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

Input:
Title: ARTIFICIAL SUBSTRATA INCREASE POND FARMING DENSITY OF GRASS CARP (CTENOPHARYNGODON IDELLA) BY INCREASING THE BACTERIA THAT PARTICIPATE IN NITROGEN AND PHOSPHORUS CYCLES IN POND WATER
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is written clearly and unambiguously. Professional English used throughout.

The manuscript includes sufficient introduction and background. Relevant prior literatures are appropriately referenced.

The structure of the manuscript conforms to an acceptable format of 'standard sections'. However, the panels of Figure 2, Figure 3(A) and Figure 5 are too small to read. Please reorganized the panles and appropriately adjust the dpi and size of each panel.

The merged sequence data have been made available.

The manuscript includes all results relevant to the hypothesis.
Experimental design: The original primary research within aims and scope of the journal.

The research question is well difined, relevant and meaningful. And the study contributes to filling that gap.

The investigation was conducted rigorously and to a high technical standard.

The methods should be supplemented. Line 82: "An explosion disc was placed in the centre of each pond." However, the authors did not specify the amount of aeration per day. Line 111-115. Please add references of the calculation of SFR and FCR. Line 141. According to what I know, QIIME1 integrated multiple softwares and algorithms.  Uchime algorithm (Edgar et al., 2011, Bioinformatics, 27: 2194-2200)  commonly used to identify and remove the chimera sequences in QIIME1. Therefore, please further elaborate which specific software or algorithm was used in the chimeric sequence analysis. Line 147. Please submit the in-house Perl scripts to a public script database or using QIIME reanalyzes the alpha and beta diversity, as QIIME1 also can analyse the alpha and beta diversity.
Validity of the findings: The manuscript reported the potential of artificial substrata to improve the efficiency of intensive aquaculture per unit area and characterized the microbiota compositions on the artificial substrata and in pond water. Considering the important role of grass carp on the aquatic products and the aquaculture production space is constantly decreasing, the manuscript provided a valuable technical method to increase the density and yield of grass carp.

All underlying data have been provided, and they are robust, statistically sound, and controlled.

Conclusions of the manuscript are well stated, linked to original research question.
Additional comments: The manuscript is clearly written in professional, unambiguous language. The organization of the manuscript is logical. I recommend accepting the manuscript for publication. However, it has some minor errors which should be improved upon before publication.
Line 82: "An explosion disc was placed in the centre of each pond." However, the authors did not specify the amount of aeration per day.
Line 111-115. Please add references of the calculation of SFR and FCR.
Line 141. According to what I know, QIIME1 integrated multiple softwares and algorithms.  Uchime algorithm (Edgar et al., 2011, Bioinformatics, 27: 2194-2200)  commonly used to identify and remove the chimera sequences in QIIME1. Therefore, please further elaborate which specific software or algorithm was used in the chimeric sequence analysis.
Line 147. Please submit the in-house Perl scripts to a public script database or using QIIME reanalyzes the alpha and beta diversity, as QIIME1 also can analyse the alpha and beta diversity.
Reference list: Please make sure the format of the references is correct. Beacuse I found the formats of reference "Administrative Administration of Fisheries and Fisheries of the Ministry ..." and those of "Fisheries Bureau of Agriculture Ministry of China. ..." seem different.
Figure 2: The panels are too small to read. I suggest longitudinally arrange the panels and appropriately adjust the dpi and size of each panel.
Figure 3(A): The panel is too small to read. I suggest appropriately adjust the dpi and size of the panel, or longitudinally arrange the panels of Figure 3.
Figure 5:  The panels are too small to read. Please reorganized the panles and appropriately adjust the dpi and size of each panel.


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

Input:
Title: ARTIFICIAL SUBSTRATA INCREASE POND FARMING DENSITY OF GRASS CARP (CTENOPHARYNGODON IDELLA) BY INCREASING THE BACTERIA THAT PARTICIPATE IN NITROGEN AND PHOSPHORUS CYCLES IN POND WATER
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: No information is provided about the chemical nature of the artificial substrata. Without this information will makes it cannot be replicated by others. So it is especially important to add this necessary information in the Materials and methods section.
Validity of the findings: no comment
Additional comments: This manuscript with clear presentation of experimental results. The authors provided adequate background of the study with references that are relevant to artificial substratum, and the hypothesis and objectives of the study are clear.

“artificial substratum” and “artificial substrata” should be kept consistancy.

Please make sure that all the LEfSe profifiles and figure 2 described in the text are visible in the figures. I suggest including high magnification inset or another figure.


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

Input:
Title: ARTIFICIAL SUBSTRATA INCREASE POND FARMING DENSITY OF GRASS CARP (CTENOPHARYNGODON IDELLA) BY INCREASING THE BACTERIA THAT PARTICIPATE IN NITROGEN AND PHOSPHORUS CYCLES IN POND WATER
Review round: 2
Reviewer: 1

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


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

Input:
Title: ARTIFICIAL SUBSTRATA INCREASE POND FARMING DENSITY OF GRASS CARP (CTENOPHARYNGODON IDELLA) BY INCREASING THE BACTERIA THAT PARTICIPATE IN NITROGEN AND PHOSPHORUS CYCLES IN POND WATER
Review round: 2
Reviewer: 2

Output:
Basic reporting: The English is clear and all the references are necessary.

All data including figures and tables are also clear.
Experimental design: This study within Aims and Scope of the PeerJ and the research question is interesting. Methods are described sufficient now.
Validity of the findings: Findings of this study is important for aquaculture and environment.
Additional comments: All my original comments and suggestion were fully addressed in the revision, I have no additional 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: THE DOPAMINE RECEPTOR D5 GENE SHOWS SIGNS OF INDEPENDENT EROSION IN TOOTHED AND BALEEN WHALES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The writing is solid and easy to follow.  References are adequate as is.  The structure of the paper is simple and fine as is.  Additional analyses would benefit the paper to further corroborate the evidence of gene inactivation or reduction in selection pressure (see below).
Experimental design: I think additional analyses should be done, but the analyses presented seem fine for documenting and confirming inactivating mutations in this gene across Mammalia.
Validity of the findings: By its nature, the inference on function in the paper is speculative, but the authors do not overstate the significance of their results.
Additional comments: Alves et al. examine inactivating mutations in the DRD5 gene, a dopamine receptor in mammals.  I am not an expert on dopamine pathways, but can comment on the comparative analyses done on various published mammalian genomes.   The authors pulled out DRD5 gene sequences from more than 100 mammal species, and describe sequences from taxa that show indications that this gene is not functional (frameshifts, stop codons, etc.).  They find that cetaceans, both mysticetes and odontocetes, are incapacitated along with the golden mole (Chrysochloris).  The authors suggest that these mammals are therefore naturally occurring knock outs for the DRD5 gene and could be used as models for the study of vasoconstriction.  The authors use synteny relationships and various other procedures to compile the published data and map mutations that disrupt the reading frame in various mammalian taxa.  The analyses are sound and the authors do not overstate conclusions, but the results are a speculative, involve only one gene KO, and the work will not overwhelm any reader I think.  Some additional analyses should be done to give the limited  work done here more weight given that no new data were described and that the inferences remain pretty speculative (see below).

Things that the reviewers might consider in improving their manuscript are listed below:
1) In the Introduction, the authors note that DRD5 knockouts have effects on renal salt balance.  Is this of importance for cetaceans that live in saltwater and is this related to hypertension?
2) In the Materials and Methods, the authors use single letter genus abbreviations for cetacean species.  These genus names should be spelled out here as many readers will not be familiar with these genera, and the reader must guess as to which species are described because of these abbreviations?
3) line 91 typo; change “flaking” to “flanking”.
4) line 107; “a reference phylogenetic close
species” is a bit awkward.  Reword this?
 There is additional awkward phrasing in this section that should be fixed.
5) For Odontoceti, all species but Physeter have a shared inactivating mutation, and all species for which data are coherent show evidence of KOs.  For Mysticeti, there is a common inactivating mutation for all baleen whale species examined.  The authors note that, “Importantly, no conserved
mutations were detected between Odontoceti and Mysticeti clades, suggesting that DRD5 pseudogenization events occurred independently after the divergence of both lineages (Figure 2)“.  The inactivating mutations alone suggest independent inactivation in three cetacean lineages: mysticetes, Physeter, and all remaining odontocetes (except Physeter).  However, the authors should try to corroborate this result by reconstructing dN/dS on their gene tree for DRD5.  What is the ancestral dN/dS for this gene where the gene is presumably functional?  For the authors interpretation, the stem cetacean branch (common ancestral branch) should have a dN/dS that suggests a functional gene with adaptive constraints (i.e., dN/dS much less than the neutral dN/dS of 1.0).  Is this the case, or is the dN/dS on this stem cetacean branch much higher than the ancestral dN/dS for this gene in mammals that have functional copies of this gene.  Also, the same question could be asked for the stem Odontoceti and stem Mysticeti branches.  Does these have low dN/dS that is indicative of functional constraints (and suggesting independent KOs of the gene in Mysticeti and also in Odontoceti), or is dN/dS not significantly different from 1.0, suggesting neutral evolution that expected following a gene inactivation?  These analyses should be done to bolster the authors’ conclusions about where these gene KO events occurred, especially considering that no new data were collected for the paper, that limited analyses were done, and that conclusions of the paper are already fairly speculative regarding functional outcomes for cetaceans given the gene KOs inferred.  Basically, is there further evidence from dN/dS that DRD5 was KOed two or more times within Cetacea, or do dN/dS instead suggest a single KO of the gene on the stem to Cetacea with lags in mutational evidence that the gene was inactivated (common in cetaceans with slow mutation rates) and perhaps evidence from regulatory regions of the gene that DRD5 is inactivated earlier?
6) line 225 and following paragraph.  The authors again refer to genera of mammals by using abbreviated genus names.  The average reader will have no idea what the authors are talking about unless these genus names are spelled out here.
7) The authors note DRD5 KO in one burrowing mammal but not many other burrowing or deep diving mammals.  Do the authors see any large differences in selective constraints, perhaps summarized by dN/dS for these “hypoxia lineages” or changes in otherwise well-conserved amino acid positions for this gene.  Ideally, the authors could compare dN/dS on these hypoxia lineages relative to the dN/dS on taxonomic lineages that are not characterized by deep diving or burrowing.  For example, what is the dN/dS in mole rats, moles, and deep diving pinnipeds (e.g., elephant seal).  Perhaps there are shifts in dN/dS in these lineages that suggests DRD5 KOs that show lags in accumulation of inactivating mutations.  The authors could either estimate dN/dS for each branch of their DRD5 tree separately or otherwise group branches in different categories (deep diver vs. not; burrower vs. non-burrower) to look for effects.  Because the inferences in the paper with regards to functional impacts are currently speculative, the authors should try to bolster their inferences by doing some additional analyses of the data that they have worked to compile for this short paper.
8) Figure 1.  Genus names of species should again be spelled out.
9) Figure 3 may not be necessary for this short paper?


