ as an alternate. ‘Time horizon’, line 274, in the results section is also a term that is not in common use. In the discussion section, the word ‘inadequate’ line 333, should be replaced with ‘inappropriate’. The term ‘inadequate’ needs to be replaced by ‘inappropriate’ throughout the text. It’s used again in line 360 of the discussion. The use of the term ‘elderly’ is not current any more (line 378) and needs to be replaced with ‘older’.
2.	Some of the background information about benzodiazepines and z-drugs is not correct and needs to be corrected. In the abstract it is stated (line 32) that long-term therapy is associated with considerable side effects. It needs to be stressed that short-term therapy is also associated with considerable side effects, including falls, language impairment, confusion, cognitive impairment and ataxia. The statement regarding risks being associated with long-term use only is also made in line 62 - intro. A statement regarding tolerance to the effects of benzodiazepines is needed in this section. There is also a line saying that z-drugs have similar pharmacological effects (Intro - line 61) to benzodiazepines. This is not true. Z-drugs have a different GABA receptor binding site and do not possess anxiolytic properties - so this statement is not correct.
3.	The results section needs review as parts are not relevant, specific outcomes are not mentioned and some statements are unclear.
4.	Table 2 is separately labelled ‘Table 1’ on its header page.
5.	Finally, the referencing of journal names is inconsistent. Abbreviated versions are used alongside fully spelt non-abbreviated namings. Capital letters are also missing when Journal names are fully listed.  A good edit for the correct style is needed.
Experimental design: The methodology of the review is a strength and well described in sufficient detail. The research question is well defined, relevant and meaningful. However,
1. In the methods section there is no inclusion of the search term ‘hypnot*’ (hypnotics) which is odd and may have resulted in some papers being missed, and
2. In the eligibility part of the methods line 121 it states that studies were excluded if they focused on BZD use for mentally ill patients (e.g. psychiatric diagnoses such as panic disorder or schizophrenia). This needs to be re-defined. Surely generalised anxiety disorder or sleep disturbance are psychiatric diagnoses as per the DSM-6 – In this case all studies would have to be excluded. Do the authors mean serious psychiatric disorders? Panic disorder would be a common diagnosis in older adults.
Validity of the findings: The validity is good and table data consistent with the studies. Interpretation is good. the conclusion links well to the introduction. Just a few points:
1. In the results section the authors confuse matters when they include antipsychotics in the discussion of the intervention results. E.g. Line 272 provides an example of this. “Meader et al investigated if an intervention could change the intake rate  of antipsychotics…they found a reduction in antipsychotic dose (including BZD).“  It is not clear what the author means here and I question why the inclusion of antipsychotics in this results section is relevant to this paper?
2. When the interventions are discussed it would be clearer if the subject matter was confined to benzodiazepine use and the resulting outcome of the intervention. E.g. In the results section line 292, more detail is needed about the size of the decrease in drug use and whether the ‘drug use’ referred to was actually benzodiazepine use. In line 307 for this study there is no specific mention of benzodiazepines at all.
3.	More detail and clarification is needed with line 310 which is the last sentence starting with 'Compared with a control group….'
4.	Line 292, Smith is Alysia (i.e. female) so ‘his’ should be ‘her’. The information provided here in this discussion part stating that there was no effect of the intervention directly contradicts the information provided on Table 2 which reports a significant reduction in BZD use. Which outcome is it? Please make this clear.
Additional comments: A good proof reading by a native English speaker would assist this submission. The discussion section should also concentrate on the subject matter at hand and summarise the outcomes.


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: PATIENT-CENTERED CARE INTERVENTIONS TO REDUCE THE INAPPROPRIATE PRESCRIPTION AND USE OF BENZODIAZEPINES AND Z-DRUGS: A SYSTEMATIC REVIEW
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a literature review of intervention studies, based on a patient-centred approach, and directed at patients and at Health Care Professionals (HCP) in order to reduce the chronic prescribing of Benzodiazepines  and Z-drugs (by discontinuing chronic use and reducing incident BZD use).

Many features of a systematic review are present, but some are missing (protocol, common primary outcome measure, mathematical synthesis).  Although this is not a Cochrane review,  some essential features of Cochrane reviews have been used (trial selection procedure, risk of bias assessment of individual studies, Prisma flowchart and checklist).

The criteria of basis reporting are met, with regard to the language, refererences, background/context. The manuscript is well structured and self-contained.
Experimental design: Research question is well defined (for as far as patient-centred approach is well defined), and within the scope of the journal.

The investigation was rigorous and described in sufficient detail.

It is a bit more difficult to identify the knowledge gap that is identified here.

As the results are described only narratively, it is maybe not so important to analyse risk of bias across studies (publication bias, heterogeneity etc).
Validity of the findings: The narrative data are robust, but their statistical properties are hard to check.

The conclusions are within the fabric of the study and original research question.

The studies with inconclusive results are discussed.  Replication and further research is encouraged.

The question of the novelty and impact of this work remains, but the overview of existing study within the framework of a theory (patient-centred approach) is useful.

The connection between theory and the simple and complex interventions is well discussed (although not with a theory of change map).
Additional comments: L 307 : positively influenced by some other variables" is not very informative.

332-334: what is the point here. That you only found studies in the setting of primary care (GPs) and nursing homes ?  These sentences are unclear and wordy.
372 : do you mean "clinically relevant" or "statistically significant" ?

