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: MELATONIN IMPROVES THE EFFICIENCY OF SUPER-OVULATION AND TIMED ARTIFICIAL INSEMINATION IN SHEEP
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: It is a good paper to state that the effect of melatonin treatment  on the efficiency of super-ovulation and timed artificial insemination in sheep. The positive results shed a light on improvement of quality of embryos and fertility of sheep by melatonin administration.


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: MELATONIN IMPROVES THE EFFICIENCY OF SUPER-OVULATION AND TIMED ARTIFICIAL INSEMINATION IN SHEEP
Review round: 2
Reviewer: 2

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


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

Input:
Title: DIATOM IDENTIFICATION INCLUDING LIFE CYCLE STAGES THROUGH MORPHOLOGICAL AND TEXTURE DESCRIPTORS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is written in a clear language, it is easy to read and to follow. I am a biologist working with diatoms and I had to skip the maths in the Methods section as I am not qualified to judge those parts. I am glad to see that the image sets used are publicly available. The same is not true about analysis code for segmentation and feature extraction or any downstream analysis – as a diatomist, I can only recommend to the authors to consider the possibility of publishing their analysis code (especially relating to segmentation and feature extraction) since the features extracted can be highly interesting for many diatomists, but hardly any of them will be able to apply them only having the mathematical descriptions.

I have one main problem with clarity of the manuscript: I am finding that the first sentence of the conclusions (l. 324-326), or the line of argumentation in the discussion quite clearly reflect the contents of the manuscript, but the Introduction and the Abstract (and even the title) do not do so quite as clearly. The way I see it is that the main novelty of this paper is a) the set of feature descriptors that it uses, and b) that it uses (at least one of the three datasets analyzed?) sets of images explicitly capturing the range of life cycle / size related morphological variability in the target taxa. As the Discussion and the Conclusions clearly state, the features extracted are used in algorithmic classification experiments. To the contrary, the title, abstract and introduction focus on life cycle modelling, a related, but different aspect. The manuscript does not provide any explicit statements about life cycle models inferred by any of the models, only about classification accuracy and discriminating power of features. One can of course argue that a classification algorithm trained on a set of images depicting life cycle related variation must implicitly model the life cycle, but to me it would make the situation clearer to call these analyses classification (in the statistical meaning) or identification (in the biological meaning) experiments. For this reason, I would suggest the authors to consider re-formulating the title, introduction and abstract, de-emphasizing the aspect of life cycle modeling and making it clear up front that what is being done is image classification.

I am finding the third paragraph of the introduction (l. 43-65) somewhat convoluted. The text rightly points out difficulties with modelling diatom life cycles (non-linearity and multidimensionality), but then the manuscript does not in any explicit manner address these problems. The text then gives a short literature review of many things related to what one could call the large topic of digitally capturing/extracting morphological information from diatom images, but in a to me, not very logical ordering. The authors could consider separating the issues of feature extraction for diatom morphometric characterization from capturing life cycle related variation and explicitly modelling the latter.

I am missing citations pointing to precise description of applied methods (for instance, the whole section on classification methodology from line 173 to 220 only includes two references, both relating to support vector machines, but none to any of the other applied techniques).

The statement that currently more than 100’000 diatom species are described (l. 24) appears rather high to me, please cite a reference on which this number is based. Also the second paragraph of the Introduction (l. 31-42) comes out without a single citation – probably no problem for a diatomist but it could be useful to cite some sources, to other potential readers not so familiar with the organisms.

It did not become quite clear to me what the differences between the analyses applied to data set 1 vs data set 2 and 3 were. Reading the Results and the Discussion, it seems that a) only data set 1 is explicitly meant to capture life cycle related variation – is this correct? If yes, it would be useful to explicitly state this, perhaps in the Methods section; b) in experiments 2 and 3, the final (supervised and unsupervised classification) steps of the procedure shown in Figure 1 were not applied – is this correct? If yes it would be good to state this explicitly in the Methods.
Experimental design: Experimental design is sound and I do not have any major criticisms related to it, only some remarks for consideration.

One methodological novelty of the paper is the use of LDA for dimensionality reduction (instead of the commonly used PCA). If this is really a novelty (as it seems since no previous examples are cited), it would be interesting to see a comparison of how this difference affects classification performance. One further remark related to this aspect is that the paper does not specify how many dimensions were kept after the LDA for downstream analyses.

The manuscript applies a workflow to three low diversity diatom image data sets in three separate experiments (8, 6 and 5 taxa, respectively). The discussion then states (l. 295-299) that the size of the data sets is rather small and testing the methods on more “realistic sized” data sets remains to future work. It appears to me that it would have been a simple extension of the study to pool the three image sets for a fourth experiment. This is still far from the diversity one sees in most natural habitats, but would nevertheless go one step further into that direction. I would encourage the authors to consider adding such an analysis if feasible.
Validity of the findings: To me the main (potential) findings of the paper are that

a) the proposed features do a good job in capturing morphological information separating different diatom species, this is convincingly supported;

b) LDA is preferable (vs. PCA) as a dimensionality reduction technique for multivariate classification – this point is not addressed in a systematic manner; if that is an intention of the authors, it would be informative to compare at least two analyses which only differ in this aspect, such a comparison is currently not found in the paper;

c) since the aspect of life cycle is given a lot of sentences, especially in the first half of the paper, one would expect to also find a conclusion that relates to this topic, but I do not really see such a finding. One might say that it is important to capture life cycle related morphological variability in the training set in automatic identification attempts, but I currently do not see anything in this paper that would make this point clearer or better supported than it already was. Perhaps this is only a question of clearer formulation in the discussion or a more explicit comparison between data set 1 vs. 2 & 3 (the former apprently capturing life cycle more completely than the latter two)?
Additional comments: The final paragraph of the introduction could be removed since it only outlines the standard manuscript structure common to the majority of PeerJ articles.

The use of parentheses in citations is unusual and inconsistent, for instance double parentheses on l. 28 or in the legend of Fig. 8; parentheses between author and year instead of before author name on l. 45, 54, 55, 59-63 (although this variant can of course be used if the author name is part of the sentence like on l. 50 or 57).

Specific epithets are capitalized inFig. 9, 10 and 12 but not in 13 – I would recommend to not capitalize them.

Figure 8 in its present form is completely incomprehensible to me; although a reference is cited, it would help the reader if the idea behind this plot was shortly explained.

I would interpret Figure 11 to say that the difference between the three clustering methods is marginal but this might be caused by the scaling of the y-axis – would it make sense to separate out Silhoutte (which lies in a different range from the other metrics) and plot all metrics with narrower y-axis limits?

Related to the same comparison, how was the final number of clusters chosen in the clustering exercises? If it is simply chosen as the known number of classes in each data set, then this cannot be called a completely unsupervised classification approach since the number of clusters present in an unlabeled data set is in general not known a priori.

The sentence on l. 200-201 seems incomplete or is otherwise incomprehensible to me.

Fig 12 compares newly generated with previously published data from identical sets of images. We see strongly improved separation of a priori known classes, but there are multiple differences between the old and new analyses which makes it difficult to pin down whether the improved results are caused by the novel set of features used vs. the different dimensionality reduction technique (PCA vs. LDA). That the latter improves separation is not surprising; it would be more interesting to see the effect of the novel features alone, like in Fig. 13, in which case it could make more sense to present LDA results on both the old and the new feature set side by side.


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: DIATOM IDENTIFICATION INCLUDING LIFE CYCLE STAGES THROUGH MORPHOLOGICAL AND TEXTURE DESCRIPTORS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Basic reporting

The paper is easy to read, but a major text revision is required for correction of mistakes. I have found word-choice, misspelling and punctuation errors. In addition, some phrases should be rewritten. Several examples were the language should be improved include lines:
41,150 Contractions are inappropriate for scientific papers.
45-46: Blanco et al. (2017) --> (Blanco et al., 2017). There are some more cases like this.
48-50: phrase is very long and can be made more understandable by adding some comma or rewritten it.
91: pues Images: text written in Spanish and the first letter of “images” written in capital letter.
103: dilatation --> dilation
110: Phase Congruency descriptors and Gabor descriptors --> Phase congruency and Gabor descriptors.
129: the amplitude of them --> their amplitude
132: combine all the information of phase congruency --> combine the phase congruency information:
135-138 where θ indicates the orientation, … and φ are the… --> where θ indicates the orientation, … and φ the… The use of is, are can be avoided here.
170: On the other hand, but you must also use “on the one hand” to describe different ideas. Do the same with similar expressions
236: On the other side --> on the other hand, but “on the one hand “is also missing.

