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 REFLECTIVE WARNING MARKERS ON WILDLIFE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Very poor writing which hinders the comprehension of the manuscript. Inconsistent  terminology and unclear/confusing relationship between the objectives, metrics described in the Methods and their usage in the Results (including Figures).
Experimental design: The study seems well design but important details about the characteristics of the sampling area are missing; which ultimately can hinder the comparison between the sites with and without RWM.
Validity of the findings: Discussion of the results in a fairly light way: 1) mainly based on speculations (with limited support from the existing literature), 2) without any discussion about the levels of reflected light in the study area, and 3) no mention to possible study limitations/ confounding factors, which may have impacted on study results and/or compromise the endorse of such strong practice recommendations by the authors.
Additional comments: GENERAL COMMENTS
This paper addresses an interesting topic: the effect that traffic warning markers may have on wildlife. The study was conducted in a mountain area in China, with cameratraps being placed in 5 sub-sampling areas to assess the relative abundance, species richness and diversity of birds and non-flying mammals in the presence (treatment) or not (control) of a reflective warning marker. There is extensive literature about effectiveness of wildlife warning reflectors (exclusively developed for altering wildlife behavior nearby roads and reducing wildlife–vehicle collisions); however, studies about the effect that regular traffic reflectors have on animals seem to be uncommon.

However, the manuscript will need major revisions in order to achieve the quality and scientific level expected for a publication. In my opinion, the most important shortfalls that need to be addressed are:
• Poor writing which makes the comprehension of the ms difficult. A revision by a native English speaker is strongly recommended.
• Problems with ms structure which hampers its clarity and impact (i.e. help guiding future research and/or the implementation of mitigation measures). E.g. formal “Materials & methods” and “Conclusion / recommendation” sections are missing.
• Inconsistent  terminology and poor interconnection between objectives/question of the study, the metrics described in the Methods, and the results presented in the different Results sub-sections. For example, methods describe two relative-abundance metrics (UCR and CR) which usage is unclear in the results (including Figures 3, 4 and 6).
• Lack of detail in the description of methods and results (e.g. poor description of study area; captions of figures very incomplete/unclear).
• Discussion of the results in a fairly light way, without any mention to 1) the levels of reflected light in the study area, or 2) possible study limitations/ confounding factors; which may have a impact on study findings and/or compromise the endorsement of such strong practice recommendations.

For more details, please check the specific comments given below.

SPECIFIC COMMENTS

Line 1 – This is a very broad title that might mislead the readers. It would be important to make it more informative (e.g. “Effects of reflective warning markers on wildlife: a case study from Cangshan
Mountain, China”).

Line 23 – besides diversity, the study also assessed species richness and relative-abundance, correct?.

Line 23 and 28 -  Replace “animals” by “mammals” or, to be more precise, by “non-flying mammals”, since bats were not included in the surveys.

Line 32  - Is should be numbered (1. Introduction).

Line 48 – “It is expected to further study on whether…”- difficult to understand (please revise grammar). Is this the first study about the effect of traffic warning markers or not?

Line 40 – delete the blank space between the parenthesis and the final period. Revise all ms accordingly.
Line 61-63 – This is somehow confusing as the reader does not know what are “independent photographs”. Revise the sentence to clarify the reader about which parameters were assessed (relative-abundance, species richness and diversity?).  It is also important to keep the terminology consistent across the different sections of the ms.

Line 65 – It would be helpful to create a formal section dedicated to the Methods (as recommended by PeerJ standards and manuscript template), which included a sub-section with the description of the study area (2.1).

Line 66-69 & Fig. 1a – A more informative map (also with scale and north) would be helpful. Perhaps, keep the same broad map (which shows the location of Dali region) but then add a second map, showing the Cangshan mountain, the Mocan and Heilong streams, and the location of 5 sub-areas (where the camera were placed). See chkeck the Figure 1 from O’Brien et al. 2003.

Line 70-73 – Provide more details about the characteristics of the study area, namely in terms of 1) shrub cover, as it can affect the amount of reflected light that reaches the animals; and 2) existence of roads (or other sources of artificial light) nearby. Since the RWM are not self-luminous, it would be important to understand what are the “expected” levels of light reflected and what is its primary source (only moon light and/or artificial lights).

Line 73  – were placed?

Line 74-80 – Information about cameratraps and their settings a part of “Data collection” and, therefore, should be moved to the next section. Alternatively, you may rename this section, for example, "2.1 Study area and camera trapping" and the next one "2.2 Data processing and statistical analysis".

Line 83 – Please add a reference for the software M-photov1.0.

Line 84-85 - delete the text box (with a letter "d") on top of the main text.

Line 85-86 – Replace “rodent rat” by “rodents”. Besides rodents, which nocturnal animals were also excluded?

Line 87 - Replace “beasts” by “species”.

Line 90-93 – These sentences are a transcription from O'Brien et al. (2003). Revise the text in order to use it in own words or, at least,  make it clear that this is the criteria defined/used by O'Brien et al. (2003).

Line 93-95 – To which “two relative-abundance indices” are you referring? UCR and CR? If that is the case, move the sentence in between (“Night-time was defined ... setting time point the sun”), in order to make it clear. Please also note that, as far as I can understand, UCR and CR are not the same two relative-abundance indices described by O'Brien et al. (2003). The way the text is currently written may be misleading for the readers.

Line 96-102 – the meaning of each formula would be easier to understand if the definition of the parameters was done immediately after each formula. E.g.
Unit capture rate (UCR) and capture rate (CR) were calculated by the following formulas:
（1）UCR=n/t×100%
where n was the overall number of photos captured by a single camera, and t was the effective (shooting)working days that were obtained by subtracting the days of camera maintenance from the total days during the study period.
（2）CR=N/T×100%
where N was the total number of photos by all working cameras, and T was the total effective working days.

Line 101- Replace “shooting” by “ working” in order to keep the terminology consistent.

Line 104-105 – It would be important to clarify that the “two communities” you are referring to, are the ones present in RWM and control sites (to avoid confusion with the bird and mammal communities).

Line 112 – and the left?

Line 119 / -  Include a reference to support the claim “The number of independent photographs is a reliable indicator of animal distribution density.”. Please also make sure that terminology is used consistently (e.g. “animal distribution density” Versus “relative abundance index”).

Line 119-122,  Figure 3 and 4 – Its not clear what are the  metrics being used in each situation (e.g. 10 ± 3.4 vs. 20.1 ± 5.3). When are UCR and CR used?

Line 101- Replace “RWM treat” by “RWM treatment”.

Line 125 – in different places?

Line 126 - The results of the Jaccard index  would be the same if they were calculated separately for birds and mammals?

Line 131-132 - make it clear that you are referring to the difference between day and night times (otherwise it makes no sense to repeat here the same results of section 3.1).