Some reference to reviews of pharmaco-epidomiological assessments of the risk of chronic BZD use and reviews of barriers and facilitators to use reduction could be made.

There is no distinction made between chronic not escalating low dosage sleeping pill use (only at bedtime) and escalating anxiolytic (or sedative) use in multiple doses per day.
These different indications should at least be discussed and also identified in the 21 selected articles.
Hypnotic* was not present in the search profiles.


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: PATIENT-CENTERED CARE INTERVENTIONS TO REDUCE THE INAPPROPRIATE PRESCRIPTION AND USE OF BENZODIAZEPINES AND Z-DRUGS: A SYSTEMATIC REVIEW
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is a better version.
-At times the English terms and expression are not correct e.g. derivates (should be derivatives) (69). The word 'seriously' needs to be put before 'mentally ill' (156). The word pharmacy should  replace the word pharmacology (332). What do the authors mean by prescribing rates were handled more appropriately in both groups? (354) This statement doesn't make sense. The words 'monthly visits' (362) should be replaced by 'medication reviews'. The word 'greater' should replace 'clearer' (387). 'More profound' should be placed between to and improvements in line 436.
-Some statements are only relevant to the author country. e.g in the abstract it is stated that the maximum treatment is 8 weeks (41). In most English-speaking countries it is 4 weeks.
-Could the authors please include the results for combined interventions as they do with HCP and patient studies? What were the numerical impacts of the Patterson and Westbury studies specifically the relative reduction in benzodiazepine rates of use? This important information is omitted here for some reason?(359-373).
-citations are missing at times. e.g. behind the bold words in line 450, and after 'still prescribed frequently' (81)
- inconsistent referencing is still evident. Journal titles are not all capitalised and there is a big mismash of abbreviated titles and fully named journal titles. Please follow the referencing rules consistently
- Tables are sound.
Experimental design: Mostly well done review
Validity of the findings: Mostly good. I think though that the authors need to qualify that patient education, although generally a more effective strategy when compared to direct HCP approaches, may not be suitable for older cognitively impaired residents of nursing homes or people with advanced stages of dementia. This is not stated and I think it needs to be.
Additional comments: the PsycInfo database search engine should be written as PsycINFO - this is written several times in the text.

The authors have accepted most of the recommendations but it still needs some more editing work. They also really need to pay more attention to referencing. It makes the work look sloppy.


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: PATIENT-CENTERED CARE INTERVENTIONS TO REDUCE THE INAPPROPRIATE PRESCRIPTION AND USE OF BENZODIAZEPINES AND Z-DRUGS: A SYSTEMATIC REVIEW
Review round: 2
Reviewer: 2

Output:
Basic reporting: no further comments, in addition to the first review round
Experimental design: no further comments, in addition to the first review round
Validity of the findings: no further comments, in addition to the first review round
Additional comments: The article has much improved. The authors have well responded to the comments of both reviewers.


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: PATIENT-CENTERED CARE INTERVENTIONS TO REDUCE THE INAPPROPRIATE PRESCRIPTION AND USE OF BENZODIAZEPINES AND Z-DRUGS: A SYSTEMATIC REVIEW
Review round: 3
Reviewer: 1

Output:
Basic reporting: Thank you for working hard on the expression in English.
Experimental design: No comment
Validity of the findings: Revision made to conclusion.
Additional comments: Thank you for incorporating the comments of the reviewers so comprehensively. Your paper has benefited greatly from it. Thank you also for doing considerable justice to this important topic.


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: ULTRACONSERVED ELEMENTS (UCES) ILLUMINATE THE POPULATION GENOMICS OF A RECENT, HIGH-LATITUDE AVIAN SPECIATION EVENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The writing here is clear and concise but most of the jargon is above my pay grade. I have no doubt that experts in genomics analyses will understand all this but it’s a daunting task for the non-expert to understand the Results section. The Discussion, however, makes it very clear what those results can and cannot tell us.
Experimental design: Seems fine and highly appropriate but someone more familiar with the methods should evaluate this
Validity of the findings: The findings seem entirely valid given the results but I do not have enough knowledge to evaluate the validity of those results
Additional comments: This looks to me like an interesting and very valuable study not only for showing the value of UCEs but also helping to determine the extent of genetic difference between McKay’s and Snow buntings. As the authors say, more work will be needed to determine where the consistent genetic difference are between these two species, especially with respect to identifying the genes responsible for the plumage difference. A few recent studies of other sister taxa of birds have been able to do this. This main value of this study seems to be in the evaluation of UCEs as a population genetics tool.
Also:
18-34 I feel there is too much jargon in this abstract. As it stands it will only be comprehensible to experts in this sort of genomic analysis. I suggest rewording or defining: orthologous, ultraconserved elements, marker sets, analysis pipelines,  30x sequencing coverage, significant genetic structure
51 this is unnecessary hyperbole. Powerful computers are not in any way strained by these datasets, they just take more time to analyze
Table 1. I would like to see more details in the figure caption as I do not understand the difference between parameters and estimates. It’s also not clear why you present lower/upper bounds and 95%CIs because they tell the same story. 95%CIs


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: ULTRACONSERVED ELEMENTS (UCES) ILLUMINATE THE POPULATION GENOMICS OF A RECENT, HIGH-LATITUDE AVIAN SPECIATION EVENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an overall well wrote and clear manuscript but a few paragraphs could be improved for readability (e.g. lines 83-87, 158-161, 169-171).