The first letter of the name of a figure or table must be written in capital letter, for example:
81 "As Fig. 1 shows"
93 “Images from Table 1”
Experimental design: The experiment design seems OK; however, I do not think the Otsu method provides a good segmentation in every image. I would like to know if I am wrong or what was done when images were wrongly segmented.
Line 177: “training was carried out by selecting a small subset”  --> Please, specify how many images or which percentage was used for the training.
Validity of the findings: The results seem good. however, the Otsu algorithm does not always produce a good segmentation of diatom images, which is important to ensure adequate contours. Likewise, phase congruence does not always provide adequate results if images are noisy or diatom are not clearly defined. Therefore, it would be appropriate to know if these cases were presented and how they were avoided or resolved.

264-265: The percentages of the variance along the axes are higher in LDA than in the case of PCA --> This is only true on the abscissa axis.
Additional comments: I feel that the authors should revise their manuscript, even though the results seem to be good. I therefore suggest to rewrite some parts of the paper, improving the quality of the text


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: DIATOM IDENTIFICATION INCLUDING LIFE CYCLE STAGES THROUGH MORPHOLOGICAL AND TEXTURE DESCRIPTORS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper proposes a method for classification of diatoms an its life cycle modeling. Features based on elliptic Fourier descriptors, phase congruency and Gabor filters are used in two supervised and three unsupervised classification methods. Three unsupervised clustering methods are compared with five validation metrics. Three datasets are considered.
Clear English language is used throughout the paper. Introduction describes the main objectives and related work is sufficiently covered. However, some equations have unclear notation or variables are undefined. See my general comments below.
Experimental design: Methods and experiments are described with sufficient detail, however the relevance to diatom life cycle modeling is not well stated. See my general comments below.
Validity of the findings: Experimental results demonstrate that on the given dataset the chosen features and classification methods achieve high accuracy levels and outperform methods based on morphometric measurements.  Unfortunately, comparison with a deep learning technique was not carried out due to the different nature of the datasets. See my general comments below.
Additional comments: 1) The algorithm for mask extraction is described in lines 101-106.  It seems to me that it is designed to work only if one single diatom is present in the image. If the system is presented as an automatic method of diatom identification (line 284) then I think it is necessary that it can also work with images that do not contain any diatoms and also that contain more than one.

2) It is not clear how many features were used in experiments after dimensionality reduction.  In line 241 a number seven is mentioned, yet it is not clear if this is the dimension of the feature space. I suspect that the number of selected features is important and therefore the authors should include accuracy results w.r.t. to the number of features or at least some discussion on this topic.

3) In Experiment 2, the same features are used and the only difference is whether LDA or PCA is used for dimensionality reduction? If this is true, it should be clearly stated.

4) The experiment 1 considers diatoms from a different genera and both experiments 2 and 3 consider diatoms from the same genera (lines 255-257). However the achieved accuracy is higher in the exp. 2 and 3 than in the exp. 1, which is not intuitive.  It is indicted later at lines 291-294 that the dataset in exp. 1 includes diatom life cycles, which make the diatom classes more variable and therefore decreases classification accuracy. I suggest to clearly state in the section “Experiment 1” the presence of diatom life cycles. Life cycles are mentioned in the title, but experiments do not discuss this phenomenon. It would then be clear why the exp.1 accuracy is lower then in exp.2 and 3. I also suggest to improve in this manner the section “Discussion”.

5) It is unfortunate that the comparison on the dataset (80 classes) used in Pedraza et al. (2017)  could not be done. It would further strengthen this paper.

6) Notation in some equations is not clear.
PC is defined in eq. (9) and then used in eqs. (13)-(15). Theta (orientation) is used as a parameter but in (9) theta is integrated out. Is PC in (9) a different PC in (13)-(15)?
In the definition of Gabor features in eq. (16), the function B(u,v) is not defined.

Minor:
- line 91: “pues” not an English word.


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: DIATOM IDENTIFICATION INCLUDING LIFE CYCLE STAGES THROUGH MORPHOLOGICAL AND TEXTURE DESCRIPTORS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Substantially, the authors added pointers to publicly available code relating to their analysis, I welcome that.
Also citations in the Methods section were added.
I commented previously on the statement that “currently more than 100’000” diatom species are described. The authors now added two sentences detailing different estimates for possible number of diatom species; I do not see these as necessary, my point was simply to say that the number of currently described diatom species (which I think should be in the 10thousands) was confused with the estimated total number (to which the figures given by the authors refer) in the original version. This has now been fixed.
The main line of argumentation in the Introduction has improved; only one comment: a sentence was now added saying “the main source of errors come 59 from the misclassification of algae through their life cycle” – if this is so, that would be a quite substantial insight for the field, but what is this statement based on? No citation is given.
Experimental design: Here I previously asked the authors to clarify how novel their use of LDA instead of for dimensionality reduction is in a supervised classification framework. This was not addressed, neither the quantification of how/to what extent this difference affects classification performance. What is given instead is a graphical comparison between a PCA and an LDA ordination which of course shows a better visual separation in the second case (but which is not a great novelty or surprise).  I would not like to push this issue further, only remark that if the authors see this as a substantial methodological innovation, it would be good to put it into a more solid (quantitative and literature) context.
My other comment (reflecting on the discussion point related to the lack of availability of higher diversity data sets) was also not addressed on l.107-120, as the authors state, but that is perfectly fine, it was only a suggestion.
Validity of the findings: My previous comments under this point have more or less been addressed
Additional comments: The minor points I raised here previously were all addressed


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

Input:
Title: DIATOM IDENTIFICATION INCLUDING LIFE CYCLE STAGES THROUGH MORPHOLOGICAL AND TEXTURE DESCRIPTORS
Review round: 2
Reviewer: 2

Output:
Basic reporting: There is a big improvement with respect on the previous version of the paper. There are only few small typographical or grammatical errors to be corrected.Some examples are:

Line 46: where it not be reduced ->where it cannot be reduced.

Line 155 gradient operator will be sensitive -> gradient operator is sensitive
Line 156: edge Canny edge -> Canny edge
Line 158 PC was not applied for contour improvement but it was used for -> PC was not applied for contour improvement but for

Line 169 where PC is the phase congruency -> and Pc is the phase congruency.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: I think the paper is good for publication. It is very well written and easy to understand. However, there are still some grammatical mistakes to be corrected.


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: DIATOM IDENTIFICATION INCLUDING LIFE CYCLE STAGES THROUGH MORPHOLOGICAL AND TEXTURE DESCRIPTORS
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have improved the manuscript and I am satisfied with their responses to my 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: EFFECTS OF COMPLEX TRAINING VERSUS HEAVY RESISTANCE TRAINING ON NEUROMUSCULAR ADAPTATION, RUNNING ECONOMY AND 5-KM PERFORMANCE IN WELL-TRAINED DISTANCE RUNNERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: I believe that with some clarifications throughout the manuscript, that this work should be published.

However, language should be improved throughout the manuscript with particular attention to: subject-verb agreement, tense, and missing prepositions or articles. Examples include: lns 71, 75-76, 81-82, 148, 185, 205, 207-208, 238, 262, 281, 285, 301-302, 319, 320 (spelling), 323, 327 (don’t need “especially” there), 329, 335, 341, 355, 368 (word choice), 372, 376, 387, 397, 428 (word choice, “transfer”). This list of examples is not exhaustive.

Also, the authors inconsistently space references within the text. There should be a space between the last word and the citation.

Other specific notes:

Abstract: Overall, I think it would be beneficial to shorten the background portion and add some more detail to the methods and results. For example, it is not clear what your control group actually is from how it is currently written, and the reader is left wondering if the control group takes part in any training at all. Also, more detail regarding how you measured performance would be helpful. At the moment “performance” is a vague description. Please add detail on the specific performance tests – even if just the test names. For the results, do you mean there are difference relative to the control group? Or are there differences between the CXT and HRT groups? You need to clarify. It would also be helpful to clarify what kind of improvements (direction & magnitude) were made.