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

Input:
Title: THE DOPAMINE RECEPTOR D5 GENE SHOWS SIGNS OF INDEPENDENT EROSION IN TOOTHED AND BALEEN WHALES
Review round: 1
Reviewer: 2

Output:
Basic reporting: I think this manuscript needs some english editing, as some passages are difficult to understand. Additionally some terminology (e.g. rightwards) should be replaced with common jargon.

I believe that the authors should make a better effort to introduce the problem. The manuscript would benefit if the authors make a smooth transition between the first and second paragraph in the introduction. As is now, the first paragraph is mainly devoted to the physiology of dopamine receptors and a little bit of evolution, whereas in the second the authors state their goal(s) and result in (mostly in) cetaceans. Something in between would be acknowledged.

The structure of the article is ok. Figures need some rethinking, in my opinion, figures 1 and 3 could be reported as supplementary material and figure 2 needs to be remodeled.

This manuscript is a self-contained unit, but in my opinion could be shortened to a "short communication" format, mostly reporting the cetacean result.
Experimental design: The research proposed is within the aims and scope of the journal

The research question is well defined.

The methods are well described and sufficient to replicate
Validity of the findings: This research is presenting a novel case of gene loss in cetaceans (and other mammals). The functional relevance is hard to assess, but some speculation is written in the manuscript.

The data they collected is sufficient to conclude that some mammalian lineages have lost DRD5.

Given the mammalian lineages they are working on, the main result is the gene loss. Some speculation regarding the functional consequences related to not having DRD5 is presented. They also highlight that cetaceans represent a natural KO system, however, it is also true that very limited research can be performed in this group.
Additional comments: General comment
This paper is trying to understand the evolution of the DRD5 gene in mammals, with an emphasis in cetaceans. I believe that this paper should be shortened to a "short communication" format. Cases out of cetacea should be removed, or commented in a couple of lines, no more. Figure 1 and 3 should be reported as a supplementary figure, and figure 2 should be extensively modified.

specific comments
In the introduction, I think there is a sort of disconnection between the first paragraph, which is mainly devoted to the physiology of dopamine receptors and a little bit of evolution,  and the second one in which they state their goal(s) and results in (mostly in) cetaceans. I think the manuscript would benefit if the authors make a smooth transition between the first and second paragraph. In the case of the results section, I believe that the first paragraph, which is mostly devoted to technical issues, should be shortened. The last two sections of this section seem to be a small detail, as most of the paper (including figures) is dedicated to cetaceans. Figures are not attractive; I think the authors should make an effort to improve them.

Line 92: I suggest changing rightwards by 3´ side.
Line 93: I suggest changing leftwards by 5´ side.
Lines 93 to 95: I do not understand what are they trying to say, maybe expressing the idea in a different way would improve the text.
Line 102: what is the meaning of LQ?
Line 133 to 134: The description of this procedure should be expanded.
Line 149: the comma after the word "available" I think should be removed.
Line 177: I believe that the manuscript should benefit if common names are preferably used instead of specific names.
Lines 242 to 243: There are other papers reporting gene loss in cetaceans and linking this phenomenon to the conquest of the aquatic environment.

For example:
https://www.biorxiv.org/content/10.1101/521617v1
https://bmcgenomics.biomedcentral.com/articles/10.1186/1471-2164-15-869

Figure 1 & 2: I suggest using common names.

Figure 1: Having the names of each syntenic gene in each species make the figure very busy. My suggestion would be to have the names of the syntenic genes only in Bos taurus, and join them using vertical lines.

Figure 2: This figure is not attractive, and it does not communicate its message. I strongly suggest that these results should be presented in a phylogenetic context. Additionally, the symbology used is difficult to follow. Maybe using bars, stars, triangles instead of just bars would maximize the message of the figure.


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

Input:
Title: THE DOPAMINE RECEPTOR D5 GENE SHOWS SIGNS OF INDEPENDENT EROSION IN TOOTHED AND BALEEN WHALES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have made improvements to the paper following the reviewers' and editor's guidance.  Generally my main concerns were addressed.  I have put comments and small suggested edits using track changes on top of the authors' edits and will submit this.  Perhaps the authors could consider some of these suggested changes and adjustments.
Experimental design: Generally, the approach taken is valid.  I would have like to have seen a bit more, but the paper is improved in revision by adding some dN/dS analyses.
Validity of the findings: Generally the results seem valid.  dn/ds values should be reported for all categories.  As currently written, I am maybe not sure that three independent KOs within Cetacea is supported, so I suggest that the authors consider this topic a bit more.
Additional comments: The authors have removed the manuscript in revision and I think with just a little bit more tweaking it will be 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: MORPHOLOGICAL AND MOLECULAR BARCODE ANALYSIS OF THE MEDICINAL TREE MIMUSOPS CORIACEA (A.DC.) MIQ. COLLECTED IN ECUADOR
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is severely lacking in the Introduction (not all significant areas introduced), Results (combination of results and discussion) and Discussion (totally absent). Furthermore, numerous unforced errors are found throughout the paper. All these lead to the paper not conforming to expected standard practices for a submitted manuscript. Please refer to the accompanied edited manuscript for additional issues.
Experimental design: The presented experimental design is in accordance with standard practices. However, significant amounts of information is lacking. In addition some information needs clarification. Please refer to the accompanied edited manuscript for additional issues.
Validity of the findings: The findings is presented in accordance with standard practices. Please refer to the accompanied edited manuscript for additional issues.
Additional comments: Please refer to the accompanied edited manuscript for all issues.


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: MORPHOLOGICAL AND MOLECULAR BARCODE ANALYSIS OF THE MEDICINAL TREE MIMUSOPS CORIACEA (A.DC.) MIQ. COLLECTED IN ECUADOR
Review round: 1
Reviewer: 2

Output:
Basic reporting: •	The manuscript is, in general, well written. The use of English language is appropriate and clear.
•	The Introduction of the article is not totally coherent with the Abstract. It should be rewritten in order to make it more understandable for the audience. There is information in the Abstract that is missing in the Introduction, for example: Some M. coriacea medical properties and previous studies in M. elengi, thus as the molecular barcodes used in the study.
•	The Background could be improved referencing the authors whom studies correspond to general characteristics of Sapotaceae family (lines 69-71) and Mimusops genre (lines 71-77). On the other hand, the Abstract is clear about the importance of the study, but the Introduction must be more explicit about the state of the art of the topic.
•	The importance of molecular barcodes, and how it complements the morphological characteristics of the plants for their identification, could be included in the Introduction since it is the basis of the work (lines 83-86).
•	 At least in the manuscript, the figures are not in a desirable quality.
Experimental design: •	As it is redacted in the letter shared by the authors, the plant material was recollected with all the permissions needed and doesn’t affects the ecology or wildlife situation of the studied species.
•	The research question and the gap their answer pretend to fill is clearly defined; however, since most of the research is descriptive work, the hypothesis is not well stablished. This point could be improved emphasizing the importance of the molecular barcode used in this study, together with the morphological tissues they measured in the species. Referencing another similar work in related species to M. coriacea.
•	Morphological values of plant tissues measured might be presented as Table (and the number of units measured), it would be clearer for the audience and easier to catch the results and characteristics of this species. The methodology to achieve this data seems suitable.
•	PCR methodology is well described.
•	Bootstrap value of the phylogenetic analysis is not shown.
Validity of the findings: •	Morphology measures and PCR information should indicate number of biological and technical replicates.
•	Poor quality in Figures 1 to 6 does not provide visually information to confirm the results indicated by the authors.
•	Molecular weight marker in gel electrophoresis does not show the ladder values (Figure 7).
•	Discussion section is poorly described. A series of suggestions aimed to improve it, are the following:
1.	The morphological values obtained from the tissues of M. coriacea should be included in the Discussion section, not in the results as it is written.
2.	Authors could contrast the findings in the study with others similar in the field (for example, molecular analysis for the genus or even the species family in order to explain the phylogenetic results) might be done in order to enrich the findings of this study.
3.	Complement the achieved results with the ecological context of M. coriacea in the studied region may be interesting to elucidate the natural history of this species, and the differences among its relatives in other regions.
4.	Molecular analysis may be the strongest results of the study, it is recommended to enrich this section and make it more relevant for the article.
5.	Improve the molecular barcode analysis speculating about other techniques or experiments (for example, recommending alternative molecular markers or comparison between sampling zones).
Additional comments: The knowledge generated by this study is an important base for other works involving common plants in traditional medicine, from a multi-disciplinary view including plant physiology, molecular biology, pharmacology, and even ecology. As a particular opinion, this study has potential for being published, if the authors attend the observations written above. In general, the methodology used in the work is really well applied; however, the manuscript (from the Abstract to the Discussion) is an important area to be improved and to show the real relevance of the 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: MORPHOLOGICAL AND MOLECULAR BARCODE ANALYSIS OF THE MEDICINAL TREE MIMUSOPS CORIACEA (A.DC.) MIQ. COLLECTED IN ECUADOR
Review round: 2
Reviewer: 1