The structure of the manuscript conforms to the journal standards. Although the introduction is very relevant in the context of UCEs, the manuscript could be improved with more detail about the target species, including the known range distribution with the locations of the used samples.

The figures seem fine although they could just be considered as supplementary material, table 3 and 4, could be included as supplementary material and not in the main text. It was supplied raw data and code of the main demographic analysis, but I would advise the authors to add the scripts used for bioinformatic analyses and all the analyses code into the figshare link they provided for the raw data, so there would be no need to include the code as supplementary material. Additionally, I advise the authors to add a figure describing the six analyzed models (see below).

Although this manuscript is showing the advantage of using population genetic methods with UCE data, the results or absence of these don't necessarily support that well that UCE data should be used directly for population genetics. Below I comment on this and provide a few suggestions on how still UCE markers can be very useful.
Experimental design: This is original research within the scope of the journal, and although it is a short paper, it has important relevance for the studied species and for the understanding of UCE limitations with population genetics analyses.

The description of the methods was very thorough from bioinformatics to the demographic analyses, which helps anyone that would like to perform similar studies. However, the description of the models was less clear and that could be improved by drawing the scheme for which model (e.g. Robinson et al. 2014, BMC Evolutionary Biology).

This figure could help simplify the detail describing these methods since this section is confusing. This is just a suggestion but the authors could refer to the simpler model as no isolation, the simple split model as strict isolation and the models with migration and growth as isolation with migration and population change, with the sixth model as isolation with bidirectional migration. Additionally, I suggest to the authors to add, even if as supplementary, a figure of the spectrum which can show how informative the folded SFS is.

The optimization procedure could be more explicit in describing how many repeats were performed when varying the parameters within bounds, and please explain why three runs with the highest observed likelihood value would be enough. The same issue for jackknifing with only 10 replicates since this seems insufficient, but not sure if the authors did more and realized the difference between running 10 or 50 times yielded similar results. Also, not sure if averaging the highest five values when getting variable likelihoods is standard practice, but if so please add a reference (line 190). For better readability, the results for model selection could also be described in a table with the model and its respective maximum log composite likelihood values.

In relation to the sequence data analyses that were not shown, I suggest the authors to either remove the recombination analysis, because it doesn’t correspond to the main findings of the paper, or to include but with a plot that shows the lack of convergence by IMa2p.
Validity of the findings: Considering the difficulty in dadi with the “split-with-no-migration model” (line 264) and the no convergence with the sequence based analyses IMa2p, I would personally, feel more comfortable in acknowledging the presence of high gene flow between these species if there was another method corroborating these results, like fastsimcoal2 which is also based on SFS. Regardless, I see great value in this work as a possible example of a study suggesting evidence that described species with a few morphological differences should possibly be considered more as highly differentiated populations than as species.
I commend the authors for being cautious in declaring the need for more work to properly infer if these should be considered as different species, but suggest the authors to mention in the discussion how this could be done. This could possibly be done using the same UCE dataset but including a few more closely related species, like a species delimitation study with Calcariidae species including validation methods like BPP, or methods that use Bayes factors to properly test different hypotheses for the number of species (e.g. *BFD SNAPP and BFD StarbBEast2). I suggest this because the big advantage of sequence capture methods, like UCEs, is the flexibility of analyzing phylogenomic data and population genetics data, so a future study that uses the same markers but including samples from different species would exemplify this advantage of UCEs.
However, the authors could discuss a bit more the limitations in applying UCEs to population genetic studies, since using one lane to sequence 8 individuals from 2 species and only be able to perform SNP-based analysis (without including an outgroup), would probably make the study not the most cost-efficient, unless the same data is being used to address other questions. For the number of samples used and given the use of only SNP data, probably RadSeq data would have been better, but I can see that the goal was to also explore demographic analyses with haplotype data, which it seems these did not converge. If more individuals had been sequenced, another possible sequence-based approach would be using Extended Bayesian skyline plots (Heled and Drummond 2008).
Given this, I suggest the authors to reconsider what they think is the main take-home message in the conclusions, personally I think it is really interesting the evidence that these two described species might have more gene flow than expected to be considered as different species implying the need of a proper species delimitation study with integrative taxonomy.
Additional comments: Some additional comments:
•	Not clear why it should be removed the FASTA sequences from non-UCE loci (lines 118-119), these sequences could provide more informative loci if they would still align across species.
•	For consistency, please add the version of used software where this is not specified.
•	I assume the description of subselecting one SNP per locus is repeated in line 146 and 166, and that this step was not done before and after converting the SNPs into a biallelic format if that is the case I suggest removing line 146 and edit the sentence.
•	Although it is mentioned in the results, I would add to the methods that a DAPC was done, and possibly complement it with a PCoA, even if it is just to refer these plots to the supplementary material, so it can be visualized the 100% diagnosable samples to clusters.
•	As suggested, rewrite the description of the models since it is not clear the difference between 4 and 5, which I assume is that one includes population change parameters. The expression "five basic and one derivative model" seems unusual to me, these models are more or less complex in number of parameters even if the 5th model was not in the example functions in dadi. For better comprehension of the alternative models, it could be better to enumerate them by complexity in parameters.
•	I acclaim the authors for explaining how they obtained each value to convert estimated parameters to time and population size, but given the filtering performed during bioinformatic processing, I think it would be helpful to mention what is the total sequence size used to convert the substitution rate. In line 290-291, it is mentioned the difference in loci size as a comment for the difference in substitution rates, but ideally this wouldn’t affect the results when you take into account the total sequence size, so this comment seems unnecessary.
•	The authors mention this is a study that adds to the utility of UCEs when looking to population data (line 271), please add a few other examples where analyses at similar scales have been done.
•	Considering the reduced number of samples used and these were from very few populations, it could be possible the DAPC result is a bit biased by lack of diversity representing the distribution of the samples, so maybe there is a need for some caution about how much it can be said about the 100% diagnosability.
•	The species names in figure 2 legend should have the genus name or at least "P.".