Key Words: I think it would be helpful to include a key word/phrase relevant to training programs

Introduction:
1.	I think the first two paragraphs should be combined and shortened so you can get to your main point faster.
2.	Line 87: please define RM (1 rep max) for the reader
3.	Lines 92-93: How so? You need to make an argument how explosive power is important for endurance running. You make this point in the discussion (lns 433-435) – but this argument needs to be made in the introduction to tell the reader why explosive power, as developed through HRT and plyometrics, is actually important for endurance athletes.
4.	It would be helpful for the authors to lay out the neuromuscular adaptations of interest earlier on in the introduction and why they are the ones of interest.
Experimental design: Methods - specific notes
1.	Who were these participants? Part of a university team? National team? Where were they located?
2.	Part of “Procedures – Preparatory Strength Training” – it would be helpful to know what exercises were employed – even just by referring to one of your already constructed tables.
3.	The authors need to make the argument for which tests they are using and why they are using those tests much earlier on in the methods and procedures. This is done to some degree in the discussion (lns 348-353), but the rationale for the different strength and running tests needs to be provided early in the methods.
4.	This is a style point, but it would be helpful visually in table 3 to bold or underline the different training group names in the first column – it is tough to pick out.
Validity of the findings: Results
1.	Throughout the results, instead of just saying significant interaction or significant difference, it would be helpful to provide the direction and magnitude of those differences to give the reader a better idea of what is happening
2.	Ln 302-305 – what about the control group?
3.	The results overall could be written more clearly to truly lay out the differences between the group. It is not always readily apparent if there are differences between CXT and HRT groups relative to the control. A short summary paragraph of where there were differences (as I state in #1 above) and the magnitude of those differences would be helpful.

Discussion:
1.	I would recommend not using the word “increment”. This word implies a regular (consistent) change in value. Using “increased” or “improvement” would be a better word choice.
2.	Lns 385-386 – instead of saying different – say how they are different
3.	Lns 401-403 – wouldn’t this be a sign of greater adaptation in Type I fibers? Endurance and efficiency improved so that type II fibers are not used? And the argument has not necessarily been made of better lactate removal – it could be that Type I fibers are more active and efficient.
4.	Ln 424 – clarity – is this for all groups?
Additional comments: The authors are to be commended on the experimental design and extensive testing the performed.


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

Input:
Title: EFFECTS OF COMPLEX TRAINING VERSUS HEAVY RESISTANCE TRAINING ON NEUROMUSCULAR ADAPTATION, RUNNING ECONOMY AND 5-KM PERFORMANCE IN WELL-TRAINED DISTANCE RUNNERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are a few spots in the paper in which it is not clear what the authors hope to convey (please see my annotated pdf where I note such spots and where I suggest grammatical and typographical edits). In terms of basic reporting, the raw data are supplied, the paper has a traditional structure, the hypotheses are clearly laid out, and the results of analyses are shown in tables. However, there are no figures showing the data. Given the many tests that were performed, I suggest that the authors should make a few figures that graphically demonstrate what they consider to be their most important results.
Experimental design: The general basis for the study is sound, and the experimental design is clearly explained.
Validity of the findings: There are a few areas in the Discussion section of the manuscript (please see my annotated pdf for specifics) where explanations for the study’s findings are hypothetical/speculative, but are not clearly stated as such. Please revise to specifically state which interpretations are speculative and which are supported by the data in this paper. Also, please revise the discussion to note that this study only examines young male endurance athletes and is therefore not necessarily broadly applicable to all athletes (such as older individuals, strength athletes, and women).
Additional comments: This is a relatively straightforward paper examining the effects of heavy resistance training and complex training on various running performance parameters in young male distance runners. My specific comments are in the attached annotated pdf. My major general comments are: 1) figures of the data are required, 2) speculative interpretations of results in the discussion section should be revised, 3) the discussion should include information on how these results could (should) be applied to female athletes, as well as what the implications are for future work, and 4) more clarity is needed in placing the study’s results within the broader context of the existing literature.


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

Input:
Title: EFFECTS OF COMPLEX TRAINING VERSUS HEAVY RESISTANCE TRAINING ON NEUROMUSCULAR ADAPTATION, RUNNING ECONOMY AND 5-KM PERFORMANCE IN WELL-TRAINED DISTANCE RUNNERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The language is much improved! I commend the authors for taking all the reviewers' comments to heart and making substantial changes to this manuscript. The only thing I would suggest is that they shorten up the methods and results part of the abstract. I know I told them to expand, but I think they expanded a bit too much!
Experimental design: The new detail and clarifications to the methods are excellent
Validity of the findings: Love the addition of the figures and clarifications in the results including magnitude and direction of individual findings.
Additional comments: The improvements are great, excellent 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: EFFECTS OF COMPLEX TRAINING VERSUS HEAVY RESISTANCE TRAINING ON NEUROMUSCULAR ADAPTATION, RUNNING ECONOMY AND 5-KM PERFORMANCE IN WELL-TRAINED DISTANCE RUNNERS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Previously I raised the concern that the paper did not include figures demonstrating the major results. The authors now include Figures 2-5 which clearly show the results among training groups. In addition, they have edited the format of the in-text references and provided clarity in areas that were unclear.
Experimental design: The general basis for the study is sound, and the experimental design is clearly explained.
Validity of the findings: The authors have addressed my concerns regarding marking speculative comments as speculation and noting the caveat that the study’s results are not broadly applicable to all athletes such as older individuals and women.
Additional comments: The authors have satisfactorily addressed all of my previous comments. The new data figures are very useful for the reader to visualize the significant results. My only question regards the use of CT (complex training) versus CPX (complex training group) throughout the manuscript. It may make the manuscript easier to read if only one abbreviation is used to refer to complex training throughout the manuscript, regardless of whether the text is referring to the method of training or the training group.


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

Input:
Title: LONG-READ VIRAL METAGENOMICS CAPTURES ABUNDANT AND MICRODIVERSE VIRAL POPULATIONS AND THEIR NICHE-DEFINING GENOMIC ISLANDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript by Warwick-Dugdale et al describes a beautifully constructed study to evaluate an approach that will be transformative in viromics. The manuscript is extremely well-written and the conscientious attention to detail in this study is readily apparent.

Minor comments:
Line 114: should probably read “viral communities in seawater”

Line 476: Is the “and” supposed to be “or”?

Figure 7: Can the authors insert units for “Total abundance” and “Estimated abundance”? (in the figure or in the caption)


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: LONG-READ VIRAL METAGENOMICS CAPTURES ABUNDANT AND MICRODIVERSE VIRAL POPULATIONS AND THEIR NICHE-DEFINING GENOMIC ISLANDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment. The article is very well put together, well written, and addresses all the goals that the study sought out to do.
Experimental design: The methodology produces novelty in the field of viromics and adds a workflow that will be useful for environmental virologists to further elucidate viral populations and communities. It would be worth mentioning something about ssDNA viruses and the ability to assess these members of the communities with these types of sequencing technologies (e.g. will the VirION workflow be able to identify these members in future work?). From my understanding this work only targets dsDNA members of the community. The research question was well-defined and addressed with the experiments and bioinformatic workflows. The methods described were extremely detailed and there is enough information to replicate the study. I would suggest thinking of ways to implement this pipeline as user-friendly for biologists, either through a GUI or some type of package. This is not necessarily required for this publication, just a suggestion to build this methodology so that other virologists using these sequencing platforms together in tandem would utilize this novel pipeline.
Validity of the findings: The novelty and results are very well demonstrated but some of the biological and ecological implications are touched on very lightly. Future directions for understanding how the VirION data can inform ecologists might be worth mentioning in the conclusions section in a little more detail (see lines 654-658). It also may be worth mentioning if this pipeline can be utilized with the sequencing of other environmental viruses that have different types of genomes, for example, RNA viruses and ssDNA viruses (i.e. this adds more breadth to the methodology for capturing the whole community).
Additional comments: I recommend to accept this article with minor revisions. My suggestion for minor revisions would be to address a few of the comments I have listed above in the "experimental design" and "validity of the findings" section. These are mostly just discussion points that could be addressed in the conclusion section.


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: LONG-READ VIRAL METAGENOMICS CAPTURES ABUNDANT AND MICRODIVERSE VIRAL POPULATIONS AND THEIR NICHE-DEFINING GENOMIC ISLANDS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This study has developed a technique to use both long and short read sequencing to overcome struggles in viral bioinformatics relating to difficulties in assembling viral genomes from metagenomes. I think it is timely and I appreciate the strategy of modifying or improving upstream aspects of sequencing and informatics as opposed to solely relying on assembly-based improvements.
Overall—great idea, addresses a known problem in viral ecology. Results seem promising in that it was successful in both mock communities and in the natural environment. My main criticism of this manuscript is that it is too long, and it is redundant in some places. I would suggest that some of the points in the Materials and Methods and Results are streamlined and that the repetition between sections is removed. Otherwise, I really appreciate this study, the novelty of the approach and the thoroughness with which the study was performed.
Experimental design: My only critique is that in some instances, the justification for using particular parameters is not well explained (eg. line 285-292). It is likely there are valid reasons for selecting particular parameters, however for broader audiences, it would be helpful for the justification, or citation to be listed.
Validity of the findings: Line 401 The indel rate is still rather high for the long read assemblies, which may bring into question the validity of this strategy for use with environmental samples. However, I still think this study is warranted and valid. The field cannot move forward with out these types of honest assessments of the current state of technology.
Additional comments: Line 95-97 Additionally, culture based approaches could improve the knowledge base.