Line 133-135 – hard to understand. Please revise the sentence grammar.

Line 140 – “leopard cats and wild roars” – correct to “boars”. Latin names of both species are also missing.

Line 150-153 – It is not completely clear why do you think that animals’ abundance was nearly 50% less in RWM sites (compared to controls). The “different features of RMW between the day and the night” are clear, but, during night-time, do you believe that the amount of light reflected is enough to explain such strong avoidance effect? This seems, to some extent, implausible taken in consideration that even the effectiveness of wildlife warning reflectors (which objective is exclusively to deter wildlife from approaching roads) is not completely clear (e.g. Benten et al., 2018). Thus, it would be important to discuss the study results bearing in mind the known (or “expected”) levels of reflected light that reaches the animals in this particular study site (given the presence roads, shrubs, etc.), how it may differ (or not) from other study areas, and the results of studies where the effectiveness of wildlife warning reflectors was tested.
         Benten, A., Annighöfer, P., Vor, T., 2018. Wildlife Warning Reflectors’ Potential to Mitigate Wildlife-Vehicle Collisions—A Review on the Evaluation Methods. Front. Ecol. Evol. 6, 37. https://doi.org/10.3389/fevo.2018.00037

Line 154-165 –The authors speculate (based on a study on owls and their better predation efficiency under moonlight) that reflective materials may help nocturnal hunters. However, it seems implausible that the amount of light reflected by RWM is enough to create such opportunistic behaviour in owls, hares and porcupines (and the opposite effect on preys). Once again, it would be important to discuss the results considering the known (or “expected”) levels of light reflected in this particular study site.

Line 175-192 – The impact of the study results could be improved through the conversion of the current 2nd-level section (4.3 Suggestion on RWM usage) into a new 1st-level section (e.g. 5. Conclusion and recommendations).

Line 190-191 – The extrapolation of the recommendation for the wind energy and power lines context seems too much. I advise you to delete this sentence or to revise properly the extensive literature (on mitigation measures of bird and bat collision with wind turbine and overhead wire) before making any recommendation on this matter.

Figures - Revise the captions of all figures to make them accurate and more informative. All metrics and abbreviations need to be clearly defined and consistent with the terminology used across the ms.
Figure 3 –“Effective independent photos number” - which metric is this?  “Number of independent photos/ p working day”?
Figure 4 – “Effective Independent photo number” – do you mean ”Accumulated number of independent photos”?
Figure 5 – replace the label of X-axis by “Number of species”
Figure 6 – replace the label of Y-axis by “Number of species”.
Figure 7-  hard to understand. It would be helpful to included the name of the species directly in the x-axis (instead of using numbers) and to identify which are birds and which are mammals (to help readers not familiar with China's native fauna).

Appendix 1 - Correct the spelling of “protection level”. Please clarify to which reference you are referring to and the meaning of level ”II”. Also include in the caption of the figure the meaning of CITES (including the meaning of “II” and “III”).


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 REFLECTIVE WARNING MARKERS ON WILDLIFE
Review round: 1
Reviewer: 2

Output:
Basic reporting: English needs substantial correction

The work by Kamiel Spoelstra and colleagues is very relevant to this study and should be included (intro/discussion) https://nioo.knaw.nl/en/light-nature
Experimental design: I think there are 5 treatment sites and 5 control sites (paired)? Please make explicit

Maybe I missed it, but was there a light source during the night that was applied to the reflective strips? Please clarify, especially if there was none, because along roads car’s headlights would periodically illuminate the reflective treatment.
Validity of the findings: x
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: EFFECTS OF REFLECTIVE WARNING MARKERS ON WILDLIFE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English language should be revised and improved throughout the article to ensure that an international audience can clearly understand the  text. This might  be done by a native English. I give some examples:

-These phrases should be rewritten to make them more understandable: L26-28, L35, L48, L51, L56-58, L84-86, L107, L116-118, L124-125, L131, L133-136, L144-148, L154-156, L176

-There are also orthographic errors in the use of punctuation marks: L41, L43, L63, L132, L159, L163, L180.
Experimental design: This paper presents data on the effects of artificial and reflective warning-signals on biodiversity, a kind of light pollution with relevance to biological conservation. Such works, linking human activities with their potential impacts on biodiversity, are needed to understand the responses of wildlife to pollution and also to define proper conservation strategies. The paper suits well to the aims and scope of PeerJ.

Field methods and design are appropriate according to the study objectives.

There are some inconsistences when using the initials of reflective warning markers (RWM). Throughout the article the authors use “RMW” many times in place of “RWM”. See for example L22-27.

According to the design of the work it would be clearer to define “control” and “experimental” (or “RWM”) sites or cameras.

Probably the observed effect of RWM is related to the smell of these markers. Could authors reject this hypothesis?
Validity of the findings: Some analyses need more information or could be improved.
For example according to the study design, it would be more appropriate to use t-test for dependent samples (pairs of control and experimental cameras) better than t-test for independent samples (L107-108).
L108. More information is needed about “proportional test” or alternatively give a reference.
L1016-118. It seems authors have performed a species richness analysis but no information is given about this kind of analysis. Did authors use specific software (for example “EstimateS”)? How did authors estimate the total richness of species)? Did authors calculate the percentage of species observed (an indicative of the sampling effort)?
L121. You might use a factorial ANOVA to test the effects of RWM and regions in the number of photos.
L121-122. You might use an analysis of covariance or a Homogeneity-of-slopes model to test for differences in the rate of accumulation of photos.
L131-132. You might use a factorial ANOVA to test the effects of RWM and period of the day in the number of species.
Additional comments: Some parts of the article could be improved to make it more clear and attractive. Please find my comments below.

L23. I guess you mean “birds and mammals” and not “birds and animals”.
L26. “Jaccard” and not “Jaccad”

L51. I guess authors are referring to “natural warning colors”. Please, include “natural”
L53. What do you mean by “destructive colors”?
L53-54. I guess authors are referring to aposematic signals.
L56-60. These sentences are not clear. Are you referring to artificial or natural warning colors?

L71-75. Then authors placed 3 control-cameras and 3 RWM-cameras in each of the 5 areas (sub-regions). Please clarify.
L91. Please explain what you mean by “consecutive”.
L102. Please clarify what you mean by “camera maintenance”.