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: ULTRACONSERVED ELEMENTS (UCES) ILLUMINATE THE POPULATION GENOMICS OF A RECENT, HIGH-LATITUDE AVIAN SPECIATION EVENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: Overall it seems to me the authors have improved and clarified a lot of the issues that I previously mentioned in the first review. However, I still have some minor points that I think need clarification.
Experimental design: The methods section about DAPC should be a bit more detailed. This is mostly a problem because of the 100% diagnosability point that is used as an argument for supporting of these species. In the DAPC tutorial (https://github.com/thibautjombart/adegenet/blob/master/tutorials/tutorial-dapc.pdf), there's a section entitled 'On the stability of group membership probabilities' that explains how 'if too many PCs are retained this will have a destabilising effect on the coefficients estimated, leading to problems of overfit. As a result, membership probabilities can become drastically inflated for the best-fitting cluster, resulting in apparent perfect discrimination.' This is also why I suggested to additionally provide a PCA/PCoA, since discriminant functions are linear combinations of variables which optimize the separation of individuals into pre-defined groups, so the potential "lack" of clustering that a PCoA would show, might be more realistic than the inflated clustering the DAPC might be suggesting, if the retained number of PCs was much higher than the number of analysed samples. If this was something that it was taken into consideration, at least I request the authors to add the number of retained PCs.

There's another issue that I should have realized in the first review. In table 1, there are two migration parameters when the best model is a split with one migration rate between populations, for which the authors get the number of migrants by multiplying by the population size of each taxon. However, the migration rate estimated from this model can only provide the number of migrants between populations without an asymmetric gene flow. This should actually be calculated in relation to Nref and not to the estimated with the population size of each taxon, I found this topic in dadi's forum to be useful: https://groups.google.com/forum/#!topic/dadi-user/AY_4NoEjsx8. Additionally, I urge the authors to include the information of how many sites were considered to make these calculations, given the concern to think about what would be the best substitution rate and generation time, this is the only information that is not explicitly stated for this purpose.
Since the authors prefer to keep the section about recombination within loci, I suggest this section be moved in the methods to be before the demographic analyses, following the order as it is in the results.
Validity of the findings: The issues still pending about the validity of the findings are related with my two comments in the previous section. Since the number of migrants per generation should be corrected, I think it's likely the number of migrants per generation would be two orders of magnitude lower, which will change the discussion of the findings. This suggests that maybe gene flow between these taxa is not as high as the 2-3 migrants, which tend to be more common between well-connected populations than between supposedly well-differentiated species.
Since I am also not so certain about the 100% diagnosability of DAPC without knowing if the sampling size was considered (or without observing these data with a PCoA or a structure plot), there might be more to comment in the discussion about evidence of why these population genetics results might support or not the validity of these species. Given the relatively recent split time and the evidence for gene flow (although now maybe low),  I think the difficulty to do proper species delimitation within this group, as referred by the authors in response to my comments, could also be mentioned in the discussion. I personally think it is also the lack of an outgroup, even to get an unfolded SFS which could be more informative, that is limiting better conclusions from the population genetics side of analyses that can be executed when trying to do proper species delimitation.
This is just another suggestion that I should have provided initially, but since the authors estimated nucleotide diversity using a concatenated alignment in MEGA, and the fact these species seem to be in the "grey zone of speciation" (sensu Reux et al. 2016), maybe it would also be worth it to provide dxy and da values between species beside the significant but really low Fst value.
Additional comments: These are just some minor issues that could be improved:
- The last sentence of the introduction reads much better but some commas would improve readability.
- The structure of subsections like 'Laboratory.—' in lines 96, 115, 148 seems to be different from most papers I have seen from peerJ.
- In line 113, I personally find it unnecessary to abbreviate 150 bp pair-end sequencing since it just adds another abbreviation that should be clarified.
- There were a few references in the manuscript for just Supplemental Information without referring to the number of table/figure (e.g. line 188, 249).
- In line 215, it is not clear what is (hit table, csv) referring to.

I apologize to the authors for not identifying these two main issues I detected by reviewing this manuscript a second time. I think this a relevant paper that will be a stepping stone for future research done with UCEs and demographic analyses, and that's why I think these issues should be clear and well done for others to follow this work.


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

Input:
Title: ULTRACONSERVED ELEMENTS (UCES) ILLUMINATE THE POPULATION GENOMICS OF A RECENT, HIGH-LATITUDE AVIAN SPECIATION EVENT
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Thank you for the thorough responses and apologies for my own confusion in calculating the number of migrants from your estimates, I seem to have confused how to use mij and Mij to get the number of migrants.