Output:
Basic reporting: Minor grammar corrections needed – refer to accompanied annotated manuscript.
References - Format not consistently applied
Experimental design: Acceptable
Minor grammar corrections needed – refer to accompanied annotated manuscript.
Validity of the findings: Acceptable
Additional comments: Abstract
1.	Minor grammar corrections needed – refer to accompanied annotated manuscript.
2.	“… and diﬀerent genus were grouped in one clade of the subfamily Sapotoideae” – unclear statement – rephrase sentence
Introduction
3.	Are all of the 1st paragraph from just one source, namely Sánchez (2011)?
4.	Minor grammar corrections needed – refer to accompanied annotated manuscript.
5.	Focus morphological descriptions only on leaves, stem and seeds –as indicated in the Abstract
6.	In Ecuador there is ? species.
7.	“Although Mimusops coriacea  (ADC – in abstract it is indicated as A.D.C.) Miq,…”
8.	In Ecuador it has a restricted distribution along the coastal regions of ….?
9.	“This species, as well as others of the genus, is used for different various medicinal purposes…” - This paper focus on LEAVES, STEM and SEEDS (see abstract) – thus ethomedicine should also just focus on these 3 plant structures.
10.	Additional issues indicated in the annotated manuscript.

Materials and Methods
11.	Acceptable
12.	Minor grammar corrections needed – refer to accompanied annotated manuscript.

Results
13.	Minor grammar corrections needed – refer to accompanied annotated manuscript.

Discussion
14.	“The morphological characteristics of the leaves correspond to that reported by Miranda and Cuéllar (2000) and Gami et al. (2012)” - for which species?
15.	Minor grammar corrections needed – refer to accompanied annotated manuscript.
16.	“The venation is a closed type, which corresponds to a reticular system (the veins branch and anastomose with each other forming a network that facilitates the diffusion of liquids); which is very common in the dicotyledons. In this case, of the   penninervia type, the vascular system is one of the most advanced systems that ensures nutrition to all parts of the leaf (Gami et al., 2012).” – remove - low quality information not suited to high quality international paper.
17.	Leaves:	The microscopic analysis of the powder drug showed different fibers and vascular
bundles, in this case belonging to the xylematic tissue, classified as scalariform. RESULTS!! Remove.

References
18.	Format not consistently applied

Figures
19.	Figure 3 – most photos out-of-focus (??)
20.	Figure 4 - Cu: cuticle, Ep: epidermis, COC: calcium oxalate crystals – no arrows to indicate location of these structures – see also others.


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: EXOGENOUS MELATONIN CONFERS DROUGHT STRESS BY PROMOTING PLANT GROWTH, PHOTOSYNTHETIC CAPACITY AND ANTIOXIDANT DEFENSE SYSTEM OF MAIZE SEEDLINGS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: No comment
Additional comments: This manuscript described the effect of melatonin in response to drought stress in maize seedlings. Two different methods including foliar and soil drench ways were used to precisely monitor the effects of varying concentration of melatonin against drought. Many morphological and biochemical parameters were thoroughly investigated to confirm the positive roles of melatonin. The experiments were well organized and provided solid evidences concerning the melatonin’s physiological roles when the maize seedlings were challenged with drought stress. Authors are missing a couple of recent articles and several typos to be corrected as follows.
1. line 27: change Foliar Application into Foliar application.
2. line 37: lowers to lower
3. line 66: Authors would be better to add the most recent review article (Reiter RJ, Tan DX, Sharma R. 2018. Historical perspective and evaluation of the mechanisms by which melatonin mediates seasonal reproduction in mammals. Melatonin Research 1:59-77. DOI: 10.32794/mr11250004)
4. line 72-73: Authors did not cited the most recent achievement of melatonin’s novel role against ER stress in plants (Lee HY. Back K. 2019. Melatonin plays a pivotal role in conferring tolerance against endoplasmic reticulum stress via mitogen-activated protein kinase and bZIP60 in Arabidopsis. Melatonin Research 1:94-108. DOI: 10.32794/mr11250006)
5.  line 79: Add a recent interesting article (Arnao MB, Hernandez-Ruiz J. 2019. Melatonin: a new plant hormone and/or a plant master regulator? Trends in Plant Science 24:38-48. DOI: 10.1016/j.tplants.2018.10010)
6. line 84: A few studies instead of A few study
7. line 88: In addition
8. line 89: melatonin’s into melatonin
9. line 90: Authors need to be add the most recent review concerning the antioxidant activity of melatonin (Reina M, Castaneda-Arriaga R, Perez-Gonzalez A, Guzman-Lopez EG, Tan DX, Reiter RJ, Galano A. 2018. A computer-assisted systemic search for melatonin derivatives with high potential as antioxidants. Melatonin Research 1:27-58. DOI: 10.32794/mr1125003)
10. line 134: whereas
11. line 145: 10 ml into 10 mL. Check out throughout the manuscript.
12. line 157: 1 mmol into 1 mM
13. line 163: Add space between number and unit. 13mM into 13 mM; 750mM; 4um into 4 uM; 0.1mM.
14. line 167: 50mM into 50 mM
15. line 172: 0.5 mMAsA into 0.5 mM AsA
16. line 175: 12.5mM into 12.5 mM
17. line 180: 15,000 g into 15,000 x g
18. line 183: 10,000 rpm into 10,000 x g
19. lines 190, 194: ul into uL
20. line 199: process into processed
21. line 221: water stress into drought stress
22. line 224: fresh into Fresh
23. line 233: In into in
24. lines 253, 255: delete “there was”
25. line 261: DM2. Which into DM2 which
26. line 301: Remove a phrase “With increasing…enzymes.
27. lines 319, 328: evidentially into evidently
28. line 377: endogenous promoters into endogenous growth factors
29. line 396: maintained into maintain
30. line 403: improve into improved
31. line 430, 431, 433: Remove parentheses
32. line 487: Proline into proline
33. lines 500, 509, 528, 554, 567, 653: Italic forms of species.
34. line 522, 531, 536, 541, 544 560, 589, 610: Change capital letters into lower case.
35. line 545: Remove 1 after Rice
36. Fig 7 legend: Ascorbate into ascorbate


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: EXOGENOUS MELATONIN CONFERS DROUGHT STRESS BY PROMOTING PLANT GROWTH, PHOTOSYNTHETIC CAPACITY AND ANTIOXIDANT DEFENSE SYSTEM OF MAIZE SEEDLINGS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment.
Experimental design: no comment.
Validity of the findings: no comment.
Additional comments: It is well-written and easy to follow. Data are properly presented and interpreted. But there is no special innovation in this paper.


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: EXOGENOUS MELATONIN CONFERS DROUGHT STRESS BY PROMOTING PLANT GROWTH, PHOTOSYNTHETIC CAPACITY AND ANTIOXIDANT DEFENSE SYSTEM OF MAIZE SEEDLINGS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The author presented a study on the impact of varied concentrations of melatonin on the physiological and morphology responses of maize seedling to drought stress. Many researches on drought stress mitigation by exogenous application of melatonin have been reported. Here, the authors targeted their study by exogenous application of melatonin in leaves and roots of maize, showing that its alleviated drought stress effect by depressing ROS burst, increasing soluble protein and proline content, activating antioxidant enzymes activity, which is an interesting topic. The results are clearly presented. But this article needs major revision, especially the abstract and discussion. After the required revisions, I will recommend publishing.
Experimental design: the authors targeted their study by exogenous application of melatonin in leaves and roots of maize, showing that its alleviated drought stress effect by depressing ROS burst, increasing soluble protein and proline content, activating antioxidant enzymes activity, which is an interesting topic.
Validity of the findings: The results are clearly presented. But this article needs major revision, especially the abstract and discussion.
1) this manuscript showed soil drench and seed drench, but in my opinion, soil drench or seed drench (L 213) is different, which one did the authors use. 2) Please carefully follow the format required by PeerJ. Such as in line 239, after DCK2 add a comma. 3) Please show the pictures of root length phenotype, it will make your results more convincing. 4) The writing should be improved and the format should be recheched.
Additional comments: Abstract: in this experiment, foliar and soil applications of melatonin were performed, but in this section, the author only showed the results of foliar application of melatonin. Why?
L18-19 Please rephrase this sentence, it may make reader confusing.
L19 “check” should be replaced by “investigate”
Introduction:
L33 Please rephrases “Drought is an important to stress” this sentence, it may make readers confusing.
L36 delete “pigments”
L38-39 Please rephrases “One of the common consequences of” this sentence
L44 Please rephrases “an increase in the ROS levels could be toxic” this sentence
L66-69 After this sentence, please added references
Material and methods:
Please add the formulas for calculating the antioxidant enzyme activity, the contents of soluble protein and proline, and the concentration of MDA and H2O2.
Results:
In this section, the results were clearly presented, however, there are some errors needed to be revised.
L205-206 Please added references.
L210 delete “in the”
L233 change “In” to “in”
L258-259 Please rephrase this sentence, it may be make readers confusion.
L267 delete “pigments”
L268 Please rephrase “In melatonin foliar application”
L271-273 please rephrase this sentence, it may be make readers confusion.
L273 “Chla” changes to “Chl a”
L274 “Chla” changes to “Chl a”
L276 “Chla” changes to “Chl a”
L283 “(Fig. 1-3)” changes to “(Fig 1-3)”
L290 “expansion” should be replaced by another word.
L332 “MD1, MD2, MD3, and MD4” changes to “’DM1, DM2, DM3, and DM4”.