L113. Please explain “second-class protected wildlife” or alternatively give a reference.
L1016-118. It seems authors have performed a species richness analysis but no information is given about this kind of analysis. Did authors use specific software (for example “EstimateS”)? How did authors estimate the total richness of species)? Did authors calculate the percentage of species observed (an indicative of the sampling effort)?
L121. You might use a factorial ANOVA to test the effects of RWM and regions in the number of photos.
L121-122. You might use an analysis of covariance or a Homogeneity-of-slopes model to test for differences in the rate of accumulation of photos.
L131-132. You might use a factorial ANOVA to test the effects of RWM and period of day in the number of species.
L140. “Wild roars”?
L151-153. I do not understand this sentence. Please clarify.
L154. I guess you mean “species” and not “animals”. Please clarify.
L159. Why “Reflective materials may help nocturnal hunters like owls at night”?
L201-203. Please include the name of the journal.
L256-260. Raap et al. should be placed after Pechmann et al.

Figure 7. Please, separate “Birds” and “Mammals” in the text.
Table 1. This table needs more information. For example columns “protection leve” and “CTIES” need to be defined.


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 REFLECTIVE WARNING MARKERS ON WILDLIFE
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1.	Language should be corrected by a native Englishman. I am not a native speaker but some sentences have grammatical errors and look strange, even for me. Corrections made by authors alone are not enough.

2.	Figures should be corrected. Night and day parts of graphs should be marked somewhere. Please, remove any raw numbers (e.g. means with sd). This can be read from axes. Keeping them is very misleading.

3.	Raw data are hard to understand. It is a large Excel file with many sheets. Actually, I could not find any column with plot ID in order to repeat GLMM. There is no description of the variables. Species names are not in a tidy format. Scientific (Latin) names should be separated from Chinese. Moreover, it would be more useful to see English names instead of Chinese ones. Remove any graphs. Explain every column. Sheet names should be clear.

Minor comments
Please, change „Cameras traps”  to „Camera traps” throughout the manuscript (e.g. lines 132, 13;5).

Line 99 „We identify…” should be „We identified…”

Lines 132-134. A vague sentence. „Significantly” what? Less or more? Please, check the order of the figures in the brackets.

Line 136: Please insert hypo after 284.

Lines 146- 147: Please change into „…while the species compositions were extremely dissimilar (Jaccard =7.6%) or moderately dissimilar (Jaccard =58.3%) between the two groups in birds and mammals, respectively.”

Line 148: Change „Independent” into „independent”

Lines 186-187: Change the entire sentence into „Therefore, the materials might be attractive to the owls because they might use light reflected by RWMs during hunting.”
Experimental design: The methodology seems to be fairly good. I liked using mixed models. However, for count data more natural choice is the use of generalized linear mixed models with Poisson error and log-link function instead of the linear mixed model. Please, check excellent guidance in papers published in PeerJ:
- Harrison XA. 2014. Using observation-level random effects to model overdispersion in count data in ecology and evolution. PeerJ 2:e616 https://doi.org/10.7717/peerj.616
- Harrison XA, Donaldson L, Correa-Cano ME, Evans J, Fisher DN, Goodwin CED, Robinson BS, Hodgson DJ, Inger R. 2018. A brief introduction to mixed effects modeling and multi-model inference in ecology. PeerJ 6:e4794 https://doi.org/10.7717/peerj.4794
	Moreover, there is no need to use ANOVA. You can use a generalized linear mixed model again. Just introduce interaction term between time and treatment to the model.
Validity of the findings: Findings are novel and can be a substantial contribution to ecology, namely the effects of light pollution on animal communities. Better statistical tests could strengthen the impact of the manuscript as it was mentioned in point 2 above.
Additional comments: The manuscript was substantially improved as compared to the earlier version. The strong side of this manuscript is a novelty. However, it still requires major revision before it can be accepted in the international journal.


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

Input:
Title: COMPARATIVE ANALYSIS OF THE RIBOSOMAL DNA REPEAT UNIT (RDNA) OF PERNA VIRIDIS (LINNAEUS, 1758) AND PERNA CANALICULUS (GMELIN, 1791)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The MS uses mostly clear, professional English and follows a standard structure.
Some references missing.
Experimental design: The methods employed are adequate and the results seem to be congruent with previously published data from other taxa.
The objectives stated at the end of the introduction were not fully addressed.
Validity of the findings: Please, see general comments
Additional comments: This article describes and analyzes the nucleotide sequences of the major ribosomal DNA repeats in two species of Perna mussels. The MS uses mostly clear, professional English and follows a standard structure. The methods employed are adequate and the results seem to be congruent with previously published data from other taxa. Thus, this is an interesting contribution and merits to be published in Peer J but needs major revision prior acceptation.

The most important issue is that the objectives stated at the end of the introduction were not fully addressed. Although objective 1 conforms to the experimental design used and the results obtained, objectives 2 and 3 are not. To accomplish objective 2 “to assess individual, intraspecies and interspecies diversity in the IGS sequence” many more clones from a single specimen and many more specimens, of different geographical origin, from each species must be studied.  Objective 3 “to explore the phylogenetic relationships of P. viridis and P. canaliculus with each and other Mytilidae species” needs wider analyses (i.e., Kartavtsev et al 2018 Rus J Genet, Liu et al 2018 Mol Phylogen Evol). Comparing the 18S and 28S rDNA sequences of those two Perna species with those described for other eukaryotes (Saccharomyces, Drosophila, mammals...) would be a more interesting and feasible objective.

Other issues that must be addressed are

All species names, and their taxonomic status, that appear in the MS must be revised following the World Register of Marine Species (WoRMS, http://www.marinespecies.org/). Some examples
Perna picta is unaccepted the accepted name is Perna perna (Linnaeus, 1758)
Perna indica is unaccepted the accepted name is Perna perna (Linnaeus, 1758)
Aulacomya atra maoriana is unaccepted the accepted name is Aulacomya maoriana (Iredale, 1915)
Mytilus coruscus is unaccepted the accepted name is Mytilus unguiculatus Valenciennes, 1858
Geukensia demissa (Dillwyn, 1817) belongs to subfamily Brachidontinae

The references along the text must be ordered chronologically (i.e. L43-L44)

Introduction
The common name for Perna canaliculus Gmelin 1791 must be checked. The classical common name for this species was green-lipped mussel and I am not sure about the TM label for greenshell mussel. See for instance Wood et al. 2007.
L45: identification instead of classification?
L50: usually? Is it any exception to the rule?
L50-63 paragraph must include some references to other members of the family Mytilidae, as well as to other bivalves, and cite Passamaneck et al. 2004 Mol Phylogen Evol.
The final paragraph of the introduction (L64-68) must be reformulated.