Line 104-107 This sentence is confusing as worded.

Line 285-292 Why these particular parameters? There is no citation, and the number of reads, as well as the quality score selected are not justified.

Lines 354-360 This block of text repeats the methods.

Line 478, artifact not “artefact"


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: BELIEFS ARE MULTIDIMENSIONAL AND VARY IN STABILITY OVER TIME - PSYCHOMETRIC PROPERTIES OF THE BELIEFS AND VALUES INVENTORY (BVI)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is clear and relevant, but some minors corrections should be made:

1. There is no reference to Figure 1 in the text.
2. In line 224 where it says  .05 it should say  0.5.
3. In line 225 where it says .09 it should say 0.9.
4. In line 366 and 367 the authors mention that, in Figure 2, there is a 5. histogram comparing the test and retest samples. However, Figure 2 does not show this. Maybe the authors wanted to refer to Figure 4. The authors should add this figure or change their reference.
5. In line 481, it says 736.  It I think that it should say 381.
6. In line 574, it says redaction it should say reduction.
7. Figure 1 says that the original sample size is 1093, but in the text, it says that it is 1035. After looking at the data in the repository, my conclusion is that the correct size of the sample is 1035 and that de the figure 1093 is a typo. Please correct this.

Additionally, I suggest some changes to improve the quality of the work:

1. Figures 2 and 3. I suggest rotating the axis 90º. With the rotated axes, there will be space in the figure for using the full name of the variables instead of their codes, improving readability. If the authors go in this direction, they could delete lines 162 to 174 from the Methods’ section.
2. The raw data is very accessible and useful. However, I would recommend, if possible, upload the scripts in raw text format and not as a pdf file which is cumbersome to use.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: BELIEFS ARE MULTIDIMENSIONAL AND VARY IN STABILITY OVER TIME - PSYCHOMETRIC PROPERTIES OF THE BELIEFS AND VALUES INVENTORY (BVI)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Thank you for an interestíng, clearly written article that was supported by professional figures. It is interesting to think the whole approach from a clinical perspective (whole introduction, especially 71-75), extending it to the whole spectrum of beliefs and values.

However, I was missing references to the psychological debate about beliefs and values, especially when it comes to fringe beliefs. You, for example, mention conspiracist beliefs (63), but do not refer to theoretical and empirical works that relate conspiracist ideations to beliefs, political and otherwise (starting with Goertzl, 1994, with Swami and Uscinski as more recent contributors). I would encourage you to go into a bit more detail here.

I would also suggest to extend your discussion of Haidt, as the nature of the beliefs and values you aim to measure is crucial to assess the validity of your findings. (see 3. Validity).

You might consider to re-visit your abstract. Right now, about one third (beginning until '... in delusions') raises expectations that are not covered in your manuscript, e.g., the mechanisms of belief.
Experimental design: no comments here, procedure and descritpiton are sound and thorough
Validity of the findings: There is one aspect in your findings that has strong implications for the validity, and that is not discussed in sufficient detail yet. You refer to Haidt as a source for your morality items, and according to Haidt, judgements stemming from the proposed moral foundations should be quite stable over time. In some publications, those moral foundations are even seen as the lasting source of more variable political beliefs.

This is at odds with your findings, where the moral theme turned out to be the least stable over the course of several months. The explanation given (570-572) might or might not hold; but it should be connected to the theoretical background (Haidt/ MFT).

An alternative explanation for the rather instable dimension might be that the questions in your condensed version suffer from inherent reliability issues, as most of them focus on emotional and physical hurt. That is just one aspect of the MFT; but one aspect that might be most susceptible to a person's current affective state. As it is now, 'Hostility' would be a more fitting name for this dimension.

All other conclusions are transparent and well-stated.
Additional comments: Thank you for an interesting manuscript. I'm interested to see how your measure will perform with a clinical sample, especially with regard to the three dimensions (A/I/R). Having the full spectrum of beliefs and values in mind right from the beginning is important -- and a promising avenue.


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: BELIEFS ARE MULTIDIMENSIONAL AND VARY IN STABILITY OVER TIME - PSYCHOMETRIC PROPERTIES OF THE BELIEFS AND VALUES INVENTORY (BVI)
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors addressed all my previews comments, so I recommend accepting the manuscript.


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

Input:
Title: BELIEFS ARE MULTIDIMENSIONAL AND VARY IN STABILITY OVER TIME - PSYCHOMETRIC PROPERTIES OF THE BELIEFS AND VALUES INVENTORY (BVI)
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment, all the points mentioned in the first round have been addressed
Experimental design: no comment, all the points mentioned in the first round have been addressed
Validity of the findings: I am still not convinced that morality, as operationalized in those studies, is on par with the other dimensions.

Beliefs and values are associated with stable predispositions. Especially, Haidt's MFT rests on the assumption of very stable underlying intuitions. When looking at the ICC confidence intervals (Koo & Li suggest this, rather than focusing on the ICC value alone), M_A with a lower bound of 0.46522 must be considered "poor to moderate"---for a rather short time interval of 48 hours. There are other hints that morality seems to be particular here (cf. line 604/ 605).

While the test-retest stability cited here by Graham et al. (2011) was not that high either, the correlation reported there was .71 for a test-retest interval of four to six weeks.

The justification given here, line 573 to 575, is not convincing, as "political or life events" that might cause attitudes to alter should not, in general, have that massive of an influence for a retest 48 hours later (for the retest 3.5 month later, that might seem more plausible). I would acknowledge that the whole politics dimension might be less stable because of current political developments reported in the news (even within 48 hours), but the items given here for morality do, prima facie, not appear to be susceptible for such short-term influence.
Additional comments: Thank you for your revised manuscript!

While everything presented here undoubtedly has great merit and should be reported, I do not share the conclusion that the BVI in its present form, with all five dimensions, is valid as an instrument that covers "beliefs and values", as there are empirical indications here that the morality dimension is on a different conceptual/ psychological level.

One possibility could be to report all the findings and propose a four-factor-BVI, while undertaking further research on the morality dimension; and/ or run a short study with just the M-items, to see if the ICC scores are comparable for a different sample (maybe it was just some unknown bias in the original sample). The "poor to moderate" ICC CI, for M_A in particular, is puzzling and should be investigated further (theoretically and empirically) before being included as a BVI dimension.


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: BELIEFS ARE MULTIDIMENSIONAL AND VARY IN STABILITY OVER TIME - PSYCHOMETRIC PROPERTIES OF THE BELIEFS AND VALUES INVENTORY (BVI)
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 very interesting manuscript!

One minor comment/ suggestion, where you should check with the editorial office in case of acceptance, as it will depend heavily on the final form/ typsetting:

The grey text in Figure 2, the plots in Figure 3, the axis labels in Figure 4, and especially the numbers/ texts inside the boxes and circles of Figure 6 are hard to read on a printout, you might want to consider a final check of your Figures regarding font color and size.


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 FIXED AGREEMENT—CONSEQUENCES OF BROOD SIZE MANIPULATION ON ALTERNATION IN BLUE TITS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is competently written, with sufficient references. Some rewording could be useful in places to explain the rationale behind some of the predictions, but overall the hypotheses and results align well. Revisions are needed in the presentation of the figures - details below. Data underlying the article is provided.

Presentation:
Figures 1 & 2: Axes labels and plot symbols need to be much larger, and units are required for prey size. Figure 2: Units or explanation (in legend) required for deviation from expected alternation score.
Figures 3 & 4: Again, axes labels need to be much larger to be easily readable. Different symbols very difficult to distinguish – these also need to be much larger. Either the figure legend or the axes need to make it clearer what the units are for the deviation from expected alternation score.

Other comments:
47: ‘Parents were expected to visit more and more consistently alternate their visits’ would be clearer wording here.
49: Not clear what ‘less relevant’ means here.
60: It’s odd for the citation here to be a chapter of a book, and then the entire book. Also, the 90% figure for biparental birds is taken directly from a single paper (Cockburn 2006 PRSB), so that paper should be cited here instead of the chapter. Furthermore, the 90% figure includes cooperative species where both parents care, which are not ‘biparental’ in the relevant sense for this paper of only having two carers (a more accurate number would be 81%).
132-4: Move this sentence down – it mentions video recordings before any description of the recording methods.
147-8: This sentence is part of the statistical methods and is not needed here.
Experimental design: The article fits within the journal aims and scope. Research gap identified, but rationale for research question and approach could be improved. No concerns about ethics or technical approach taken. Methods need some additional details for reproducibility.