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: POTENTIAL IMPACTS OF FINFISH AQUACULTURE ON EELGRASS (ZOSTERA MARINA) BEDS AND POSSIBLE MONITORING METRICS FOR MANAGEMENT: A CASE STUDY IN ATLANTIC CANADA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is geneally well-written and easy to read and follow – there are few typos, which I have pointed out in the last section of the review, but these can be easily fixed.
I do have issues about the structure of the manuscript in its present form – in particular there is information that is missing in the methods:
	-notably, a lack of information about the 9 additional sites
	-and details about the sites that are brought up only in the discussion, but should be moved to the methods section.

In addition, I was surprised to find that discussion jumped so quickly into the use of metrics. The title, abstract, introduction, methods, and results are very focused on the present study, but the discussion mostly focuses on using metrics for determining impacts. This needs to be better balanced throughout the manuscript (starting with changing the title to indicate this)! I LIKE that the authors are discussing how these metrics could be used, but this should be clear.

I would also like the 9 additional study sites to also be listed in Table 1, and shown on the map in Figure 1. I wonder also if it would be appropriate to add this data to the raw data, as there it only shows your four sites.

There are also legends missing from some graphs (I point this out below).
Experimental design: The experimental design is ok. It would be nice to have more than one control site, but you do make up for this by using the 9 additional sites (and I like that you pulled in additional data to fill the gap).

As I mentioned above, the manuscript addresses two important but different questions: First the impact of this particular fish farm, and secondly the use of metrics, but the title, intro, and abstract do not accurately reflect this.

I would like it to be pointed out explicitly in the methods that the fish farm did not actually have any fish in it when you did your sampling. This is stated, but it seems a bit hidden in the methods.

I also have a suggestion for the stable isotope analysis: you have the delta C and delta N of your eelgrass in all the sites, and you seem to also known the values of the potential sources (lines 354-358 and 370-373). Have you considered using a diet-mixing analysis which could possibly tell you where the nitrogen and carbon is coming from?
Validity of the findings: I have a major issue with how you are interpreting your non-significant results:
In lines 243-245, you say that there was lower shoot density, but at the same time say that it is non-significant. You do the same in lines 255-257 – either the results are lower/higher or they are non-significant, you cannot state both!

Again in the discussion (lines 297-299, line 384, lines 417-419) you say that you found impacts on shoot density, cover, biomass, and then on macroinfauna community metrics – but the only significant finding in this is the eelgrass biomass! You are severely misrepresenting your results here!

That said, I cannot quite interpret the figures in line with your statistical results – based on the error bars, it seems there should be significant differences between: SI and OW in Figure 3a, and then CB and the other sites in Figure 3b, CB and the other sites in Figure 4a, and between PJ and the other sites in Figure 4b.

I suggest you have another look at your statistical approach, and then properly report your results (and write your discussion) based on significance.
Additional comments: Note: some of this may repeat what I've said above, but I hopefully it is valuable to see it line-by-line

Title: As previously mentioned, change the title to give some idea that this paper is about the use of metrics for analysing impacts

Abstract: As with the title, revise the abstract to match the scope of the manuscript

Introduction:
Lines 32-33: citation for "most diverse of the soft-bottom marine communities"?
Lines 43-44: some expansion on the actual effects seen on seagrass meadows (increase/decrease, etc.)
Lines 51-53: Need some references for this
Lines 66-67: Expand on these potential impacts
Lines 75-77: Expand on this section to match the discussion

Methods:
Line 96: Explicitly state here that at the time of sampling there were no fish.
Lines 99-101: State the distances of each site here
Line 111: How far apart were the transects?
Lines 125-126 and 128: This is confusing, you state several taxa and then mention only hydroids? If you are only using hydroids, then remove the references to the others.
Lines 166-175: Move this section to the beginning of the Methods section or integrate with the last paragraph of the introduction.
Lines 174-175: Expand upon these 9 sites. Where are they (add them to the map)? And how are they used statistically? Are you taking the mean of all of them and treating them as a fifth site?

Results
Lines 242-245: These are not significant, do not state them as if they are
Line 255-257: Not even close to significant (according to the p-values above), there is no trend.
Line 260: Fix typo: “no overlap” to “noT overlap”
Line 262: change “lied” to “lay”
Line 278: “also” should not be capitalised
Line 280: remove “beach”

Discussion
Line 297: The only significant result is eelgrass biomass!
Line 298: Also not significant!
Line 302: Name the site
Lines 306-307: As I mentioned above, you need to better integrate these two goals (impacts in your site AND using metrics) into your whole manuscript. I would also suggest that based on your non-significant results, eelgrass cover and density are NOT appropriate metrics, not is infaunal biomass and abundance.
Lines 310-334: Most of the this section should be moved into site description in the methods (except for lines 326-328 abd kubes 332-334)
Lines 344-346: A sentence or two of the ecological consequences of anoxia/hypoxia would be good here.
Lines 354-357: As I mentioned above, it seems you would have the data to do a nice diet-mixing analysis here!
Lines 366-369: This is good! I am wondering if the cold event could have had an effect on the eelgrass community also?
Lines 384-385: NOT significant, exept biomass!
Line 386:  The “TWO” closest sites
Line 416-417: once again, you cannot state “our results indicated lower biomass” especially as your say right before that there were no significant differences

Table 1
-“Wharf” is misspelled
-Add the 9 additional sites to this table (or at least their mean and SE)
-What exactly is the difference between muddy sand and sandy mud?
-Highlight that PJ is the control site

Table 3: Change the last sentence to : “Species identified in SIMPER analysis”

Table 4: Same as Table 3

Figure 1:
-Add the 9 additional sites to a map of Nova Scotia.
-Remove the “disappeared” sites

Figures 2, 3, and 4:
 Note in the captions that the distance from the fish farm increases from left to right, this makes it much easier to interpret the results. Also, please add the colour legend to the actual graph, it’s much easier to read that way.