Material and Methods
What’s the rationale for using so many pairs of primers? How long were the amplified fragments?
Considering the structure of the major (45S) rDNA in eukaryotes, was not enough using two pairs of primers, one for the IGS and another one for the coding region?
Taking into account the nucleotide sequences provided as raw data, the primer pair IGS-ar1 and IGS-br-2 must work in both species, was that not the case?
Is something missing in L90 (Fr Three)? Were three different clones for each specimen sequenced?
L90: How were rDNA units assembled? Were consensus sequences for each species determined?
L93-94: Which sequences were used to determine boundaries between regions? Those mentioned in L100-101?
L104-111: Why selecting only those species of Mytilidae? Why using an abalone as outgroup? Why not using other Pteriomorphia bivalves, i.e. Ostreidae? I would suggest removing this part from the MS

Results
The results are not clearly and unambiguously displayed. There are some sentences that belong to discussion.
Figure 1 is not informative enough and must be changed to include the localization of the primers. The nucleotide in which transcription starts and the nucleotide positions (from the consensus sequences for both P. viridis and P. canaliculus) in which all regions (5’ETS, 18S, ITS1, 5.8S, ITS2, 28S, 3’ETS, NTS) start and end must be also signaled.
Maybe a single figure including this information and that provided in Figure 2 would be more adequate.
Were three different clones for each specimen (and each amplicon) sequenced?  Were the sequences for each specimen identical or showed differences? Were the six sequences deposited in GenBank (not released) consensus sequences or identical?
Information in table 2 is somewhat misleading. As one of the implicit objectives of the work (L47-49) is the possible use of variation in IGS nucleotide sequences in intra-specific analysis, in my opinion pairwise identities and distances for each species must be also provided.

Discussion
I wouldn’t use the word gene for the IGS.
I suggest focusing the discussion in the comparative analysis of the rDNA unit, including the more conserved coding regions, both between the two Perna species and with respect to other eukaryotes.
L173-185: Instead of centering the paragraph only in the differences in length of the whole repeat, I suggest also compare the “conserved” sequences (18S, 5.8S and 28S)
L209-223: I suggest removing, or drastically reducing, this part from the discussion


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

Input:
Title: COMPARATIVE ANALYSIS OF THE RIBOSOMAL DNA REPEAT UNIT (RDNA) OF PERNA VIRIDIS (LINNAEUS, 1758) AND PERNA CANALICULUS (GMELIN, 1791)
Review round: 1
Reviewer: 2

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: Even when the paper is valuable because of the results are information that practically does not exist in molluscs. However, they should include more analysis to the sequence data as for example secondary structure analysis and diversity. I also suggest that they analyze a higher number of individuals or clones per individual for each species.  My suggestion is that they complete the data and analysis, and they submit again the 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: COMPARATIVE ANALYSIS OF THE RIBOSOMAL DNA REPEAT UNIT (RDNA) OF PERNA VIRIDIS (LINNAEUS, 1758) AND PERNA CANALICULUS (GMELIN, 1791)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript submitted by Guo et al deals with the sequencing and analysis of the rDNA cistron in two species of Perna shellfish, with special regard to the IGS and ITS region. The analysis describe in detail the IGS sequences, including variation of sub-repeat arrays, and shows a phylogenetic analysis based on the ITS sequence.
While the analysis is interesting and potentially leading to efficient application of IGS as species recognition marker, I think the whole manuscript need improvement of language and analyses before resulting acceptable.

The English language need to be improved: in many instances incorrect/weird sentences and /or words are used. I listed some go them in the General Comments for the authors.
References are sufficient and the background appears complete. Article structure is in line with current standard; tables and figures are well done, self-explicative and complete. Raw data are shared and submitted to the relevant on line repository.
The analysis is rather descriptive and somewhat limited, although envisaging the use of obtained data as a basis for future application. Unfortunately, this remains only a hypothetical use, and no practical tests have been carried out: this makes the analysis a little bit incomplete.
Experimental design: I can’t see any flaw in the experimental methods: data isolation and elaboration are in line with current standard. I may question the use of the Neighbour-Joining for phylogenetic tree reconstruction, as it is greatly outdated and error-prone, but for the limited dataset presented in the analysis could be acceptable.
Validity of the findings: The manuscript is rather descriptive, but this is OK. I think that it could be greatly improved if methods addressing the aim of species recognition and results are presented. I think that that’s could be not a great deal to perform and would demonstrsate the really utility of IGS analysis for Perna species identification.
Additional comments: As said, the manuscript is rather descriptive and, to some extent, limited.  I would suggest to authors to improve the level of the analysis to better addressing the aim stated in the background section.
Moreover, I think that language need to be improved; while a commercial editing service of the help of native speaker colleagues are necessary, here I listed a bunch of examples where word/sentences need to be fixed:

line 43: weird notation CoI. it is more generally indicated as COI or cox1;
line 51: "arrayed" should be "arranged";
line 57: "fragments and promoter and enhancer" should be "fragments, promoter and
enhancer";
line 65-66: IGS is not a gene;
line 136: please, motivate why those regions were considered TTSs;
line 140: please, define "common zone". Could it be a shared/conserved region?;
line 147: "better" should be "more"
line 150: IGS is not a gene;
line 157: it is unclear if the difference was attributed to SR copy number or it means that the difference is actually due to SR copy number variation. Please, explicit this point;
line 165: "cluster analysis" should be "clustering pattern analysis" (the fact is that the former is the name of a statistical method which, I assume, has not been used for this purpose);
line 170: I think that "relatively closely related", in this instance, should be simply "more related";
line 174: why "notably"? I don't think there were hints of different expectation;
line 175: avoid the use of terms such as "type of organisms": terms like "taxa" or the same "organisms" is enough descriptive and more scientific.
line 185: please, define "higher evolutionary level". Probably, author want to refer to the fact that IGS appear longer in vertebrates. The sentence "higher evolutionary level" is, technically, non-sensical: I suggest to avoid it.
line 188: base substitutions do not affect sequence length;
line 198: please, define "rules";
line 200: "this phenomenon could" should be changed with "the repeat pattern could";
line 219: please, replace "phylogeny tree" with "phylogenetic tree";
line 227: IGS is not a gene.


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: COMPARATIVE ANALYSIS OF THE RIBOSOMAL DNA REPEAT UNIT (RDNA) OF PERNA VIRIDIS (LINNAEUS, 1758) AND PERNA CANALICULUS (GMELIN, 1791)
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: PeerJ-36103-v1 Guo et al. 2019
Comparative analysis of the ribosomal DNA repeat unit (rDNA) of Perna viridis and Perna canaliculus

Although the reviewed version of the article addresses most of my previous comments, the MS still needs further revision before being ready for publication.

I am still not sure about the convenience of using a trade mark as the common name of a species, the editor has to make a demission but I suggest using green-lipped mussel instead of greenshell (TM) mussel.