Comments about research question:
50-1: Why would alternation lead to more weight gain? I would expect visit rate and prey size to be all that matters here. Unless the suggestion is that parents of offspring that gain more weight should both visit more and be better-coordinated… but in that case why not just predict that visit rate and alternation should be correlated with each other?
95: What does a ‘stable parental strategy’ mean?
99: Is a brood size manipulation changing ‘environmental conditions’?
109-12: This explanation is clearer than the one in the abstract, however the causality is still unclear to me. The brood size manipulation is intended to manipulate visit rates (which may then alter the level of alternation), but here it reads as if it’s alternation driving visit rates. Is this meant to be a prediction that in general one should expect pairs that alternate more to have higher visit rates, as they have lower levels of sexual conflict?

Comments about reproducibility:
136: Source company of camera should be added.
138-9: Some information about how these prey size scores were assigned should be included in the text rather than just cited, as this is a key variable in the following analysis.
145-6: With L125 this suggests that all your visit data are from between 4pm and 5pm, but with L144 this suggests you analysed up to two hours from 4pm. Which is accurate?
162: To clarify (since you stopped at 10 visits from either parent), are the visit rates for each calculated over different observation windows?
Validity of the findings: Conclusions clearly linked to original question, discussion and speculation reasonable. I have a number of concerns about the statistical approach that might influence the results. The most important of these are (1) the comparison of observed values to the mean of randomised values, without accounting for the variance of those randomised values; (2) analysis of the ordinal prey size variable as if it were continuous, (3) some missing details on how the statistics were implemented.

Comments about statistics:
143: How are you judging whether these constitute ‘very similar’ visit rates? The Pearson test just tells you that the values are correlated (which will obviously be the case) – did you have some threshold r value you used to choose the number of visits to analyse?
170-3: The variance of the randomisations is also important here – the absolute difference between the observed alternation and the mean of the randomisations does not tell you much without knowing the spread of the randomised data.
173-4: I don’t think it is appropriate to calculate a mean from ordinal data in this way – only the median and mode are defined. Depending on the distribution of S/M/L items, ‘fraction of small items’ or similar might also be suitable.
180: Was there a reason for not including visit rate as a predictor of alternation? This would seem relevant to your question.
181: Do you know whether alternation was related to prey sizes? This is mentioned as being important above but I couldn’t see any analysis of it.
185: A cleaner analysis here would be to have a model with both male and female visit rates as the response variables, and your existing factors as predictors.
195: This package only adds p-values to models that are run by other packages (e.g. lme4)
196: Not clear what you mean by this: you don’t present minimal models in the results, just the significance levels of each fixed effect.
198: What is ‘total provisioning’? Why is this in the model as well as visit rate, when they are presumably highly correlated?
212-4: I don’t follow the statistics used here. Is this a sign test (or similar paired test) comparing the observed values to the means of the randomised values?
215-6: I don’t think the deviation is a meaningful value without an estimate of the variance.
229: I don’t understand the rationale of any potential effect of alternation on mass gain.

Comments on discussion points:
242: Why does this change need to occur via alternating more, rather than just directly as a response to increased demand?
249: I don’t think Johnstone et al. suggests that there is really a ‘feedback loop’ – just that one parent increasing investment encourages the other to do so immediately and to the same degree, rather than the (potentially delayed) incomplete compensation predicted by other models.
Additional comments: Overall, I think this is a competent piece of work that mainly requires 'polishing' to be suitable for publication. While I made a relatively large number of comments above, many of these are minor problems of detail, clarity, or presentation. The more substantial issues should at most require relatively simple reanalyses, so I encourage the authors to persist in improving the article.


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

Input:
Title: A FIXED AGREEMENT—CONSEQUENCES OF BROOD SIZE MANIPULATION ON ALTERNATION IN BLUE TITS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an interesting study that investigates the effects of experimental manipulation of provisioning behavior (via brood size manipulation) on parental coordination (i.e. alternation of feeding visits). The manuscript is generally well written, but improvements are necessary at some places (see below).
Experimental design: There is no explanation for the rationale of the experimental design chosen. Why were brood sizes manipulated at an advanced stage of the nestling period (days 8-10)? What was the goal of the repeated-measure design, especially, that no within-nest analyses were performed? Given this short-time manipulation, it is hard to expect any differences in the nestling mass, and actually the study did not find a treatment effect here.

The description of the methods is sometimes confusing or not detailed enough.
Validity of the findings: I think the statistical analyses could be improved - although I don't think that it would alter the conclusions.
Additional comments: 1. I'm not 100% convinced by the conceptual framework of the study. First the description of the theoretical background is a bit superficial. McNamara et al. 1999 were not the first to suggest a bargaining process reciprocally settling mate efforts at limiting values (see e.g. Chase 1980). Next, Johnstone and Hinde (2006)  did not simply predict that "parents should invest below the most efficient level of care". But most importantly, one can argue that while higher alternation may lead to higher overall feeding rate, the reverse should not necessarily be the case, i.e.  increasing the offspring need would not lead to higher alternation (and as the study finds, it does not). Put it simply, if the chicks are hungry, the parents are busy searching for food and as soon as they found a food item, they should bring it to the offspring. If they are waiting for their parents to feed first, that would limit their provisioning rate. Actually, one may argue that alternation should be higher when the offspring need is decreased, because in that case parents can afford bargaining about provisioning.

2. I also think that the way alternation is calculated is too restrictive, because it only considers immediate reciprocity, and disregards any other temporal pattern in the data. To illustrate this point, imagine a nest, where males (M) and females feed their young in this sequence:

MFMFMFMF - this is 1.0, i.e. maximal alternation score.

Now consider

MMFFMMFF - which is also alternated, but follows the rule "you feed twice, then I feed twice", but the alternation score is merely 0.42

And now consider

MMMFMMMF - which also yields an alternation score of 0.42, despite being much less egalitarian than the sequence MMFFMMFF.

Considering temporal autocorrelation in the data may be a more sensitive way to analyze the data.

3. The structure of the Methods should be improved. For example, the description of the brood size manipulation experimental design is hard to understand (lines 125-131). Only from line 178 we figure it out that each nest was subjected to all treatments. This should be made much more clear.

In line 133, you mention "during the video recording", but this is the first time we learn that video recordings were used at all. The description of the video analyses needs more details. For example, who analyzed the videos? Was that the same person? Was that person blind to the treatments? How was the repeatability of measurements (e.g. prey size)? How did you determine the sex of the parents using IR cameras?! The data file contains a variable PIT tag number, but there is no mention of this in the text. When were the parents captured? What were the PIT tags used for? Etc.

In line 147, you say that alternation scores were calculated, but we don't understand how until we reach the next section - this is confusing.

4. Statistical analyses. Since visit rates are actually counts, with usually a skewed distribution, you could consider analyzing this variable using a Poisson model. The alternation scores are actually proportion variables, so these should be analyzed in a binomial model. The third model should be a multinomial model (only 3 categories). Analyzing these variables in a Gaussian model is not correct.

Brood age should be controlled for in the models.

5. Sample sizes
line 117 and throughout: the sample size should be denoted with a lower case 'n'. 'N' is usually the symbol of the population size, the the sample size. Sample sizes should be explicitly included in the figures. It is not explained why sample sizes differ between Fig.3 (I counted 17, 18 and 20 circles, triangles and squares respectively), and Fig 4 (18, 19, 16)?


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 FIXED AGREEMENT—CONSEQUENCES OF BROOD SIZE MANIPULATION ON ALTERNATION IN BLUE TITS
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1. The manuscript is structured well, but is still in need of revisions to ensure it conveys its key points clearly, particularly in the introduction and discussion. Specific points I think are in need of clarification:
L42 – Not immediately clear what ‘This’ is referring to.
L43 – Not clear what ‘alternation’ is.
L62 – While it is obvious what you mean, this sentence reads as if birds are not animals.
L78 – Not clear what ‘efficient’ means in this context (and this is important for your argument later).
L88 – This sentence needs rewording to make it clear this is an extension of the previous sentence attempting to define conditional cooperation in this particular context.
L102 – Not clear what ‘whether alternation represents a parental strategy’ means. From the context it seems like you mean a specific level of alternation, but this needs to be clear.
L107 – I would add in ‘through increasing individual costs of parents’ to the end of the line here, to ensure the point is obvious later in the paragraph.
L115 – I don’t know what this last sentence is trying to convey.
L281-3 – I don’t follow the argument of this sentence at all – wouldn’t difficulty tracking prey size make it easier for parents to cheat?
L297 – Was perfect alternation expected? If so, why? If not, what is this section trying to convey?
L325 – While looking at fitness more broadly is a reasonable suggestion, in the context of this study the point could be made more clearly. The key take-home of conditional cooperation (as discussed in the Johnstone et al. model cited above) is that the outcome is more efficient than a negotiated or bidded outcome, i.e. closer to both parent and offspring optima. Presumably the argument here is that one should hence expect both (a) better outcomes for offspring and (b) higher costs for adults when cooperation between parents is stronger?
L329 – I think it would be clearer to say something like ‘thought to be a strategy by which parents can avoid exploitation by their partner during care, potentially leading to higher visit rates’.