Figure 2
2a: Change to “Sediment Organic CONTENT”
2e: Flip the axis to make it consistent with the others graphs (just note it in the caption)

I pointed these out above, but the error bars in Figures 3 and 4 do not match the stats:
3a: Based on the error bars, SI and OW should at least be significantly different?
3b: It looks like CB should be different?
4a: Same here - looks like CB should again be different?
4b: Here it looks like PJ should be different
4c: OW and PJ look different

Figure 5.
There is missing a legend here: Which curve is the abundance and which is the biomass?


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: POTENTIAL IMPACTS OF FINFISH AQUACULTURE ON EELGRASS (ZOSTERA MARINA) BEDS AND POSSIBLE MONITORING METRICS FOR MANAGEMENT: A CASE STUDY IN ATLANTIC CANADA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This MS reports results from a study in which the authors have looked at how finfish farming affects eelgrass beds and associated infauna. They show that the effect of farming is obvious on some of the plant and faunal variables. The language is clear and professional throughout the MS.  The introduction is straight-forward and gives a good background to the study with relevant literature referenced. The structure conforms to the journal’s standards. The figures are for the most part well labelled, though a few things could be improved (see my further comments for the authors). The raw data is also provided and structured coherently.
Experimental design: The research is original for this particular study organism (Zostera marina). The research aims are defined and relevant, though to clarify I would like the authors to give some examples of hypotheses (see my comments). The methods are described with sufficient detail and can be replicated. Some details can be clarified (see my comments).
Validity of the findings: The data is robust and the authors have used proper statistical methods in the MS. The conclusions are for the most part well-stated but I would like the authors to clarify and justify some of their statements based on the results (see comments). I believe that the authors could speculate more instead of stating facts that are not necessarily supported by their results.
Additional comments: I think the MS is generally well-written with concise methodology, good reporting of the results and discussion, and thus adheres well to the standards of Peer J. However, there are some issues that I think should be clarified and improved before acceptance. Please see my 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: POTENTIAL IMPACTS OF FINFISH AQUACULTURE ON EELGRASS (ZOSTERA MARINA) BEDS AND POSSIBLE MONITORING METRICS FOR MANAGEMENT: A CASE STUDY IN ATLANTIC CANADA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have done an excellent job addressing the gaps that I pointed out in the original submission. They have added thorough information about the other sites across Nova Scotia, and the modified title and abstract much better reflect the scope of the paper.
One minor suggestion on this point would be to explicityly state the development of metrics as an objective of the paper in Lines 78-90.
I also really appreciate the added information in Table 1! This really adds puts the results of this study into a wider context.

A few minor corrections to make in the text and figures:
Line 21: change “identify” to past tense “identified” to match the previous section
Line 99: change “4” to “four”
Line 131: change “date” to “data”
Line 506: change “above 192” to “>192”
Figure legends: In Figures 2-5, there is a sentence stating “pairs of the same letter in bold would be further different a p<0.01”. First, I think there is a typo here and it should be “at” instead of “a”? Secondly, I’m not quite sure what this actually means – in Figure 5b, the highest ‘b’ and the lowest ‘b’ are bold, but not the one in the middle. If there are ‘marginally significant’ (p<0.1) values perhaps they should just be mentioned in the legend rather than confusing the figure text.
Figure 6: Maybe add the site abbreviation on the graph panels. This makes it easier to read.
Figure 7: What is the “distance” in panel C referring to?
Table 4: I think this would benefit from being turned into a Figure, especially for the important species that are discussed in lines  490-510.
Experimental design: As mentioned above, the authors did an excellent job adding information about the additional sites and re-analysing the data.

Minor improvements:
Line 115:  Do you have anything to put this 4000 uM number into context? What is considered high or low for this?
Line 147: Add which years the other sites were sampled (these could be also added in Table 2).
Validity of the findings: The authors have softened some of the conclusions, and instead separate “statistically significant” and “ecologically relevant” results. This is an improvement, though I still think it needs to be made clearer which results are more statistically supported versus which show a potential trend and need to be followed up on with larger scale studies of different fish farms (this last point is not a criticism of the paper – I think this manuscript is an excellent first step towards quantifying aquaculture effects on eelgrass!)

In line 355 (and line 23 in the abstract), the authors mention that there is higher algae % cover is an effect of the fish farm. However, when looking at Figure 2c, I completely disagree - only site CB is higher than the Nova Scotia average and the control site. The other two sites in the bay with the fish farm are the same or even lower than the control site. Either remove this completely, or discuss why there might be an algal biomass increase at interediate distance from the fish farm, but not closer or further away. From the graph in Figure 2c, it looks like algal biomass is random and not linked to the fish farm at all.
Additional comments: The authors did excellent work on improving the manuscript. There are a few areas in the discussion that I think could still be improved (in terms of discussing significant and non-significant results), and a few minor points to modify, all of which I have outlined 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: INFLUENCE OF MUTATION BIAS AND HYDROPHOBICITY ON THE SUBSTITUTION RATES AND SEQUENCE ENTROPIES OF PROTEIN EVOLUTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English was clear and easy to read. I only found minor changes to be made:
1. Page 17 line 14: "The result robust with respect" might be "The result *is* robust with respect"
2. Page 17 line 31: "likely caused both both by" has an extra "both" in it.