The terms rRNA, rRNA genes and rDNA are incorrectly used along the MS. See for instance, the start of the second paragraph in M&M (L71-73)
The rDNA genes were amplified using the primers listed in Table 1. The primers used to amplify the 18S and 28S rRNA regions were designed based on partial sequences corresponding to the 18S rRNA of P. viridis (EF613234) and 28S rRNA of Geukensia demissa (AY145405), respectively
The rRNA genes (or rDNA fragments) were amplified using the primers listed in Table 1. The primers used to amplify the 18S and 28S rRNA genes (or 18S and 28S rRNA coding regions) were designed based on partial sequences corresponding to the 18S rRNA genes (or 18S rDNA) of P. viridis (EF613234) and 28S rRNA genes (or 28S rDNA) of Geukensia demissa (AY145405), respectively.

The wording of the last paragraph of the Results section (L164-170) must be revised and minor spelling mistakes corrected. Why not include Geukensia demissa (Dillwyn, 1817) ITS sequences (AY621983.1, AY621962.1) in the tree?

The caption of Table 4 (the species were collected with rDNA repeat unit) does not make sense

Many more mistakes appear along the MS, i.e.
L10: P.canaliculus
L14: thisvariability
L17: the copy numbers of repeat units?
L20; Species of the same genus were easily clustered in a single clade?
L38-39: information for classification based on geographical identification or among strains (or even individuals)?  Those mussels are already classified, we only assign specimens to a taxon or identify them
And so on.


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: COMPARATIVE ANALYSIS OF THE RIBOSOMAL DNA REPEAT UNIT (RDNA) OF PERNA VIRIDIS (LINNAEUS, 1758) AND PERNA CANALICULUS (GMELIN, 1791)
Review round: 2
Reviewer: 2

Output:
Basic reporting: Revision to the ms. “Comparative analysis of the ribosomal DNA repeat unit (rDNA) of Perna viridis and Perna canaliculus” (#36103) from Guo et al.

Title: I suggest to add the authority after the scientific names.
Lines:
14: Could you change “thisvariability” to “this variability”.
17 and 18: Whereas sizes of the CpG islands are indicated for P. canaliculus, they are not inidicated for P. viridis.
15-17: It would be illustrative if you include the length of the repeats. In the same way the CpG islands can be better described adding the length.
Introduction
37: ITS should read ITS (ITS1 and ITS2).
37: Gardner et al. (2916) says that the length for P. viridis in COI and ITS are 650 pb and 670 pb. respectively. Besides, the data of length reads a little odd there, maybe this data are better in line 36.
38: After “Perna species” include the reference (Wood et al., 2007).
39-41: This may be not true as microsatellites of sizes 1-6 pb can effectively distinguish species and individuals based in the number of repeats.
41-42: This is a contradiction as you say in lines 37 and 38 that ITS does not supply enough information for intraspecies comparisons and you say you are looking for a more informative marker in a region that includes ITS.
52-54: Please include references.
Material and methods
76-78: This information is repeated in the table.
Results
114-115: Do you refers to the assembled sequences, clarify. This sentence fits better in methods.
123: Must be “one and five” instead “five and one”?
142-143: Not clear. The TIS is not in the middle.
146-148: The information of individual, intraspecies and interspecies variation for NTS, ETS and SRs could be described in a table. It is at your choice. It would be interesting. Also, in the same regard, to include the sequences obtained for ITS and RS as figures. I think they can enrich the manuscript.
Discussion
187: I suggest a dot after Bhachiola algare.
         Instead “in rDNA length” maybe is better “in the entire rDNA unit length.
191-192: This line sound odd. Sequences differences are not responsible for variations in length bur indels.
190: In length and sequences?
201: After “…evolution of rDNA units” can you add a reference?
208: “…marine invertebrate” should read “…marine invertebrates”.
210: Change rRNA to rDNA.
218: Delete genes.
220-223: It is not clear in the document in what degree are the SRs different. To perform phylogenetic reconstruction as you say you need some degree of conservation.
224: Delete sequences.
228: At you choice. You can explore a little bit more the data, if realize diversity (π) analisys using the dnaSP software, in ITS, ETS and NTS.
References
233: Change 12,1-14 to 12:13.
Line 294: Change “4:293-298” to “4,293-298”.
Tables
Table 4. Heading. Something is missing. It is not clear.
Figures
Fig. 1. Figure caption. Change “in Perna species” to “in P. viridis and P. canaliculus”.
Fig.2. Figure caption. In “methods and based”, delete “and”.

For the rebuttal letter:
The clones you sequence can serve for secondary structure analysis in ITS, this has been done in other mollusks, then you have other structures to compare. Is at your choice, as is not the main objective, but these kind of analysis can enrich the document.
Experimental design: -
Validity of the findings: -
Additional comments: The article is ok. It is a good job. But I'd like that you exploit more the data. They have a lot of potential.


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: COMPARATIVE ANALYSIS OF THE RIBOSOMAL DNA REPEAT UNIT (RDNA) OF PERNA VIRIDIS (LINNAEUS, 1758) AND PERNA CANALICULUS (GMELIN, 1791)
Review round: 2
Reviewer: 3

Output:
Basic reporting: Overall, the manuscript is improved with respect the previous version. Authors accepted and properly addressed all my remarks. Still, I think that English needs some corrections here and there: I suggest the use of either a professional English editing service or the help of a native speaker colleague.
My only point main concern is that the analysis for species identification has been not more taken into account: that could have been a nice part of the work.
Experimental design: The manuscript fit well the scope of PeerJ; I can't find any fatal flaw and it appears to me scientifically sound.
Validity of the findings: Findings are ok, data appears robust and the their discussion appropriate.
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: COMPARATIVE ANALYSIS OF THE RIBOSOMAL DNA REPEAT UNIT (RDNA) OF PERNA VIRIDIS (LINNAEUS, 1758) AND PERNA CANALICULUS (GMELIN, 1791)
Review round: 3
Reviewer: 1

Output:
Basic reporting: 17. I think is more correct “the copy number (singular) of repeat units (plural)”.
38-39. COI and ITS can distinguish populations (in different geographic regions). And ITS can serve to asses intraindividual variation. You can change the sentence to say “…not always can distinguish”, or if you are speaking exclusively in Perna populations clarify.
170. “the individuals of them” sounds odd. I suggest to go back to only “the individuals”.

Table 5. Is sounds better: “The rDNA ...used for comparison purposes in this publication.
Or if these are all the sequences published for eukaryots to this date can be: The rDNA…of eukaryots published to date.
Also, include the subregions in the title: “The rDNA unit length (nrDNA), 18S rRNA gene, ITS1, 5.8S rRNA gene, ITS2, 28S rRNA gene and IGS lengths (bp), and GenBank Accession numbers of…”
It would be good to add the data for the two Perna species at the end of the table.
In the subheadings GenBank Accessions NO. should read Genbank Accession No.
In the subheadings, 18S rRNA should be 18S rDNA or 18S rRNA genes, the same for 5.8S and 28S.
Fig. 2. Even when the sequences are so different, it is better to add the alignment, using dashes for deletions and dots for identities. Also the values for diversity, intra- and interspecific identities in table 4.
Experimental design: -
Validity of the findings: -
Additional comments: The article has been significantly improved. Hovewer there are still some minor points that should be corrected. I would be happy to do a final revision to the document.