2. Existing literature is reasonably well covered, however I would include at least one reference to an existing blue tit brood size manipulation study around L104-6 where the method is first mentioned, and again in the methods. In addition, I think the discussion would be improved by mention of some other studies, particularly those in which manipulations of parental effort have been attempted but failed to result in theoretically expected responses (perhaps around L275). This would provide context for the later interpretation of the results as suggesting a ‘fixed agreement’.  For example, Schwagmeyer et al (2002) Behav Ecol 13:713-21 report an attempt to induce parents to alter their visit rates by handicapping partners, and found no appreciable response, suggesting a fixed ‘bid’ rather than a negotiation. Contrast could also be made to Santema et al. (2017) Anim. Behav. 123:117-27, another study on blue tits, in which a begging playback failed to elicit increased provisioning rates. Does this suggest brood size manipulations are more reliable at eliciting changes in feeding rate?

3. I suggest boosting the point sizes in figure 1 - the circles and triangles are too small to be distinguished from each other (and perhaps altering the size in figure 2, although all points are circles, to maintain consistency). In figure 5, 'gram' can be shortened to 'g'.
Experimental design: 1. A few details are missing from the methods / results
L124 – Were the PIT tags part of a ring, injected, or attached some other way?
L200 – Presumably this was a LMM or GLMM as well?
L207 – Remove ‘linear’ here as you also presumably used lme4 for your GLMMs
L255 – Was there an effect of visit rate on nestling mass gain?

2. Some reordering would aid comprehension
L164-6 – This sentence belongs somewhere earlier, as currently the methods reads as if the cameras were set up, and then the following 10 visits were the ones analysed. In general this methods section should be carefully looked at to ensure it progresses in the most logical order.
L254-5 – I think it would make sense to present the overall effect of alternation before whether this effect differed between treatments.
Validity of the findings: On final revision, check speculation is clearly signposted.
Additional comments: Once all remaining reviewer comments are dealt with, I recommend the authors make a final pass through the article with a view to improving the readability of the text. There are numerous points at which sentences and/or topics either start or end abruptly, or are written as if related to or following from a previously unrelated point. Examples include L42, L115, L132, L140, L284, and elsewhere.

Minor corrections to wording:
L46 – remove ‘about’
L50-1 – ‘On the contrary’ -> ‘In contrast’
L148 – ‘that were actually biparental’ would be clearer as something like ‘at which both parents visited’.
L227 – ‘in function’ -> ‘as a function’
L238 – ‘amount’ -> ‘proportion’


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 FIXED AGREEMENT—CONSEQUENCES OF BROOD SIZE MANIPULATION ON ALTERNATION IN BLUE TITS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors satisfactorily addressed the comments raised by the reviewers and edited the manuscript accordingly. I think the manuscript improved a lot from the previous version. I think there are still a few outstanding questions and issues open for debate, but these do not affect the scientific integrity of the current manuscript. From a theoretical perspective, future articles may provide more specific hypotheses about when and how alternation is expected and future empirical studies may improve the way alternation is calculated. This paper may be a stimulation for these future works.

I only have a few minor issues that may need attention at the proofs stage:

1. Use consistent spacing following the journal's style recommendations. Currently, all possible combinations of using/not using space can be found sometimes just a few lines apart (e.g. 'n= 21' in line 149, '1=small' in line 154 and 'n = 55' in line 163). Sometimes an extra space is found (e.g. lines 227, 276). Please check the text throughout.
2. Fig. 5. The description of alternation in the legend does not match the description of the same variables in the previous figures. Please check. Also, the y-label should read 'mass gain of the nestlings' and not the 'nest'.


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: PERIODONTAL CONDITION IN GROWING SUBJECTS WITH MARFAN SYNDROME: A CASE-CONTROL STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: In their work Venza et al report the association between Marfan syndrome (MFS) and increased periodontal conditions, namely plaque and inflammation, in paediatric patients.

The language is acceptable, although different grammar mistakes are present in the text (e.g. lines 125, 132: “founded” is not the past tense of “find” – “found” should be used; line 57: “do not” should be used, and not “don’t”; line 65: the whole sentence is not properly formulated).
The introduction properly inserts the study in the actual situation, reporting what is currently known concerning the bases of MFS, and its correlation with periodontal diseases. The authors should improve the description of the relevance of their work already at the end of the introduction section.
The three figures provided could be simply merged in one panel. Figure 3 is of very poor quality, and it is not suitable for publication in big scale: this should be either changed, or published in a much smaller size.
The discussion is poor. The authors fail to show the relevance of their study for any clinical application. For example, the prognostic value of their finding is not discussed at all, as well as the potential importance of earlier treatment/preventive approaches in MFS patients before the onset of more severe periodontal affections.
Experimental design: The authors should improve the description of the relevance of their work already at the end of the introduction section. At line 73, the specification of the nationality of the patients does not really increase the impact of the manuscript, and it is not relevant at this point. Similarly, the statement that “the causal relationships among MFS, aortic aneurysms, and periodontitis have not been elucidated yet” (line 76-77) at the very end of the introductory paragraph does not really fit, as this is not the central aim of the manuscript. The authors should focus more on the only aspect of novelty of their article, that is, the study of the correlation between MFS and periodontal conditions in young patients.

The experimental design is properly described. The study is extremely simple, as it consists of a reduced set of clinical observations in MFS and control age-matched patients. The results are properly described.
Validity of the findings: The data appear solid, even though all based on subjective clinical evaluations.
The conclusions are solid, while very poor is their discussion in the light of their potential implications for the current clinical practice.
Additional comments: no comment


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

Input:
Title: PERIODONTAL CONDITION IN GROWING SUBJECTS WITH MARFAN SYNDROME: A CASE-CONTROL STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: English needs some revision being unclear in some sentences.
For example: lines 79, 146-147.

The article includes sufficient introduction which is unprecise/not correct in one point (lines 47-49). FBN1 gene is at the moment the only gene associated to Marfan syndrome

Litterature is mainly appropriately referenced except for one point where is inappropriately referenced (lines 250-251). (substitute Summers with Cook JR, Carta L, Galatioto J, Ramirez F. Cardiovascular manifestations in Marfan syndrome and related diseases; multiple genes causing similar phenotypes. Clin Genet. 2015;87(1):11–20 to show that FBN1 is the only major gene involved in Marfan syndrome).

An important Table reporting clinical features that allow the diagnosis of Marfan syndrome and the statistical P value for the plaque index (PI), bleeding on probing (BPO), medial periodontal screening and recording (PSR) for all the 16 patients is missing.

When the Authors say .	“severe periodontitis is sometimes observed in MFS patients, but no in-depth
information has been reported in Italian groups of MFS growing subjects” (lines 72-73) do they mean that “in depth information” have been reported for different geographic groups? If yes, please, specify which groups and add references/, otherwise erase the adjective “Italian” to avoid misunderstanding.
Experimental design: If, as the Authors say, severe parodontitis has been already reported in Marfan patients the Authors recruited 16 patients from the Centre for Rare Diseases for
Marfan Syndrome and Related Disorders of Tor Vergata University Hospital, from November
2017 to April 2018.  with the aim of investigating early signs of periodontitis in growing patients (age: 7-12 years). The number of recruited  patients in 6 months is pretty low for this purpose in fact, at the end of the screening, the informative patients are 5. Five patients out of 16 (about 30%) display early signs.

In the experimental design there is an important weak point: the Authors base the diagnosis of Marfan syndrome on the clinical manifestations (but they do not show the clinical features). It is very difficult to make the diagnosis at 7-12 years without confirmation by mutation analysis.

It is not clear what the Authors mean by "controls made of 20 non-syndromic subjects" (line 90) and the exclusion criteria they use for both MG and CG (lines 91.92) are not clear also.
Controls are not supposed to have a systemic features score >4. If the controls display systemic features of Marfan syndrome this should be reported.

It is not clear if these are the first data reporting early signs among world MF patients or among Italian Marfan patients, please clarify.
Validity of the findings: Results.

The main result which consists in finding an altered periodontal index  and a higher presence of plaque and consequently a generalized inflammation in the oral cavity of 5 adolescent Marfan patients is made weak by the absence of a Table displaying the clinical features that allowed the diagnosis of Marfan syndrome, the absence of data on related FBN1 pathogenetic mutations, the absence of statistical data for each of the 5/16 patients with parodontitis, the very small number of patients.