I found the background to be sufficient and the references clearly cited.

The structure of the manuscript including the figures was professional and I could find the raw data at the github repository. The figures are sufficient, but I found a few small improvements:

1. Figure 1: The Entropy x axis major tick labels overlap between the WT and MF subfigures. It looks like 1 and 3 are being printed on top of each other. I believe the correct fix is to make the MF x-axis range (currently 1 to 3) the same as the MSA and WT, which appears to be 0.5 to 3.

2. Figure 1: I noticed a light blue line between the graph legend and the figure caption. It is there on 3 of my PDF viewers, but it is not in rate_entr_v3.eps, so I am not sure if it is a rendering problem or perhaps something in the LaTeX file.

3. Figures 3-5: The font size used in these figures is much smaller than the font in previous figures and the rest of the paper. I suggest increasing it for readability.

The article seems like a reasonable unit of publication and is completely self-contained.
Experimental design: The article fits in the Aims and Scope of PeerJ.

The research question is well defined, however I am not convinced the knowledge gap it fills. The primary question addressed is "whether the sequence entropy and the substitution rate are equivalent measures of the evolutionary variability of a position". The authors then show that they are not equivalent. Was there confusion in the field of their equivalence? Did a previous group publish evidence that they may be identical? Perhaps the focus could be shifted slightly to say something new about sequence entropy, substitution rates, hydrophobicity, and mutation bias.

The research seems to be done with a high technical standard using up to date models and software.

The methods are clearly and completely described. I appreciate how all of the terms and models are thoroughly defined.
Validity of the findings: The data and sources seem robust and well documented.

The conclusions followed clearly and directly from the results, in order to answer the question at hand. I found very little speculation in the conclusions.
Additional comments: The article is well structured and clearly laid out: the data are solid, the question is clear, the figures support the results which answer the question. As I stated in section 2, my main concern is the relevance of the question. Three of your five figures focus on hydrophobicity's influence on the sequence entropy and the substitution rate. A few of your conclusions describe the effects of mutational bias. Perhaps restructuring your question to include hydrophobicity and mutational bias would more clearly describe the knowledge gap that your article fills.


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: INFLUENCE OF MUTATION BIAS AND HYDROPHOBICITY ON THE SUBSTITUTION RATES AND SEQUENCE ENTROPIES OF PROTEIN EVOLUTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Developing a mechanistic model of protein evolution is crucial in creating a null model for the observed patterns of genetic variation among extant sequences. In particular, the evolutionary variability of an amino acid site in protein families is an important indicator of the selective constraints experienced by the site in the course of evolution. However, the observed substitution variability is a function both of mutational input (or bias if it exists) and selection. Decoupling the relative contribution of these two is an important and challenging problem in protein evolution.

In this work, Jiménez-Santos et al. study the site-specific substitution rates of proteins under selection for protein stability and misfolding. They demonstrated that substitution rates vary depending on the exchangeability of mutations, which in turn depends on the hydrophobicity of residues.  They found that, on average, polar sites evolve faster unless the sequence is evolved under mutational bias that prefers hydrophobic residues. Importantly, they link the mutational bias to the biophysical properties and per-site evolutionary rates. Overall, I think the paper has merits and provides a relevant connection between biophysical properties of proteins and the observed rates of molecular evolution. However, I have several technical and conceptual points that need to be addressed prior to publication.
Experimental design: See comments below.
Validity of the findings: See comments below.
Additional comments: Major comments:
1.	The authors claim, “more hydrophobic proteins substitute more slowly, but mutation bias towards hydrophobicity increases the substitution rates".  But an alternative explanation could simply be selection for PPI. Proteins that have more interaction partners have higher hydrophobic surface patches, thus are enriched in their total number of hydrophobic residues. Moreover, the interaction sites are also under stronger selective constraints and will evolve slowly. Thus, the claimed trend might be due to PPI and not due to mutational bias. How many of the 230+ proteins analyzed are involved in PPI and what are their centrality in the PPI networks? See for example the following works: Qian, et al PNAS 108(21):8725-30; Heo et al. PNAS  108 (10) 4258-4263; Zhang Nature Reviews Genetics 16, 409-429 (2015).

2.	In the introduction, the authors make the distinction between two kinds of models/approaches that rationalize why sites that form many contacts are subject to stronger selective constraints. The first kind is the “stability-constrained fitness models” that assume a fitness function f=1/(1+expDG/kT) and that estimates the change in free energy upon mutation “assuming that the native structure does not change”. The second model is the “structurally constrained model”, which in contrast to the first, include the effects of how random mutations might perturb the structure of the native state using the GNM.

However, I think this is a false dichotomy. Several studies that model the fitness landscape as f~1/(1+exp(DG/kT))  also account for the structural changes in the native structure because they use a physical force field and the protein 3D structure to estimate the mutational effects on folding stability DDG (e.g.  Jackson & Wilke PeerJ  2013 Nov 12;1:e211; Serohijos & Shakhnovich Mol Biol Evol. 2014 31(1):165-76). Moreover, in other approaches where the fitness changes are derived from experimental data (Bloom Mol Biol Evol. 2014 Aug;31(8):1956-78; Mol Biol Evol. 2014 Oct;31(10):2753-69) the fitness effects already account for selection for folding stability and effects of mutations on structure.