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

Input:
Title: COMPARATIVE ANALYSIS OF THE RIBOSOMAL DNA REPEAT UNIT (RDNA) OF PERNA VIRIDIS (LINNAEUS, 1758) AND PERNA CANALICULUS (GMELIN, 1791)
Review round: 4
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
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: EFFECTS OF MECHANICAL AND CHEMICAL CONTROL ON INVASIVE SPARTINA ALTERNIFLORA IN THE YELLOW RIVER DELTA, CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: I think this work is helpful for S. alterniflora manaement around the world. But some of problems should be clarified and revised before it could be published on PeerJ.
Experimental design: 1, The research question should be further clarified. At now stage the research question is not defined.
2, The methods, i.e., the map of the waterlogging, mowing, tilling, should be illustrated with the in situ map, to show how the experiments were performed.
Validity of the findings: The author should provide the costs of each approach proposed in this research. Futhermore, it should be compared with other studies to show the novelty of their work.
Additional comments: It is an interesting work. I think it could be accepted if my concerns were reponded carefully.
1, The research question should be further clarified. At now stage the research question is not defined.
2, The methods, i.e., the map of the waterlogging, mowing, tilling, should be illustrated with the in situ map, to show how the experiments were performed.
3, The author should provide the costs of each approach proposed in this research. Futhermore, it should be compared with other studies to show the novelty of their work.
4, The author should provided a figure to show the spatial position, and the distribution of S. alterniflora at YRD.
5, The discussion part should be further improved to show the novelty of 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: EFFECTS OF MECHANICAL AND CHEMICAL CONTROL ON INVASIVE SPARTINA ALTERNIFLORA IN THE YELLOW RIVER DELTA, CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Spartina alterniflora is a worldwide invasive species. Many countries and local governments are trying to control its invasion and restore native ecosystems. However, the controlling approaches largely depend on local situations. This work was preformed to control this species in in the Yellow River Delta, where is an important ecological zone. Authors successfully applied an integrated approach to control Spartina according to previous studies and gave suggestions on ecological restoration. The results have great reference significance for different places to carry out effective restoration. The paper was well wrote in English and provided clear figs/tabs. Here, I suggest authors to add another cases in other places in the Introduction section, such as Yangtze River estuary, and they can show a big picture of Spartina control.
Experimental design: In the Fig. 1, I suggest authors to add more information about experiment design, such as mechanical and chemical controls, timing, etc.
Validity of the findings: Validity of the findings is good.
Additional comments: No additional comments


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

Input:
Title: EFFECTS OF MECHANICAL AND CHEMICAL CONTROL ON INVASIVE SPARTINA ALTERNIFLORA IN THE YELLOW RIVER DELTA, CHINA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English of this article need to be revised by native speaker, because many of the sentences are too hard to understand.
The background of the study in the introduction part need to be added.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The article studied different approaches used to control S. alterniflora. This is useful for the protection and management of coastal wetland ecosystem. However, the artilce need to revised carefully, including the language and the structure or logic. For example, the "Material and method“ and "Results" should be revised to make them more concise. Specific comments are listed in the attacted file.


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: PROTEOMICS AND BIOINFORMATICS ANALYSIS REVEAL POTENTIAL ROLES OF CADMIUM-BINDING PROTEINS IN CADMIUM TOLERANCE AND ACCUMULATION OF ENTEROBACTER CLOACAE
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this manuscript, the authors report the potential cadmium-binding proteins in Enterobacter cloacae, a gram-negative bacteria. The manuscript is well-written and the authors provide sufficient background information and discussed the results clearly for the understanding to broad interest readers.
Experimental design: The authors provide a complete detail description of the methods in the manuscript.
Validity of the findings: No comment
Additional comments: Few Minor issues:
1. The title of the manuscript is too long. It should have a clear, precise scientific meaning.
2. Change "folds" to "fold" in the manuscript.
3. I suggest improving the introduction section; should be more clear. Some sentences in the introduction section seem to be incomplete.
3. Re-write the line 168-171: not clear.
4. Explain in detail the method used for the estimation of cadmium accumulation in the bacterial as mentioned in line 183-184.
5. Line204: Change ' labeling' to 'labeled'.
6. In Figure 4 legend, the % of protein gel written is 12% whereas in the text it is mentioned as 12.5%. Write the correct % of gel used.
7. The authors mentioned all the 80 proteins spots in Table 3. I suggest to include a graph also showing the number of the protein identified on y-axis versus protein family on x-axis for easy understanding of the result.
8. Similarly, include a graph showing the max fold change between control and cadmium-treated group for qRT-PCR result as shown in Table 6.
9. Re-write the line 486-488; not clear.


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: PROTEOMICS AND BIOINFORMATICS ANALYSIS REVEAL POTENTIAL ROLES OF CADMIUM-BINDING PROTEINS IN CADMIUM TOLERANCE AND ACCUMULATION OF ENTEROBACTER CLOACAE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors Chuanboob et al. have presented a detailed study of cadmium-binding proteins involved in inducing cadmium tolerance in different species of Enterobacter cloacae isolated from industrial and agricultural sites around central Thailand. The study is well-designed, and the authors have presented conclusive results corroborating their claims. The manuscript is easy to follow with overall correct use of language and grammar. The manuscript is structured as per PeerJ requirements and the authors have presented relevant and adequate background information and references to set up their research question.
Experimental design: The figures and tables presented corroborate the conclusion made by the authors.  The reviewer is thankful to the authors for making the necessary raw data available. The authors have following few suggestions that will improve the quality of manuscript
1.	The figures are of high quality, easy to follow, and have the necessary legends. The authors should please consider adding legend to figure 1. Though the figure description is adequate, having a legend indicating what open and closed symbols mean within the image will make it very easy for readers to follow.
2.	The Venn diagram presented in figure 5 is insightful in differentiating protein expression in the three E.c species. As per the diagram, there are no common protein(s) between ATCC 13047 and intolerant strains used in the study. It is a bit surprising as there is 1 common protein between the tolerant and intolerant strains. Can the authors elaborate or provide an explanation for the same?
Validity of the findings: The authors have presented a solid data supporting their claims about the mechanism of cadmium tolerance observed in EC01 strain due to over expression of cysteine-rich DUF326. The study is suited for publication in PeerJ with a few minor suggestions mentioned above.
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: STANDARDIZATION AND VALIDATION OF A PANEL OF CROSS-SPECIES MICROSATELLITES TO INDIVIDUALLY IDENTIFY THE ASIATIC WILD DOG (CUON ALPINUS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: •	The language is appropriate and clear, but with several minor flaws noted below.
•	The cited literature gives sufficient field background for the study, although some additional info that are relevant for the analyses carried out by the authors should be briefly included in the text
•	Tables are clear and complete, the figure is appropriate but needs minor improvements, raw data in the form of multilocus genotypes have been shared.
•	The body of the work is coherent and complete
Experimental design: •	The research falls well within the aims and scope of the journal.
•	The research aim is clear, relevant and meaningful. It is stated how research fills an identified knowledge gap.
•	The investigations carried out look appropriate, but lack some relevant information on the results obtained
•	Methods are mostly clearly described with sufficient details to be replicated – a few details indicated below are lacking
Validity of the findings: •	The benefit to literature are clearly stated, although the actual improvements compared to previous research should be better motivated.
•	Data appear robust and controlled, although a few unexpected features of the results (genotype recaptures and sex ratio) should be better motivated and supported by additional analyses
•	Conclusion are well stated, linked to original research question & limited to supporting results.
Additional comments: The paper is mostly well written and clear, tries to fill the current limitation to better investigate an endangered canid species, taking advantage from the existing literature on domestic dog genetic features.
In my opinion, the two main issues that need to be improved or better justified in order to increase the robustness of the results are the following:

-	The very low recapture rates (1/102 genotypes) look potentially suspicious to an external reader: I strongly suggest to either better explain how, given your sampling strategy, this result could be expected, and/or perform an easy mismatch analysis on the obtain genotype in order to evaluate how many of them differ for a small number of alleles that could be due to erroneous electropherogram interpretation (and also to better exclude the possibility of observing a ‘shadow effect’  that could occur when PIDsibs is low). Similarly, the observed sex ratio 4:1 should be better motivated in the light of the species biology or of the methods applied.
-	The resulting PIDsib is good, but does not represent a strong improvement compared to previous literature. This should be better explained in the text, and its appropriateness explained taking into account the whole estimated population size (not only mature individuals)

Other points that could be improved:
L. 108-111: “Research permits and ethical considerations”: what about the 4 blood samples?
L. 133-135: please specify (if possible) how many km have been linearly investigated, or how many different latrines have been surveyed.
Figure 1: please include a section that represents the position of the study area within the Indian subcontinent. The Y-axis of the histogram should be labelled with ‘number’ only, since it also refers to scats and not only individuals. Please also indicate ‘genotypes’ instead of ‘individuals’ – which is formally less correct.
L. 161-161: ‘low PIDsibs value (3.3x10-4), indicating a potentially inefficient panel for unambiguous individual…’. This PIDsib value is in line with what obtained in your study. Please be consistent in the way you evaluate the efficacy of your results compared to theirs – and be objective in how you report your improvements compared to previous literature.
L. 173-174: just a reasoning: given the extensive application of those markers to wild canids as well, did you base your selection on the variability observed in wolves? Maybe they could be less biased by domestication and be more representative of the dhole variability…
Table1: please explicit which markers (if any) were included in the panel applied by Iyengar et al., 2005
L. 199: Please briefly summarize the methods described in Modi et al., 2018.
L. 205: Please briefly summarize the methods described Miquel et al., 2006 and explain what Q.I. threshold you applied per locus and per sample, and also the minimum number of loci per individual with a reliable consensus genotype (I think you mention this in line 271, but I think it should be reported here instead)
L. 212: Please briefly summarize the methods described in Modi et al., 2018.
L. 262: please indicate the results for False Alleles as well.
L. 280: please specify if it refers to M:F ratio (or viceversa). Possible reasons (biological, technical, etc) for this high skew should be explained in the discussion.
L. 283: please indicate as clearly as possible the correspondence (if any) between these six clusters (subclusters within areas?) and the 5 sampling areas.
L. 284. ‘The clusters indicate no genetic sharing between MTR and PTR’. Please explain the reasoning behind this statement (in the figure the correspondence of cluster with sampling areas is not indicated, please add labels if possible)
L. 286: please explicit the values for deciding that K=4 was the best clustering. Do you think that individuals with traces of ancestry from a different cluster could represent dispersers? This would be highly relevant for connectivity and future population dynamics (you can just raise the point here, then talk about this in the discussion)
L. 289: please explicit the formal results from Bottleneck for each model.
L. 292: are these results significant
L. 301: ‘with high statistical power’: please see the second main comment above
L. 323: see comment above on PIDsib vs. census size (not only mature individuals can be considered in order to reach a good resolution of the panel)
L. 332: I think the reasoning here is not powerful enough, unless you better motivate the reason why when sampling a given latrine for multiple scats you still don’t get resampling of the same individual (e.g.: maybe you selected only very fresh samples, therefore it is unlikely that you picked up two fresh faeces from  the same individual? This would be important to know in order to exclude the possible doubts raised in the first main comment
L. 338: do you think that the limited amount of blue components in PTR individuals –if relevant - cannot be due to gene flow from MTR? Also, from Suppl fig 1b I see NNTR as a separate cluster, not intermediate between the two, except for a few individuals with blue component…what are you referring to? Please try to be much more rigorous and clear here
L. 355: please the consider the term ‘great’ in the light of the not-much-improved PIDsib compared to previous studies

Minor issues:
L. 29-30: not clear what ‘and landscape level’ means here
L. 76: ‘and included in Appendix II…’
L. 77-83: these two paragraphs could be improved in their fluidity
L. 88: ‘are ineffective’
L. 91-95: please avoid embedded parentheses
L. 96: ‘provides’
L. 103: ‘this this’ ?
L. 120: ‘consisting of’
L. 125: ‘present’
L. 130: ‘sample-containing’
L. 150: ‘respectively,’
L. 176: please explicit what genome build you used as a reference (canFam 3.1?)
L. 219: newline character to be removed
L. 264: ‘relatively high’ instead of ‘higher’?

Vertebrate animal usage checks:
•	Please provide additional info on the blood sampling procedure


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: STANDARDIZATION AND VALIDATION OF A PANEL OF CROSS-SPECIES MICROSATELLITES TO INDIVIDUALLY IDENTIFY THE ASIATIC WILD DOG (CUON ALPINUS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Basically, the article is written in professional English but an additional overhaul is recommended to improve the readability.