If the Reviewer understands well 30% of patients and controls display thick marginal gingival. Then, they affirm that three clinical parameters show the presence of early parodontitis in 5 Marfan patients.The statistical P value for the plaque index (PI), bleeding on probing (BPO), medial periodontal screening and recording (PSR) should be reported for all the 16 patients.

Discussion.

The Authors say "Ganburged and coworkers revealed that capillaries were dilated or enlarged in PDL cells of mice
underexpressing fibrillin-1 (Ganburged et al., 2010). Thus, MFS patients may be susceptible to
periodontopathic bacteria, that invade from the periodontal area with a connective tissue
abnormality."

In Gambured et al 2010 capillaries resulted dilated in mice homozygous, not heterozygous, for FBN1 mutations. Marfan patients are heterozygous for FBN1 mutations. Therefore, the Authors cannot state the above correlation.

Conclusions (lines 182-183)
Authors write "to the best of our knowledge, this is the first study analyzing the periodontal index in a group of
Italian growing subjects affected by MFS".

It is a result based on a very small number of patients (5/16) displaying these early features. It is not clear to the Reviewer if these data are the first reported in Italian Marfans or in world Marfans. In the first case the Authors have to mention the articles reported for the other Marfan patients and compare the data.

In conclusion this article has some important weak points:
1.	A table with the clinical features allowing the
        clinical diagnosis of the 16 MF patients is
        missing.
2.     Also a table (or the same table) with the statistical
        results regarding the three dental parameters is
        missing.
2.	FBN1 mutational analysis is not reported
3.	Patients are too young to be definitly diagnosed
        affected by Marfan syndrome on the bases of
        clinical manifestations unless they  are strongly
        direct towords this diagnosis
4.	The results are based on a total of 5 patients
         which is  a poor number
Additional comments: No additional comments


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

Input:
Title: PERIODONTAL CONDITION IN GROWING SUBJECTS WITH MARFAN SYNDROME: A CASE-CONTROL STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: The authors satisfactorily addressed the comments and issues raised during the first round of revision. I therefore consider the manuscript ready for publication.


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

Input:
Title: PERIODONTAL CONDITION IN GROWING SUBJECTS WITH MARFAN SYNDROME: A CASE-CONTROL STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Abstract. Page 1. Line 25: "non-syndromic subjects" do not represent  a correct "control group".

Introduction. Page 2. Lines 49-51. It seems that the Authors mixed up the Berlin nosology with the Ghent nosology. It would be better to erase "Ghent nosology" and substitute the reference "Ha et al 2007" with the original one of Beighton and coworkers. (1988)
Experimental design: Are you saying that 16/16 patients were positive for FBN1 gene mutation? It is a very high score, 100%. Maybe you selected these patients with positive FBN1 mutation among a larger group of young patients.
Validity of the findings: Results. In the reviewer opinion results have to be based on a correctly selected group of patients. The reviewer is confused by the fact that the Authors do not report, as previously requested, a table with at least major clinical data on each patient confirming, together with genetic data, the clinical diagnosis.
This reviewer believes that a simple Table displaying the clinical manifestations of each patient (ocular, CVS, SF) that, together with the genetic data, brings to the diagnosis of Marfan syndrome is necessary.
Additional comments: No additional comments


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

Input:
Title: PREFERENCE CLASSES IN SOCIETY FOR COASTAL MARINE PROTECTED AREAS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Introduction

Context to study and background to DCE clearly explained. Objectives clearly stated.

Method

Generally reads well and research approach appears clear though I have to caveat that I’m not an expert in the statistical modelling being undertaken. Questionnaire is supplied in the supplementary information. I noted a few minor statements in the main text that will need checking and might need editing:

Line 130: should this read “random taste variation”? I’m unfamiliar with this terminology.

Lines 190 – 193: check sentence structure. Should line 192 read “level of protection they were offered…”?

Lines 252 – 254: can you show sample locations on Figure 1 Map? Or highlight post code areas on map in terms of where participants came from?

Results

Look comprehensive. Though note that the sample may not be representative of the Welsh population as a whole (the author’s do acknowledge this). I have one specific comment:

Lines 298 – 302: number of children also significantly lower in sample than census data (0.5 vs 1.7), this could potentially have some bearing on the result? Would this be taken into account in the modelling of latent classes?

Discussion and Conclusion

Clearly written and results discussed in the context of the broader literature.
Experimental design: This study used a Discrete Choice Experiment to measure peoples’ willingness to pay for various options of MPA design. The use of a focus group to help define the attributes for the survey will have improved the reliability of the results. The questionnaire appears to be carefully designed, and the authors consider the various factors that may influence the results explicitly in their questionnaire design.

The authors should be commended for administering the number of questionnaires they did. However, the only slight weakness for this study (and this is acknowledged by the authors) is that the sample chosen may not be completely representative or random. This may affect the wider generalisation of the findings to the Welsh population as a whole, though the identification of Latent Classes will still be informative for policy makers making decisions on MPAs.
Validity of the findings: The authors don’t overstate their results and critically appraise the validity of their findings. Sample representativeness, environmental awareness of respondents, and how the DCE questions were asked have all been taken into account in the interpretation of the results. The only omission that came to mind was how much would the outcome of the DCE reflect the time period when the survey was taken?
Additional comments: The authors should be commended on what appears to be a very thorough approach to better understanding people’s willingness to pay for protection of the marine environment, and some of the demographic factors that may influence this. I found the paper interesting to read and the results paint a nuanced and insightful picture of different classes of society's attitudes towards the conservation of the marine environment. The results of this paper will be useful for policy makers making decisions on protection of the marine environment and allow them to rationalise decisions on the basis of the wants of segments of society rather than those of narrow interest groups.


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: PREFERENCE CLASSES IN SOCIETY FOR COASTAL MARINE PROTECTED AREAS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper is very well written and covers a very interesting and hot topic - the societal views on MPA designation around Wales. I found it a pleasure to read and review this article. I thought the references used were good however there are other references which could have been included for example:

Ressurreição et al., 2012. (Biological Conservation, 145, pp. 148-159) showed that cultural diversity was just as important as biological diversity when the public are valuing marine biodiversity conservation – the public don’t just value the charismatic organisms the highest. This could be cited around line 77-78.

Sian Rees and colleagues published a nice paper which looked at “The socio-economic effects of a Marine Protected Area on the ecosystem service of leisure and recreation”. Marine Policy, September 2015. This could be cited around line 87.

The paper is very well structured which made it easy for the reader to follow the story that was being told. The figures and tables included were useful and added value to the paper.
Experimental design: The experimental design is sound, with focus-groups and pilots being used to develop and test the attributes before undertaking the actual survey. Using a face-to-face approach on trains is a novel approach but ensures you have a captive audience! I am pleased to see that the limitations of the approach used have been highlighted as papers often neglect these which can lead to the wrong conclusions being drawn by the reader. The sample size is good (n=354 usable responses). I am not a statistical expert but I understood the analysis which had been undertaken and the outputs from it.
Validity of the findings: Despite the data being collected 10 years ago, I think the findings are still valid today and part fill a data gap which currently exists within the UK. The authors recognise the limitations of the survey but nonetheless the findings are valid and could be used to further support the MCZ process in Wales and throughout the UK. The methods employed and the findings could also be applied elsewhere (outside of Wales) and therefore this paper will be a good addition to the literature.
Additional comments: I would have liked to see the findings being discussed in relation to the outcomes of the MCZ process in Wales – this was introduced at the start of the paper however things have progressed since this data was collected. It is actually a shame that this paper was not published earlier as I believe that the findings from this paper align with the concerns that were raised in the public consultation on the proposal to increase the protection of a number of existing MPAs in Wales following the introduction of the MCAA (2009). A few sentences on this in the discussion would add strength to the paper as it would bring things up to date with the MCZ process.

It would also be interesting to see how people’s opinions may have changed over the last decade given for example the impact of Blue Planet II and the plastics issue in raising the awareness of the current threats to our seas. Do you think the general understanding of MPAs will have increased as a result? I appreciate this is outside the scope of your project but it would be very interesting to undertake a similar study 10 years on to see how public opinion has changed.

I have identified a few minor issues that will need to be amended:
Line 47: socio-economic
Line 189: I believe Marine Nature Reserves and SSSIs are UK designations not European designations – please check and amend if required
Line 235: Table 1 (should have a capital)
Line 282: Tables 4 & 5 (should have a capital)
Line 289: ‘Size 30’ – perhaps explain this more clearly in the text as I had to cross reference the tables to understand what you meant by Size 30
Line 391: Sometimes Class is written in italics and other times not – this needs standardising throughout
Line 459: This does not make sense – should ‘and’ be included before ‘the permission’?
Line 681: (2008) SPSS – what does this refer to?