Discussion:
L356 Please rephrases this sentence
L359-360 This sentence may be replaced by “During our study, the protective role of melatonin was investigated in maize seedling under drought stress.”
L375-376 Please added “Table 1”
L376-378 Please added references
L388-389 Please rephrases this sentence and added references.
L400 “amount” should be replaced by “content”
L400 delete “content”
L401 “These results indicated that melatonin treatment” should be replaced by “These results indicated that high concentrating of melatonin treatment in leaves”
L426 “transportation” changes to “transpiration”
L435-437 After this sentence, please added “(Fig 4, Fig 5, Fig 6, Fig 7)”

Conclusion:
L481-482 Please rephrase this sentence, it may be make readers confusion.
L487 “Proline” changes to “proline”

Table 1:
The unit of root length is not correct, please revision.

Supplemental:
The unit should be indicated after each measurement indicators and the font need to be Times New Roman.


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: CORRELATION BETWEEN SUBSETS OF TUMOR-INFILTRATING IMMUNE CELLS AND RISK STRATIFICATION IN PATIENTS WITH CERVICAL CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript is very well-written. The main points are clearly stated. Sufficient background is provided with citation of appropriate literature. The manuscript is well structured and the figure and tables are presented professionally. The article is concise and self-contained with a hypothesis and relevant discussion.
Experimental design: The manuscript describes primary research on a small cohort of formalin-fixed paraffin-embedded cervical cancer samples. The work relies on pathologists’ assessment of staining of tissues sections with various markers of infiltrating immune cells, and on interpretation related to a good range of clinical data. The research question is well defined and meets a gap in knowledge. However, data are related to “risk stratification” in cervical cancer patients but I could not find any definition of what the authors consider low, intermediate, or high risk. Is this metastasis, recurrence, death before 5 years, or something else? This should be stated in the manuscript.

Generally, methods are well described but there is no description, or reference to, the methods/tests used to originally determine variables such as HPV status (see below) or physiological data such as renal and liver function.
Validity of the findings: The article is valid. The only negative for the study is that the cohort is small, but the authors acknowledge this, and the p-values are significant.

The HPV typing data is striking. It is unusual to find 16% of cervical cancer patients are HPV-negative (expected value ~<5%). This is why it is important for the authors to state the method used to determine HPV status.

The authors state that they see membrane staining for the majority of their immune markers in the IHC analysis (Figure 1). However, although they might expect to find membrane CD8, it looks to be cytoplasmic in the picture shown (Figure 1 E). It is very hard to be certain of the cellular position of staining with these markers at the magnification shown. It might be best to simply state that positive staining was observed.
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: CORRELATION BETWEEN SUBSETS OF TUMOR-INFILTRATING IMMUNE CELLS AND RISK STRATIFICATION IN PATIENTS WITH CERVICAL CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is well written and in plain clear English. It is easy to read and understand.
References sufficient. Data is well organised.
No typographical errors were detected and
Experimental design: Experimental design is well suited for the research question. methods described in sufficient detail. Two further clarifications in methods will improve the manuscript .
1. Lines 124-126 – Patients are classified based on their risk based on prognosis. It will be useful here to have a bit more detail on how the patients were classified.
2. HPV test was used- details of what test was used can be added.
Validity of the findings: findings are clearly shown and look valid based on the data provided.
Additional comments: This is a simple and clear paper that shows the assessment of TILs cervical cancer tissue. I would like to recommend that the paper is accepted for publication.


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

Input:
Title: EFFECTS OF RESEARCH DISTURBANCE ON NEST SURVIVAL IN A MIXED COLONY OF WATERBIRDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is logically and clearly written and provides sufficient background information to be informative. The study covers a clear and important question.
Experimental design: The experimental design is clearly detailed.

There are two areas where I would appreciate further detail. It was unclear to me what was meant by the 'ringing' variable on line 133. Please add a more detailed description.

Based on the text it was unclear to me how the 72 candidate models were developed, by digging into the code I see that the dredge() function was used, a function that does automated model selection. This should be detailed in the methods, along with an explanation of why this method was chosen, instead of creating a smaller set of candidate models a priori.
Validity of the findings: The findings are solid, and are an important investigation of a large assumption that is made frequently in field ornithology. I think the communication of those results could be improved with additional figures/tables, but I acknowledge that is personal preference to some degree. I personally find paragraphs of text with parenthetical reporting of parameters to be more difficult to synthesize.
Additional comments: The term waterbirds is used in this manuscript, which is a term used by different folks to mean different groups of species. Please define what you mean by waterbirds at its first use.

I reviewed the code, a few comments, overall its good.

Remove the setwd() commands from the top of the code, those are specific to your machine, and will not work on anyone else's

Please move all the library() commands to the top of the script, so that the user can ensure they have all the necessary packages installed/loaded at the beginning

There are several places throughout the script where comments are made (which is good), but they are not in English. My understanding is that PeerJ requires them all to be in English. Please carefully review the code and update those 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 RESEARCH DISTURBANCE ON NEST SURVIVAL IN A MIXED COLONY OF WATERBIRDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language should be improved to increase the article's clarity. The current phrasing of certain sentences make comprehension difficult. For example, the sentences starting on lines 39, 57, 60, and 62 could be improved. I suggest following IOU guidelines whenever using common names, capitalize ‘Ibis’ and ‘Egret’ https://www.worldbirdnames.org/bow/pelicans/

A detailed field background supported with strong references is provided.

The article adheres to the professional standard of current scientific articles. The raw data is present and well organized, but species names and heights are not able to be interpreted in the raw data (codes are used without a key). Figure 1 was difficult to interpret. The clarity of this figure could be improved by including a description of what the black circles represent. I was also confused as to how the number of nests that were abandoned appears to decrease over the nesting season. Can an abandoned nest change it's fate? If so, how? Some of the annotations in the R code are in French only.

The hypothesis is clearly outlined, tested, and discussed using the data collected.
Experimental design: The research question is well defined, relevant and meaningful. A knowledge gap, does disturbance resulting from research impact the daily survival of colony nesting birds, is identified and addressed in this article.

The investigation is rigorous and followed a large number of nests throughout two nesting seasons, following a high technical and ethical standard.

The methods are lacking a detailed description of the floating blinds used to monitor nests. The article did not make it clear if the floating blinds were mobile or stationary. The citation provided (Shugart et al 1981) in regards to the floating blind pertains to tunnels used to enter blinds. The construction, size, and mobility of the blinds used in the article are unknown. Shugart et al 1981 notes that the methods used resulted in altering the focal species behavior. The article in review does not address this.

The statistical methods used are not fully described in the article. A Tukey's post hoc test was used in the R code, but not outlined in the statistical analysis section. The R packages used in the statistical analysis are not presented or cited in the article.  Program R can be cited using the code "citation()", while packages within R can be cited using the code "citation(package = "PACKAGENAME")" for example "citation(package = "lme4")".
Validity of the findings: Resulted are accurately presented.

All underlying data have been provided, they are robust, statistically sound, and controlled. I suggest an adjustment to the statistical analysis that is outlined in general comments.

Conclusions are linked to original research question. The authors thoroughly describe the ringing methods that cause the disturbance in the methods section, but I would suggest that this is reiterated in the conclusion. The ringing methods used focused on minimizing disturbance, which resulted in a total of 2 research related disturbance events totaling 2 hours (or less) over the entire nesting period. The authors second model (presented in figure 2) found that, while not statistically significant, there was a signal that DSR decreased in the disturbed area. Other studies may not implement such a well managed, and limited amount of disturbance, which could result in a larger impact to DSR. While this is speculation, it highlights the fact that disturbance relatively minor in this study and it was tested as a binomial factor.
Additional comments: The overall structure of the paper is great. The paper flows logically, ideas are presented and supported with literature. The article does a good job of outlining knowledge gaps and presents how it aims to address them. Improving the language will substantially increase a reader's ability to interpret the article. The statistical analysis is good, but it could benefit from some additional explanation. The article would benefit greatly from an improved description of the floating blind.

It is interesting that the top five models pulled nest height as an important factor in DSR. A quick investigation into nest height by species showed that species 'AG' is responsible for a majority of the nests at height 'a', but this species rarely nests at height 'c'. Similarly, species 'GB'  is responsible for the majority of nests at height 'c'. I would suggest adding 'height * species' as an additional interaction effect into the model. Including this interaction would allow for the effect of height and species on DSR to be better understood.