3.	For Equation 10, its derivative with respect to the change in fitness is zero when Fa=Fb as pointed out by the authors. This implies that not only selected mutations but also neutral mutations would contribute to a higher substitution rate. This seems in contrast with the conclusion that the empirical exchangeability produces the highest substitution rate because it contains selection.  I think a clarification of how neutral mutations might clarify this point to the readers.

4.	Since the authors adopt a solid mathematical approach in this paper, I strongly advise that they also work out the analytical relation between sequence entropy and substitution rates. They can take the logarithm of Eq. 11 and re-factor sequence entropy. The evolutionary rate should be exponentially dependent on sequence entropy, which seems to be the behavior depicted in Figure 1.

5.	One interesting observation in Figure 1 is that the difference between different exchangeabilities diminishes at higher entropies (i.e., all curves coincide). This suggests that at low sequence entropies either (i) the natural sequences tend to have more neutral substitutions or (ii) more mutations are under selection. Perhaps the authors can elaborate on this point.

6.	I might be wrong but I think the main determinant of the shape of site-specific sequence entropy versus hydrophobicity is the equilibrium probability of amino acids rather the hydrophobicity. At higher/lower per-site hydrophobicity we have more/ fewer mutations fixed. Therefore, the equilibrium distribution is shifted towards the extreme (i.e., one mutation with P=1 and hence entropy=0). I would like to see the same plot with varying charge or hydrophilicity.

7.	With regards to the previous point, don’t the authors maximize the likelihood of the WT sequence as explained in Eq. 5, which already contains the background distribution Pa? If so, it should not be surprising that the maximum substitution rate, when the background is more hydrophobic, occur in sites that are more hydrophobic?
8.	There are several instances where claims are made, but no data is referred:
	*“The WT models show even better performance on several data sets (Arenas & Bastolla, In preparation).” (Page 3, line 32)
	* “Thus, in theory, the WT model is more suited for short evolutionary divergences and MF model is more suited for long evolutionary divergences (Arenas & Bastolla, In preparation).” (Page 4, line 2)
In my opinion, these are very important and relevant claims, which require that either the data be included as supplementary figures or as an accompanying manuscript.
11) It is an important observation in their work that substitution rate Ri and sequence entropy do not correlate, which is in contrast to the more intuitive claim by (Halpern and Bruno 1998). Can the authors provide an explanation on why might this be the case?
12) Interestingly, the data in Fig 2 is only for one protein (PDB code 1PYL). Although, I do not challenge the claim that this is representative of “all of our data set.” I think the figure could be made stronger by showing for all 200+ proteins.

Minor points:
1)	The manuscript can benefit if authors add a schema of the methods/models as Figure 1.
2)	Also, the discussion is solely focused on the immediate consequences of the results (e.g., the effect of mutational bias on the substitution rate). As a matter of fact, sequence entropy is frequently used to detect selection in the evolution of influenza proteins such as haemagglutinin. I suggest authors comment on the applicability of their methods in a broader context.
3)	In Figure 2, there is an overlap of white circles (<6 contacts) and black circles (6>= contacts) suggesting that some sites, despite having the same sequence entropy and less than 6 contacts evolve with a lower rate. Are these active site residues? It would be interesting to comment on these sites.
4)	I could not access the GitHub page.


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: INFLUENCE OF MUTATION BIAS AND HYDROPHOBICITY ON THE SUBSTITUTION RATES AND SEQUENCE ENTROPIES OF PROTEIN EVOLUTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: See below.
Experimental design: See below.
Validity of the findings: See below.
Additional comments: I think Bastolla and co-workers have already addressed the points I raised.


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

Input:
Title: A NEW LIMESTONE-DWELLING SPECIES OF MICRYLETTA (AMPHIBIA: ANURA: MICROHYLIDAE) FROM NORTHERN VIETNAM
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article is very well structured, and most of it is well written in good English; only in the introduction did I identify a number of grammatical errors, which I have taken the liberty of rectifying. The manuscript is well referenced and covers the breadth of literature on the topic of these frogs. The figures are nice, though a few adjustments are necessary, and the tables are also adequate, though at least one caption needs to be slightly adjusted. The authors should note that PeerJ does not expect references to be italicised, so they should check that their citation format fits that of the journal.
Experimental design: An exemplary taxonomic study, with apparent rigour and care taken. The description of poorly known taxa from threatened areas in southeast Asia (and indeed globally) is important. The authors should note some comments I have made on their methodology regarding measurements and repeatability for future studies, but these do not require action for the present study.
Validity of the findings: Findings appear robust, and the authors have clearly endeavoured to provide an accurate description of the new species and provide an exceptionally detailed diagnosis against other taxa that confirm its distinctiveness. The conclusions do a great job of framing the study in a conservation context. The authors should consider my comments on the conservation status of the new species, but need not change it if they prefer to leave the species as Data Deficient until further surveys can be conducted.
Additional comments: This paper is very well written and will require only minor revisions before it is acceptable for publication. I have made adjustments to the .docx file of the manuscript, and additional comments can be found there. I would like to see slightly more accurate colour terminology, and a few figures need to be slightly adjusted. Other changes and comments in the text are even more minor. In summary, I find this paper to be of high quality, and after these small revisions, I look forward to seeing it published in PeerJ.

Best regards,
Mark D. Scherz