Finally, I would like to add that it is great to see this research published – often data sets that are 10 years old are forgotten about and never written up and published. It is also great to see the research that Gareth was part of still getting published today – great 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: PREFERENCE CLASSES IN SOCIETY FOR COASTAL MARINE PROTECTED AREAS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The author has taken into account the reviewer comments.
Experimental design: The author has taken into account the reviewer comments.
Validity of the findings: The author has taken into account the reviewer comments.
Additional comments: I have suggested to the editor that the article is now accepted based on the revisions made.


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

Input:
Title: PREFERENCE CLASSES IN SOCIETY FOR COASTAL MARINE PROTECTED AREAS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Good
Experimental design: Good
Validity of the findings: Good
Additional comments: I am happy that my comments have been taken on board satisfactorily by the authors and fully support publication of this manuscript.


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

Input:
Title: INTROGRESSIVE HYBRIDIZATION IN A SPINY-TAILED IGUANA, CTENOSAURA PECTINATA, AND ITS IMPLICATIONS FOR TAXONOMY AND CONSERVATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript describes genetic clustering patterns in iguanas in Mexico, and the results have taxonomic implications for the species. The study provides both mitochondrial DNA sequences and microsatellites.
The conceptual basis of the study is to compare clustering patterns between nuclear and mtDNA data, and to quantify introgression. This is accomplished by 1) defining genotypic clusters, 2) examining concordance between markers, 3) examining introgression levels, and 4) recommendation of taxonomic changes.
The study meets these goals, but the results are not always clear, which leaves the conclusions open to interpretation.
Experimental design: 1) I got lost trying to figure out how lineages are diagnosed when no lineage-based approaches are used in this study. Genotype clusters are not lineages. I recommend adding a phylogenetic analysis to this study to provide a direct and transparent avenue for discussing putative lineages. Unfortunately, microsatellites are not well-suited for estimating phylogenetic trees, but the mtDNA data are available. A single locus analysis leaves a lot to be desired, but it is better to have one a one-locus gene tree versus no phylogeny at all

2) Provide an ethics statement and cite formal documents approving animal care and use, and scientific collecting permissions from government agencies.

3) The haplotype network is not a phylogenetic estimate. Replace and/or augment the supplemental figure with a phylogeny for the same data. I realize that the same analysis is likely published in one of the previous studies of the same data, but it is easier to include the phylogeny here than to require readers to find the information in a previous paper.

4) The analysis of the microsatellite data is rather confusing. The description of the Structure analyses is unclear, and it is doubtful that the study could be replicated. Some people use “hierarchical” analyses to dissect Structure results, and it seemed like that might be what these authors are doing, but it is not clear. I was left with the impression that the microsatellite data might support more populations but that the analyses were insufficient to make a firm determination, therefore the authors picked their favorite value, and then performed additional analyses for some groups and not others. The haphazard nature of these analyses should be replaced with a clear and easy-to-follow description of the methodology.

5) The accounting of samples is confusing. It seems like this study only adds two new samples to the existing mtDNA dataset. Is this accurate? If so, there needs to be a justification for why these two new samples merit additional reanalysis. Are they from a geographic area of particular interest? Are they from individuals that appear to be hybrids? What motivates the publication of a study that increases sampling from 342 to 344 specimens, which is a negligible increase?
Finally, the influence of the two new samples on the phylogeny/ clustering/introgression analysis is not stated clearly in the results. Did these two new samples expose something new or interesting in this study that is worth reporting?

6) The proposition of taxonomic changes without illustrating the resultant changes on a phylogenetic tree is highly problematic. I recommend annotating a phylogenetic tree with current and proposed taxonomic changes. These changes need to be transparent and identifiable on a phylogeny. The discussion needs to address taxonomic changes in the context of a phylogeny and not genotypic clusters, which are not equivalent to evolutionary lineages.

7) Is it necessary to include copyright information for GoogleEarth/GoogleMaps images, which the authors seem to have masked in the figures? PeerJ staff should have an answer for this.

8) I found it difficult to identify introgressed/admixed/hybrid/backcrossed individuals on the figures, because this information is never compared directly in a single figure. The maps convey important information, but it is difficult to tell which samples are the same in the different maps, because they have different sampling. Figure three provides a side-by-side comparison, but the this figure does not include a detailed geographic component (only group names). It would be useful to see a mtDNA gene tree with structure/microsat. results plotted on the tips of the tree. Alternatively, it would be useful to plot the microsat structure groups on a map, and then superimpose the mtDNA groups/clades. This would make it much easier to identify areas of concordance and/or discordance.
Validity of the findings: Minor issues:
- Supplant the definition of introgression in the introduction with one found in the population genetics literature. The use of a definition from a recent and somewhat random empirical study seems awkward.
- A recent study of Xenosaurus lizards by Nieto Montes de Oca in Molecular Phylogenetics and Evolution that used SNP data should be cited in the introduction.
- Scientific names need to be in italic font throughout, check the reference section for errors.
Additional comments: see above.


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

Input:
Title: INTROGRESSIVE HYBRIDIZATION IN A SPINY-TAILED IGUANA, CTENOSAURA PECTINATA, AND ITS IMPLICATIONS FOR TAXONOMY AND CONSERVATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are a number of areas where the clarity of the English could be improved. The use of appropriate punctuation (specifically an en dash versus a hyphen) needs to be corrected throughout the manuscript. Also, proper abbreviation of generic names needs to follow appropriate conventions throughout, such that the first occurrence of a generic name in a paragraph is spelled out and any occurrences thereafter are abbreviated. Lastly, there are numerous cases of incomplete or improperly formatted citations.

Generally, the intro and background are well-crafted, though the taxon-specific information needs to be reorganized for clarity.

The figures have a number of problems, specifically Figures 1 and 2. The inset on figure 1 is (1) too small an doesn't make full use of the "dead space" below and to its left. The inset should not cover up what it is depicting. Also, there are numerous places in the manuscript where specific named localities are referenced, yet none of them are depicted on either map. This is very confusing for readers that aren't familiar with the geography of Mexico. As point of convenience and added clarity, I also recommend better depicting the specific sampling localities for which admixture was detected and shown in Figure 3.  Indicating on the map/s which sampling localities contained the admixed individuals would be quite helpful.
Experimental design: This study represents original research that is within the scope of the journal. The research question is well-defined and relevant, and fills an identified knowledge gap.

Unfortunately, some of the authors' approaches require further justification. The clustering analyses applied are plagued by subjective thresholds that lack any language of justification. Without justification, one cannot know that the authors fully explored the patterns present in their data.

The methods are described in sufficient detail, and are replicable.
Validity of the findings: The impact and novelty is somewhat assessed in the discussion, specifically with how the results impact conservation of the study organism.

The authors support further studies that address the questions and hypotheses raised in their own manuscript.

The data appear to be robust, statistically sound, and controlled
Additional comments: Generally, this is a well-written manuscript and a very interesting study.

I have commented thoroughly throughout the manuscript where necessary. The manuscript requires a thorough read to address a number of inconsistencies and formatting issues.

The figures are generally well done, but their clarity could definitely be improved.

My only real concern with your analyses is that you seem to have arbitrarily chosen values of K to test, rather than exploring a wider range of values and fully exploring the variation in the data. While this may not prove fruitful in practice, it is vital to ensure that analyses and inferences aren't biased.


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: INTROGRESSIVE HYBRIDIZATION IN A SPINY-TAILED IGUANA, CTENOSAURA PECTINATA, AND ITS IMPLICATIONS FOR TAXONOMY AND CONSERVATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: This manuscript continues to be plagued by improper presentation of generic names, which I have tried to note throughout.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comments


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

Input:
Title: NOBLELLA THIUNI SP. N., A NEW (SINGLETON) SPECIES OF MINUTE TERRESTRIAL-BREEDING FROG (AMPHIBIA, ANURA, STRABOMANTIDAE) FROM THE MONTANE FOREST OF THE AMAZONIAN ANDES OF PUNO, PERU
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The manuscript is solid and well-written. I only have one major comment. The species is described based on a single specimen; the authors should briefly discuss the problems associated with the low sample size of the description (e.g., sexual dimorphism, intraspecific variation). Also, I suggest adding a few sentences explaining why no additional specimens were included in the description... Is the species rare? Please describe the sampling effort at the type locality and nearby areas. Finally, add information about the conservation threats (if any) that the species might be facing.