Is it possible that ringing events and/or nest observations from the floating blind disproportionately effected nests nearest the water?


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

Input:
Title: A NEW FROG OF THE LEPTODACTYLUS FUSCUS SPECIES GROUP (ANURA: LEPTODACTYLIDAE), ENDEMIC FROM THE SOUTH AMERICAN GRAN CHACO
Review round: 1
Reviewer: 1

Output:
Basic reporting: Abstract:
The abstract could be revised to reflect some additional points that is already present in the body of the text (results and discussion).

1.	For e.g. line 26. Advertisement calls can be mentioned in the list of criteria examined. Behavior is mentioned in the introduction (Line 83). Consider replacing multiple character system with one of the established terminology.
2.	Also, the results pertaining to the phylogenetic position of L. mystacinus, recovered as sister in their analysis – one of the crucial findings – could appear in the abstract.
3.	Finally, if authors prefer, they could insert a statement of comparison between the new lineage and its extant sister.
4.	Line 31-32: consider paraphrasing “males call on tree branches.” Also check whether it is a unique habit (according to results it also calls on logs)
5.	The main cephalic index character that separates this new lineage from its sister could come in the abstract.

Introduction:

Paragraph 1:
Line 36: consider removing of before Country names.
Line 40 – 41, contains some repetition on the geographical coverage that has already been mentioned in the first sentence (Line 35 – 39).
Line 68: Since you start the introduction with the definition of Gran Chaco region, mention the species range within this region might suffice.

Results:

Line 213: Since you are dealing with individual lineages here, consider using the term “lineages” instead of “clades”.
Line 219 – 221: Exact p-distance with the focal species of comparison (L. mystacinus) might help.
Line 247: “lineages or species” instead of “clades” might be appropriate here.
Line 409: In the figure 9, the individual in 1st row* 3rd column, doesn’t possess reddish brown dorsum. Similarly, in figure 10, the individual in 3rd row*2nd column shows reddish brown dorsum. This suggests coloration might be an overlapping character for the two lineages.
Line 411 – 414: It could be helpful, if the skeletal parts mentioned are named in the Fig 6 and 7. For example, the lateral edges of the middle ramus of prevomers can be labelled in the figures.
Line 445: comma usage
Line 447: “teeth present” instead of  “teeth presents”
Line 449. To maintain consistency consider including the ratio in closed brackets.

Discussion
Line 603: It is unclear what they mean by terminals (1-2 and 4-7).
Line 620: they consider it as a cryptic lineage. Considering the moderate levels of divergence and potential geographical isolation, this pattern observed could be expected. Authors could consider expanding on this.
Line 627: The general shape alluded here as an unequivocal character needs to be mentioned in the result.

Reference:
695: 2001 (bold)
Line 1066: check Sabaj 2019 -> Sabaj 2016
Line 1048: specie -> species
Line 719: Baker MR, Vaucher C. 1984. (check Line 265: it says 1986)
Line 267: de la Riva (2000) -> de la Riva et al. (2000)
Line 842: Gallardo JM, de Olmedo EV. 1991.
 check Line 269:Gallardo & Varela de Olmedo (1992)

Line 968 and Line 970: one of the citations the year could be different. See line 273-274
Line 976 – 980: a & b missing against year 2000.
Line 1046: Pinto-Viveros et al. 2017 (check line 276: Pinto-Viveros et al. 2015)
Line 973: Lavilla and Manzano 1993 (check line 280: Lavilla and Manzano 1995)
Line 881: Heyer et al. 2005 (check line 389: Heyer et al. 2006)
Line 399: Heyer et al. 2006 (as above)
Line 402: Heyer 1996 missing
Line 729: Blotto et al. 2013 (Check line 588 Blotto et al. 2012)
Line 1054: year 2012 (check line 639 – 640 year 2010)
Line 738: year 1956 (check like 650 – year 1968)
Line 651: de Carvalho & Giaretta (2013) missing
Line 813: missing in the text
Line 1090: Swofford 2000 (check line 140, Swofford 2002)

Tables and Figures
Table 3: caption: consider using “individuals” instead of “specimens”
Fig 1: It is unclear what the colors on the branches refer to. Also, please provide nodal support values.
Figure 11: Most of the ecoregions plotted on the map might not be relevant for the paper. Highlighting the Gran Chaco (dry vs humid) might help improve the readability of the map. Also, a few reference localities could help in understanding the text.
Experimental design: Materials and methods:
Line 91 -92: It is unclear why two different sets of primers were used for both 12S and 16S.
Line 107 -108 – was it a single sequence (KM091585) or, multiple sequences? Consider revising the line accordingly.
Line 112: These appear like part of the ingroup. Please check.
Line 121 – 122: Here its mentioned as Bayesian approach while in the introduction (line 84) it appears as Bayesian criteria. Maintain consistency.
Line 128: use an alternative word: “studied”
Line 149-150: it is unclear: “currently deposited in the MACN collection”
Line 178: Please check the model number of directional microphone: Sennheiser LR 6?
Validity of the findings: Line 204 – 205. According to the Fig1a, there is no nodal support for this statement. The information provided in the main figure is insufficient to evaluate the inferences provided by the authors. Since, the focal new species along with its sister form a well-supported clade, the above issue is unlikely to affect the overall outcomes of the study. Nevertheless, the authors should update Fig 1. I could see that their Bayesian tree in the appendix shows appropriate support values.
Additional comments: First, I thank for the opportunity to review this manuscript. “A new frog from the Leptodactylus fuscus species group (Anura: Leptodactylidae), endemic from the South American Gran Chaco” by Schneider et al.  The research is based on an extensive dataset (>780 individuals for morphological comparison) and multiple-criteria including phylogenetic analysis, external morphology, osteology and, advertisement calls, to discover, describe, and diagnose a new lineage of frog in the Gran Chaco region of Argentina. I congratulate the authors for this work.


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

Input:
Title: A NEW FROG OF THE LEPTODACTYLUS FUSCUS SPECIES GROUP (ANURA: LEPTODACTYLIDAE), ENDEMIC FROM THE SOUTH AMERICAN GRAN CHACO
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper is very clearly written, detailed, and adheres to the journal’s format.
Experimental design: This is a taxonomic paper. They hypothesize the presence of a new species (Leptodactylus apepyta sp. nov.) within the L. fuscus group and they support their hypothesis with several different lines of data. They use well established molecular and morphological methods to confirm the existence of this new species and bolster their findings with additional data like call and behavioral data. Overall the paper is extremely thorough and uses high sampling across the species group distribution in order to establish the full variation and distribution of L. apepyta sp. nov.
Validity of the findings: The authors were very thorough in evaluating each line of evidence in order to establish this new species. In addition they follow ICZN rules to insure that the species name will be valid according to the code.
Additional comments: This is a solid species description. I think it would be helpful to denote on the map which samples were sequenced for this description. Are these samples well distributed across both species ranges? You provide locality data for L. apepyta sp. nov. in Appendix 1 and you should provide similar detail for all of the sister taxon, L. mystaceus.

Minor Notes
Line 126, PartitionFinder is one word.
Line 481: Variation. Discuss variation of dorsolateral folds (1 vs 2) within L. apepytat. Is it random? Do most of 1 fold or 2? This features seems important for distinguishing species so it may be good to discuss this.
Line 435, remove period following (Fig. 8C,D)
Line 549: Natural History notes. Please describe the habitat in a bit more detail. Are the males calling over water? If so is this running water like a stream? Or are these ponds? Are the permanent bodies of water or ephemeral?


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: REALISTIC SCENARIOS OF MISSING TAXA IN PHYLOGENETIC COMPARATIVE METHODS AND THEIR EFFECTS ON MODEL SELECTION AND PARAMETER ESTIMATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: there are many mistakes in references.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: there are many mistakes in references.


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: REALISTIC SCENARIOS OF MISSING TAXA IN PHYLOGENETIC COMPARATIVE METHODS AND THEIR EFFECTS ON MODEL SELECTION AND PARAMETER ESTIMATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: # "Clear and unambiguous, professional English used throughout."
Yes. The article is very well written.

# "Literature references, sufficient field background/context provided."
Partly.
- The article is well referenced, and sufficient context is given in the introduction on comparative methods.
- This is a simulation study, but no details are provided regarding the specific simulation tools and methods used. For instance, from the provided R script, I can see that packages "ape" and "castor" are used, respectively, for tree generation and MK model evolution, but they are not cited.
- No comparison is made with previous simulation studies on similar topics. For instance, Cooper et al. 2016 could provide a comparison point for the base case with no missing values.
- Nothing is said about multivariate comparative methods. It might be good to specify at the beginning that this study is limited to univariate methods, as this confused me a little bit at first.
- No reference is made to the classical work of Rubin 1976 (see also Little and Rubin 2002) on inference with missing data. This could be useful, as the "phylogenetically clumped" scenario could actually be MAR (Missing At Random, it does not depends on the trait value), which might explain why it has little impact on the inference. On the other hand, the "correlated" scenario seems NMAR (Not MAR, depends on the trait value), and is typically a case where ignoring the source of missing values might impair the inference, as shown in the analysis.

# "Professional article structure, figures, tables. Raw data shared."
Partly.
- The article is well structured, and easy to follow.
- The scripts to produce the simulation are provided (but not the generated data actually used for the figures).
- Table 1 provides a nice summary for the various conditions.
- This is maybe a matter of personal taste, but could table 2 be transformed into a plot, at least in the main text ? Then the trends might appear more clearly.
- Same remark for table 3.
- Figure 1 is informative. A few comments:
    * Please precise in the legend the percentage of missing values.
    * Would putting an actual example of the simulation (with 300 species) really impair the illustrative power of the figure ?
    * Also, Fig.2C-D might be misleading, as it seems that the "blue" regime does not really represent "no fewer that 25% [...] after dropping the tips representing missing taxa." (l.190-191). From this figure, I have the impression that almost no tips from the "blue" regime are left, which might explain the high bias on theta_1.
- I find Figure 2 not really informative:
    * Maybe add facets for each of the scenarios, so that the reader knows what to compare. You could even repeat the base scenario with no missing data to give a comparison in each case.
    * No information is provided on the legend on the two numbers of each plot.
    * Instead of putting numbers, maybe you could draw points, with lines linking them together to show the trend better ?
    * In order to compare the different parameters better, it might be good to *normalise* the bias scores. This is an important remark in my opinion, because theta is around 10, while sigma^2 is around 0.5, so the raw bias on the small parameter might appear smaller, but the scaled one is actually similar, if not larger. This could change the interpretation of the figure given in the text.
    * This could be a matter of taste, but maybe violin plots would be easier to read than the dotted empirical distribution.
    * Maybe rank the rows according to the model, not the parameters (as they do not mean the same thing in different models anyway). Then maybe show a visual separation between each model's block of parameters.

# "Self-contained with relevant results to hypotheses."
Yes. The article is well focussed, it clearly states the hypothesis, conduct the analyses, and then draw conclusions on these.
Experimental design: # "Original primary research within Aims and Scope of the journal."
This is an exploratory simulation study. I don't feel competent to asses whether this is within the "Amis and Scope" of the journal, and leave that to the Editor's judgement.

# "Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap."
Yes. The research question is well identified, and relevant to the field.

# "Rigorous investigation performed to a high technical & ethical standard."
Partly.
    * Simulation scenarios:
- The simulation scenarios are well described, easy to follow, and seems rigourous.
- I really appreciate that the selection strength is given as a phylogenetic half-life, as this is very informative (and not always well specified in other studies).
- Tree generation: as shown in the scripts, the trees are generated with a birth-death process, and then rescaled to unit high, which is different from simulating them directly conditionally on their age (see e.g. function `sim.bd.ntaxa.age` in TreeSim, Stadler 2011). This could have an impact on the tree shape, and hence on the result, so it might be important to mention for comparison / reproducibility reasons.
- Regimes generation: please justify the use of a MK model here. Could the fact that the minor regime has a varying percentage of tips in different analyses bias the results ? In particular for the "correlated" scenario, since tips with high values are typically in the minor regime. See previous remark on Fig.2.
- Latent trait L: maybe give the variance of the BM used.
- corMT scenario: maybe give more precisions on the way the probabilities are chosen. How do they vary with the trait exactly ? Does the fact that the mean probability is the desired proportion of missing data ensure that you actually get the right number ?
- Minor note: notation "T" is usually used for a "Tree", so this confused me a little. But that might be a matter of personal taste.
    * Scores used:
- AICc score: please give a citation for the score. Also, it might be good to give a comparison point to what is a "low" delta AICc.
- Bias: as mentioned previously, I think it would be more informative to normalise the bias with the true parameter value, unless there is a good reason not to.
- Precision: maybe give the formula, or more details ? I could not see whether this one was normalised or not.
    * Results
- 30% error rate in the BM case seems quite high at first sight. If I'm not mistaken, Cooper et al. 2016 find in similar scenarios (with no missing data, and no measurement error) an error rate that is not above 10% (see their table 2).
- AICc scores: why is less than 2 good ? See remark above. Maybe a citation would help.
- bias: see remark above about the normalisation. E.g., for the OUM, the bias on theta_1, as stated in the text, goes from -0.254 to -0.454. For the same scenarios, the bias for sigma_0 goes from 0.025 to 0.036. But the true parameters are, respectively, 11 and 0.5, so the normalised bias are -0.023 and -0.041 (theta_1) and 0.05 and 0.072 (sigma_0). So the relative bias on theta_1 is multiplied by 1.8, which is about 0.02 units, and the bias on sigma_0 is multiplied by 1.4, which is about 0.02 units too. One could argue then that the two parameters do not behave that differently, and that sigma_0 in not much more robust to bias. But maybe I am missing something, or misunderstood a point ?
- For theta_1, could the high bias be explained by the fact that this regime has fewer tips, and that they are more affected by the correlated missing values, since they are higher ? See previous remark about Fig.2.
- Results on precision are not interpreted.

# "Methods described with sufficient detail & information to replicate."
Partly.
- Methods are described in good details, and the procedure is very clear.
- See section above for some minor caveats on the scenarios.
- Functions used for simulation are provided in two R scripts. However, the master script to call these function and produce the output is not given.
Validity of the findings: # "All underlying data have been provided; they are robust, statistically sound, & controlled."
No actual data. But see above: functions are provided, but not actual scripts, nor the data generated and used in the article.

# "Conclusions are well stated, linked to original research question & limited to supporting results."
Yes. The discussion and conclusion are well suited to the analysis, draw appropriate interpretations. Some comments:
- l.279-290: Maybe a reference to Rubin 1976, as previously mentioned, could help understand this point.
- l.291-297: See previous remarks on normalised bias for sigma^2 and alpha, that might change the interpretation here.
- l.298-314: I'm not sure I understand fully the point about BDPR. Is the message that it's better to use a tree with missing data, than one with inferred data ?
- l.259-363: I don't understand the "multivariate" latent trait approach. Maybe I missed something, but could you explain a bit further what you would do, and why it would be better ?
- l.364-372: The scenario you describe, although not realistic, would be really easy to simulate. Maybe it could be worth to include it in the analysis ? Also, the interpretation of the correlated scenario is highly dependent on the way the probabilities are generated (see remark above). Maybe you could include several functions to do that, giving a more or less high "missing probability gap" between the low and high values ?
Additional comments: Overall this is a well written paper, that is well focussed on a specific research question, and try to address it through simulations. If well conducted, those analyses could be of good use in the comparative biology field. However, in addition to the technical points raised above (that could change the interpretation of the results), I think this paper mostly lacks references, both on the statistical side (MAR/NMAR sampling scheme) and on the simulation side (comparison with previous simulation schemes).


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: REALISTIC SCENARIOS OF MISSING TAXA IN PHYLOGENETIC COMPARATIVE METHODS AND THEIR EFFECTS ON MODEL SELECTION AND PARAMETER ESTIMATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: Excellent writing. Good reference to literature but there are a couple additional citations that should be included (especially Ho and Ane 2014, see below).
Experimental design: Good to go.
Validity of the findings: I have no problem with the validity of the findings.
Additional comments: I commend the author on pursuing an area of strong interest to biologists interested in phylogenetic comparative methods (PCM), and in evolution more generally. This manuscript was extremely clearly written, especially in the introduction. I thought the author did a great job of explaining the need for this kind of study in terms that should be digestible by biologists with a variety of areas of expertise. I was very interested in understanding the outcomes of the simulation studies.

As I found the simulations to be valuable, and to highlight some important areas of concern for PCM, my criticisms mostly pertain to interpretation (and presentation) of the results.

1)	The simulated data were generated under simple evolutionary models, in which the true model is known. The author has added a dose of realism with respect to sampling (and has also mimicked recent extinction - see below). Thus the author has found that somewhat standard methods used to distinguish between evolutionary models are in a variety of cases not misled under more realistic sampling regimes, but that parameter estimation suffers exaggerated bias in some cases under these more realistic sampling regimes. As the data are still ideal data despite incomplete sampling, I interpret the main results as highlighted (that statistical inference regarding evolutionary processes tends not to be misled in model selection across these cases) as a demonstration that the statistical inference framework can clear a pretty low bar. I agree with the author that these results demonstrate that particular statistical inferences are robust to incomplete sampling for ideal data, where the model is known. However, as evolution does not in reality proceed according to these simple models (e.g. evolutionary processes are likely far more heterogeneous), as we don’t know the generating model, and as empirical data are not ideal (e.g. we have measurement errors), as a biologist interested in using PCM for empirical data I can only take so much confidence from these results. As the author points out in the discussion, we don’t always know the trait values of taxa that are missing from such analyses (especially true of extinct taxa), and thus we can’t know if, for example, we’re missing taxa that belong to a different OU regime.

Consequently, I think the most valuable results from the simulations are those showing where we could be misled in the inference process, even for ideal data. I wanted to know more about how we are misled. E.g., what explains the systematic downward bias in the estimation of theta1 in the OUM models? Is it related to the “intrinsic inference difficulties” in OU models raised by Ho and Ané 2014 Methods Eco Evo? Are the biases likely to be worse for higher theta values? Also, what explains the inference issues when BM is the model generating the data? Has this issue been noted before, e.g. in Beaulieu et al 2012? Which models receive better AICc scores when the generating model does not have the best AICc score? I wondered if you might include an additional table or figure showing which models were preferred by AICc when the generating model was something different.
The discussion touched only briefly on the results, and pivoted to discussions of other issues in the literature, including BDPR. I felt it would be more relevant to put your simulations into the context of previous work on inference difficulties for continuous trait evolution – the discussion regarding sample size was interesting.

2)	The manuscript and simulation study addresses studies of extant taxa in large
clades. Extinction looms large here. Sampling for all studies of extant taxa in large clades is incomplete because of it. But the consequences of extinction are first considered at the very end of the discussion. I’d suggest that you devote some space in the intro, discussion, or both to the topic of extinction, in part because you’re mimicking recent extinction by removing tips. There are extinction scenarios that are likely similar to your “clumped” and “correlated” missing taxa scenarios. For example, studies of extant birds only would severely underestimate the number of transitions to flightlessness, as compared to studies including recently extinct taxa (this is discrete obviously, but the bias would likely impact studies of continuous traits like body mass or size-corrected wing length). You might cite papers using e.g. the fossilized birth-death model in studying traits, and peruse some more of the literature for relevant papers.

Some minor issues:

Abstract:
I think you’ve understated your case here when you say that “often the tree and trait dataset contain missing taxa.” I suggest re-phrasing to say that these analyses have rarely, if ever, contained all taxa within a clade. This is most obvious when considering extinction, which occurs even in young clades and results in incomplete sampling.

Lines 111 – 112: I’d add in a phrase to indicate you’re saying these things are heritable at the lineage level, just to help people who don’t usually think about heritability at the macroevolutionary scale.
Line 167: Suggest rewording from “It is worthy to reiterate” with “I reiterate”
Line 232: sample-size corrected?

Lines 287 – 290: I think this interpretation relies on large trees and relatively homogeneous evolutionary processes (which are true for your simulations but won’t be for a great deal of empirical data), and that merits mentioning here. Having phylogenetically clumped missing taxa in a smaller tree or where processes are more heterogeneous could have consequences for model selection problems and parameter estimation.

Lines 307 – 309: Jetz et al 2012 published both genes-only and BDPR trees, which made me wonder whether Rabosky 2015 needed to prune the BDPR trees for that study?

Lines 320 – 322: I had trouble understanding the end of this sentence. Consider rephrasing for clarity.

Lines 362 – 363: You might examine whether it would be possible to do what you’re describing using the “phenotypic tango” model, Arnold and Houck 2016 Am Nat.

Lines 370 – 372: To me, this caveat about extinction is really important, to the point where the scenario is really not unrealistic at all.

Table 1: I’d re-phrase the description of L at the bottom of the table for clarity.

The end of the Table 3 legend appears to be truncated. (oh – it’s on the next page so there’s a floating Table 3 legend). The figure 2 legend in the manuscript I read was truncated.


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: REALISTIC SCENARIOS OF MISSING TAXA IN PHYLOGENETIC COMPARATIVE METHODS AND THEIR EFFECTS ON MODEL SELECTION AND PARAMETER ESTIMATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: See below.
Experimental design: See below.
Validity of the findings: See below.
Additional comments: # General Remarks:

I thank the author for his answers to my questions, and the editor for this opportunity to review the revised version of the manuscript. I feel that the manuscript is now stronger, and easier to read. However, there are still a number of points that, in my opinion, remain problematic, as summarised below (see more details in the line by line comments).

- Figure 3 is a major limitation of this article in my opinion. Not only is it very hard to read, but it might also be misleading. Since it is one of the main output of this simulation study, and central to the paper, I would strongly advise for a major revision of this figure before publication. This is a key point, because it drives all the interpretations that are made of the results (see various points below).

- Discrepancies between these results and previously published ones are stated but not explained. I find this lack of explanation troubling, as it could indicate, at best, a design difference that is worth noting, or, at worst, a flaw in one of the studies. In my opinion, finding the roots of this discrepancy is essential in this work.

- I found that the statistical notions about missing data were presented in a confusing way, and as such did not help the interpretation of the results. I would suggest either clarifying this, or completely remove it.

# Reproducibility and additional script on GitHub

On the version I consulted (dated June 12th on GitHub, and most recent when last consulted on July 10th), it is said:
"I freely admit that my scripts are poorly-annotated, clunky, innefficient and just plain ugly."
I understand this remark might have been made with a humorous goal, but is that really the standard we want to ensure reproducibility ? Reading this caveat, I refrained from inflicting a careful review of this material on myself.

Just browsing through the file, I found a few inconsistencies, e.g. one of the main functions ("missing_data_corr_function") is never called in what appear to be the master script (named "run_simulations.R"), while some un-provided files are sourced (e.g "remove_rows_with_all_nas.R" in "sumarize_results").

(Permanent link to the version used: https://github.com/rafmarcondes/Missing_taxa/tree/348c7b738974f767ab8609e50baf7374d4ecd1c6)

# Detailed comments

(Line numbers refer to the "peerj-37170-tracked_changes.docx" file.)

## l.94-104 and 386-388
- I think there might be a small confusion here on the concept of types of missing data. Data is missing completely at random (MCAR) if missingness does not depend on the value of the data, and is missing at random (MAR) if it only depends on observed components, and not on the components that are missing.
- For a rigorous introduction, see e.g. Section 1.3 (p.11) in Little, Rubin, Statistical Analysis with Missing Data, Second edition, 2002, John Wiley & Sons, Inc., Hoboken, New Jersey.
- Usually, if the data is MCAR or MAR, ignoring the sampling mechanism will yield unbiased estimates. However, if it is NMAR (non MAR), then ignoring this mechanism will induce some bias.
- I mentioned it because it seemed to fit nicely to interpret some of the results presented here. However, I did not write down everything, so this is just an intuition, that might be irrelevant. Using this terminology implies precise meaning, that needs to be carefully checked (in particular with respect to the status of the trimmed of complete tree as data). I would suggest that the author mention this literature only if he feels confident that this is indeed relevant.
- In the cited study (Ingram & Mahler 2013), isn't the data even MCAR ?

## Table 2
If space is an issue, I would suggest to remove or move to the supplementary, as the information is redondant with Fig. 2.

## Figure 1
This is just a suggestion: maybe delete (D), but colour the branch of each of the trees in (A-C) according to the regimes ? That way the impact of the regime switch on the trait value and missing data might be seen more clearly.

## Figure 2
In each bar plot, it might be helpful to highlight the colour corresponding to the true model (e.g. by shading or fading the other colours). That way the proportion of "right answers" would appear more clearly.

## Figure 3
* I agree that this figure is key to the paper. However, I disagree that the goal of the figure is to present "the rawest possible depiction of the results". The interested reader can always go look at the raw results (in the supplementaries), but the figure should help him or her understand the results. As such the figure is really hard to read, and could even be misleading (see below). Choices are made anyway in the way to plot and order the data, and I think that there is much room for improvement.
* I still think that adding facets for each scenario would help, precisely to enable a "top-to-bottom looking".
* The trend does not appear clearly, maybe a line would be helpful.
* Raw numbers in the figure are very hard to interpret. I still think a visual representation of them would be better. (Tools like box plots or violin plots precisely being typical ways of showing the dispersion of the estimates graphically.)
* Bias numbers are now normalised, but not the representation of the results. This discrepancy could be misleading. Taking the same example as previously, the normalised bias on theta_1 and sigma_0 increase by the same amount (0.02, for OUM corMT), but on figure J the drop looks much bigger that on figure D, because the raw values are represented.

## Regime size
Thank you for the extra figures and explanations.
However this was not exactly my question, sorry if I was unclear.
In the paper, the case is being made that the bias on theta_1 for OUM corMT is the one with the most significant change. Although this can be nuanced when using the normalised bias (see other remark), my question was, could this drop be also partly imputed to the mere drop of tip observed in this particular regime ? If I understood correctly, under the corMT scenario, missing values will tend to be concentrated on high values, which will tend to depopulate regime 1, and hence increase the bias on theta 1.
Would that interpretation make sense ?
If it does, I don't think it shows a flaw in the simulation design. However it could potentially be used to nuance the claims about the drop in performance for OUM in the corMT scenario.

## l.263-282
- I'm not sure I understand what is meant by "relative probability". Those numbers do not seem to sum to one.
- I'm still unclear about the sentence: "R native function sample.int, which rescaled it to have a mean equal to my desired proportion of missing taxa and used the rescaled vector to sample tips to be dropped from the tree". Which mean does this refer to ? For the help of the R sample function, I have: "The optional prob argument can be used to give a vector of weights for obtaining the elements of the vector being sampled. They need not sum to one, but they should be non-negative and not all zero. [...] If replace is false, these probabilities are applied sequentially, that is the probability of choosing the next item is proportional to the weights amongst the remaining items. The number of nonzero weights must be at least size in this case." From what I understand, the right proportion of missing data is obtained because the right number of sampled integers is passed to 'sample.int' (specifically, 'PMISSING*nrow(tipdata)' l.108 of file "correlated_MT.R"), not because of a rescaling of the mean. But maybe I misunderstood something ?
- In the rebuttal letter it is explained that "However, that is not true for the rMT (random MT) scenario, where the actual proportion of missing taxa is probabilistic." Maybe I missed it, but I could not find a mention of that in the manuscript. Why is the proportion probabilist in this case ? It might be worth explaining this in the description of the simulations.

## l.290
Please provide a citation for the definition of AICc.

## l.336
Burham & Anderson (2004) cannot be found in the references at the end.
Also, is it delta AIC, or delta AICc ?

## l.346
0.2 -> 0.02 ?
A lot of time is spent discussing small differences, but maybe it should be stressed that there is a factor 10 between the biais on alpha and all the other parameters.

## l.364-367
How is the drop for theta_1 from -0.0231 to -0.0413  worse than the one from -0.007 to -0.055 for sigma_1 BMS, or from -0.0032 to -0.0374 for sigma_0 BM ? I might be wrong, but I think Figure 3 is misleading on this, or not carrying the proper information (see remarks above).

## l.413
"I have no compelling interpretation to explain this discrepancy between mine and Cooper et al.’s (2015) studies."
I find this statement a bit worrisome. If there is no design difference (in the simulation, or in the inference) that one could point out to explain the difference, then couldn't one worry that one of the results is misleading ?
If this comparison is included in the paper, I think it is essential to provide at least an intuitive explanation of why they differ.

## l.521
"then L" -> "than L" ?


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: REALISTIC SCENARIOS OF MISSING TAXA IN PHYLOGENETIC COMPARATIVE METHODS AND THEIR EFFECTS ON MODEL SELECTION AND PARAMETER ESTIMATION
Review round: 3
Reviewer: 1

Output:
Basic reporting: See below.
Experimental design: See below.
Validity of the findings: See below.
Additional comments: # General Remarks

I thank the editor for this opportunity to review this manuscript, and the author for answering my questions. I think that the most problematic issues previously pointed out have been addressed, and that this manuscript could be suitable for publication.

However, and as a personal opinion, I should say that, despite the absence of compelling obvious flaws, I have a rather low confidence in the work presented here, and I think it could improve on the three main following points: 1- Proper formatting of the code; 2- Informative representation of the results; 3- Integration with the existing literature.

I explain these points in more details below. However, I would perfectly understand that they could be taken as opinions rather than actual limiting flaws. As illustrated by the author's recent witty tweets on my previous review (yes, twitter is a public place, and grumpy reviewers can also read what's on it), we might not share the same vision on what an informative graph should look like. I would hence recommend the editor to take an extra opinion from another researcher, and I leave the decision to his discretion. Whatever his decision should be, I will not review any further version of this manuscript.

1- Proper formatting of the code.

Thank you for trying to clean the code between the previous version and this submission. I appreciate your effort, and I think clear and reproducible code is essential in the modern publication process, especially for such a study relying essentially on simulations.

Browsing the code, I still found it somewhat hard to read. It might benefit from a more rigorous formatting, based on a commonly accepted style (see e.g. https://style.tidyverse.org/). Applying the "lintr" package (see reference on the webpage above), I for instance found 767 notes and warnings on the file "correlated_MT", which is only 320 lines long, making for more than two per line. I'm not saying this shows a fatal flaw in the code, but this does illustrate the fact that this code might be hard to read for a human, which makes it more error prone.

This could extend to the use of base R functions themselves, as illustrated by the previously somewhat confusing explanation of the sampling procedure (see also comment below).

2- Informative representation of the results.

I discussed this point at length in my two previous reviews. I still think that this paper lacks one figure presenting the results in a clear way, summarising the main important features so that the reader could get a quick and clear grasp of the main trends. I just think that tables and raw data points are hard to interpret, and some inaccuracies in the exposition of the results in the main text by the author in previous versions tend to demonstrate that I might not be completely wrong.

This would not prevent the author to give the full results in whichever form he likes. I would however suggest to put the easy-to-read summary in the main text, and the more detailed, raw-data information in the appendix. But, again, that might just be a matter of personal preference.

3- Integration with the existing literature.

I have also already discussed this point, and the apparent inconsistency with some of the results existing in the literature. The general reason for this discrepancy given by the author (AIC vs LRT) could be a valid point, but it is quite broad, and not backed up either by literature or data. It made my strong suspicion go away, but I'm still not entirely convinced (see detailed comments below).

# Detailed Comments

(Line numbers refer to the "peerj-37170-trackedchanges_31july2019.docx" file.)

## l.262
Why do you apply a different sampling method for rMT and corMT, leading to a non-fixed number of missing tips in the rMT case ? Instead of using "sample" on a binary outcome with a probability equal to the percentage of missing (l.132 of correlated_MT.R), it seems that one could just apply again "sample.int" with the correct size (as in l.127), but with all probability weights equal to one (or prob=NULL). But maybe I am misunderstanding something ?
I agree that averaged over 1000 simulations, this should not have a strong impact, but I'm wondering whether there is a reason for the use of a different method in the two cases.

## l.422
I agree that the discrepancy could be due to the use of a different score (AICc instead of likelihood ratio test). This would be easy to check. I would suggest including the likelihood ratio test for model selection as an extra analysis for the sake of comparison, at least in the supplementary. That would be reassuring, and would easily settle this discussion. Also, for the comparison between AIC and LRT, the following reference might be of interest:
Lewis, F., Butler, A., & Gilbert, L. (2011). A unified approach to model selection using the likelihood ratio test. Methods in Ecology and Evolution, 2(2), 155–162.

## Figure 3
It's fine if the author does not like violin plots, but I still think that at least adding a summary statistic (e.g. a boxplot) to the raw data could be useful, so that the reader could follow easily the main trends. I also still think that separating different scenarios by panel could help. But see my above general comment. Plotting the normalised estimates, instead of the raw ones, could also help grasping the trends better.

## Table 4 and 5
Replacing, or at least complementing, these tables by graphs could help the reader to see the main point. But, again, that might be a matter of personal preference.

# Typo

## l.256
Missing period after the formula, or extra capital letter.


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: EVIDENCE FOR A NON-LINEAR CARBON ACCUMULATION PATTERN ALONG AN ALPINE GLACIER RETREAT CHRONOSEQUENCE IN NORTHERN ITALY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is clearly written throughout. I have a few comments/suggestions for this category:

•	Figure 3 has JPEG compression artefacts and should be re-produced at higher resolution. In addition, the unified scale of Figure 3 visually flattens the response curve in T1 in July… although I’m not sure this is worth re-drafting the figure over.
•	Figure 6 has positive soil depth values! I assume these are aboveground vegetation measurements or have missed a key point, but clarification would be welcome.
•	Figure 7 legend should probably have a wider legend key to show the distinct line-types.
Experimental design: The authors clearly state their research goals and explain their methodology. I have no major comments for this section, but I would suggest that, in the case of flux measurements, more measurement events during the year would have been welcome and, ideally, would have been performed for several years.
Validity of the findings: The authors clearly present their results and include all underlying data in the supplement. My main concern above about the frequency/duration of measurements does not invalidate their findings in any way, but simply reflects a desire for a more complete picture of the ecosystem.
Additional comments: Some specific comments below. The last comment (regarding lines 502-504) I believe would greatly improve the perceived impact and importance of post-glacial colonization ecosystems to other fields.

•	Lines 92 to 97 describe Figure 1, which are three hypothetical flux trajectories following colonization. All three show a similar, fundamental trend of increasing respiration with increasing assimilation of C. However, keeping the fundamental trend in mind (as A increases, R should also increase due to plant maintenance R as well as soil R), more than these three cases can be imagined. In addition, ‘time’ here is clearly meant to be at least somewhat hypothetical, but the x-axis could be labeled with phase of succession rather than arbitrary numbers. Likewise, I would recommend removing the arbitrary numbers from the y-axis altogether.
•	Unpacking the above strictly for Scenario 1: The authors claim this scenario depicts a soil-dominated outcome where maximum sink capacity (A-R) is achieved in the later stages, where soil is most fully developed. However, given that soil R is a function of (among other things) the size of the soil C stock, we might imagine that in later stages of succession where a steady-state is achieved with maximum plant cover and fully developed soil, that the sink capacity would actually be less because it is under these conditions where soil R is maximized (due to the large stock).
•	I am not looking for a specific response from the authors, as they do mention (lines 121-122) that the overall trajectory is likely to be driven by an interplay between plant and soil development. I would, perhaps, consider emphasizing that the scenarios presented are idealized ‘end-member’ hypotheticals somewhere in the paragraph on lines 92-97.
•	Line 151 (and subsequent mentions): The third transect site is named ‘Conoid’, but I don’t quite understand the significant of this moniker? Is this a discipline-specific term? If it is arbitrary, I might suggest using a more descriptive term for the third transect (e.g., ‘old growth’).
•	Line 297: This should reference Figure 5, right?
•	Line 306-307: This series of estimates of BG stocks is difficult to read. Consider editing or removing and focusing on the reference to the Table.
•	Line 374-376: How much of this could be attributed to changes in phenology? I am not familiar with the site type and species, but shifts in plant resource allocation that occur during a season could account for some of this change, right?
•	Line 433: This thought has occurred several times during reading… but how certain are you that T3 represents primary succession from T2? Presumably there are not many disturbances that may have occurred, but since there is no age estimate for T3, it may not present the final stage of primary succession for the T2 (or T1, for that matter).
•	Line 477: Another thought… if we wanted to calculate total C storage on previously barren land, then we should measure soil C to bedrock, yes? While 0-15 cm may be sufficient for estimating total C storage in T2, this may not be the case for T3, right?
•	Line 502-504: I am glad the authors returned to this point. The yearly accumulation flux is fine, but better would be the following: compare the glacial albedo radiative forcing to the radiative forcing of the CO2 captured and stored as C stock within the new ecosystem. This would allow a direct estimation of just how much of the glacial radiative forcing that is lost by retreat is offset by plant colonization. If data are available, a further comparison to forest ecosystem C stocks in the region could provide a forward-thinking estimate if the tree line is actually likely to move into the current study site.