The structure of the article conforms.
Figure 1 should by revised. An overview map of India should be included to show the location of the sampling area within the country. Additionally, I suggest to show the inserted bar graph independently (maybe as Figure 2). This would improve clarity and gives better visibility of the results.
The legend to Supplementary Figure 1a does not explain the correlation between the cluster numbers and the Sampling sites. This makes the corresponding section of the manuscript (lines 281 to 285) not verifiable. Text and/or Figure 1a should be revised.
An additional Supplementary Table showing the genotypes of all samples should be included to make raw data available.
Experimental design: The authors state that “in this study, we address key methodological issues…” (Line 99). Taken this self-chosen key area into account more methodological details should be given. The authors should consider the following questions and objections:
As reference samples only 4 dhole individuals were used. This small number should be explained. Are these individuals unrelated? Was the genetic diversity sufficient?
All markers were originally developed for the domestic dog. During the validation of the genetic tests samples from domestic dogs and other canids should have been included. Can the authors exclude that DNA from other species affect the genotyping results. Is a mix up with domestic dogs possible?
Are the primer sequences shown in Table 1 taken from the corresponding references listed in the Table or are they newly designed/modified?
Table 1 does not show which markers are combined to the 4 multiplexes. This reduces the reproducibility of the method by other laboratories.
Line 186: 50 cycles is very high; please comment; was contamination an issue?
Line 194: specify the CE model used
Line 279-280: Molecular sexing is not described in Methods
Validity of the findings: The genotype data were processed with different statistical methods to draw conclusions. This was done in an appropriate manner. But the following issues should be considered:
The authors refer PID values based on 12 loci. As a minimum of 7 loci was considered as sufficient for individualization PID values for this reduced number of loci should be given and evaluated.

The conclusions drawn from Supplemental Figure 1b (lines 285-287) are not comprehensible. Please describe in more detail and/or improve the Figure.

In the Discussion I miss a consideration of possible relatedness of the sampled individuals. From the social behavior of dholes sampling of scats from related individuals is probable. Have you found individuals most probable closely related? And can closely related animals compromise the accuracy of individualization?
Additional comments: The authors present an appealing contribution for non-invasive DNA analysis of wildlife animals. This publication should be revised to improve the usability for other scientists working in the field by adding more methodological details and by expanding the validation approach (i.e. by including other species like the domestic dog)


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: STANDARDIZATION AND VALIDATION OF A PANEL OF CROSS-SPECIES MICROSATELLITES TO INDIVIDUALLY IDENTIFY THE ASIATIC WILD DOG (CUON ALPINUS)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The current version of the manuscript is much improved and the explainations given by the authors are mostly satisfying.
Nonetheless, the use of English still needs some polishing, especially in the newly added sections (e.g. matching the new section tens with the rest of the ms).
The caption for Suppl. Fig 2 is not clear enough.
Experimental design: All relevant information previously missing from the methods is now available to the reader.
Validity of the findings: The conclusions on genetic structure and connectivity are still counter-intuitive given the relative distance among the study areas and the possible reasons for this (e.g. geographical barriers? known patterns of deforestation?) need to be clarified to the reader. This section looks like the least robust of the paper
Additional comments: Specific comments:
(suggested changes are indicated in capitals)
L. 156: "roadS/trails"
L 160: "once a latrine WAS found"
L. 161: "One bolus from each fresh scat was collected, ASSUMING IT TO DERIVE from one individual"
L. 174: "once PER SITE"?
L. 184: please indicate how much blood was collected
L. 267: "bands" can be misleading (more commonly referred to gel testing of amplification success), maybe use "allelic peaks" instead?
L. 273-283: please adapt the tens to the rest of the paper (the past should be used)
L. 303: see above regarding "bands" (consider rephrasing)
L. 386: Is this value calculated based on the seven best-performing loci, the 7 worst-performing ones, or as an average of any combination of 7 loci among the total 12?
L. 411-412; "assigment OF each sample TO" ?
L. 529: you mean "fragmentation"?
L. 542: I would not use "great", maybe "valid" is more appropriate?


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

Input:
Title: STANDARDIZATION AND VALIDATION OF A PANEL OF CROSS-SPECIES MICROSATELLITES TO INDIVIDUALLY IDENTIFY THE ASIATIC WILD DOG (CUON ALPINUS)
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comments, see general comments
Experimental design: no comments, see general comments
Validity of the findings: no comments, see general comments
Additional comments: The authors made great efforts to improve the manuscript according to the reviewer’s suggestions/comments. They added important details of the used methods and the lab work carried out. This improves the traceability of the results and is know more helpful for other researchers planning to perform similar analyses.
However, some potential for improvement remains. Therefore, I recommend this manuscript for publication if the following point are considered, which in my opinion could easily achieved at this stage:
L381-382: the numbers of unique individuals do not correspond to those given in Figure 2, please check.
L384-386: Figure S 1a shows clearly attributable genetic clusters for MTR and NNTR, but for PTR I cannot see obvious differences to UKWLS and TATR.
Figure S2: Please add sampling areas to each of the listed samples and insert column headings. The meaning of the colour coding is not explained.
Suggestion: as Figure S1b and Figure S2 both base on DAPC maybe change numbering (S2a and 2b)


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: STANDARDIZATION AND VALIDATION OF A PANEL OF CROSS-SPECIES MICROSATELLITES TO INDIVIDUALLY IDENTIFY THE ASIATIC WILD DOG (CUON ALPINUS)
Review round: 3
Reviewer: 1

Output:
Basic reporting: The manuscript has further improved and most of the previous observations have been addressed, including language polishing and clarity
Experimental design: no comment
Validity of the findings: I still think that the presentation of the population structure analyses could be further improved by including some of the reasonings on possible weakpoints regarding discrepancies between the two methods, possibly unsampled populations, etc. reported in the cover letter also in discussion, especially regardin the PTR-TATR group.

Beside this, it is not clear to why "the low success rate might be one of the reasons behind the skewed sex ratio in this study."...if you expect to find a differential amplification success, please clearly state so with a specific reason/reference, or delete this sentence.
Additional comments: I also agree with the other reviewer on the opportunity to reorder Suppl. Figures as to improve their logical flow (TESS vs. DAPC all together)


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: STANDARDIZATION AND VALIDATION OF A PANEL OF CROSS-SPECIES MICROSATELLITES TO INDIVIDUALLY IDENTIFY THE ASIATIC WILD DOG (CUON ALPINUS)
Review round: 3
Reviewer: 2

Output:
Basic reporting: No comments, see general comments
Experimental design: No comments, see general comments
Validity of the findings: No comments, see general comments
Additional comments: The manuscript of the second revision shows only minor changes compared to the preceding version although both reviewers and the editor raised a number of concerns. The authors provide extensive and mainly comprehensible explanations in their answer letter, however it would have been beneficial to include these in the manuscript.
In the following I want to focus on the points raised in my preceding review:
L313: There is still a discrepancy between text and Figure 1. TATR=4 instead of TATR=8
L314-317: This section was not edited and is still misleading although the authors state in their answer letter that no difference between PTR; UKWLS and TATR was found. Generally, the population genetic analyses show some pattern, but it would be better to shorten the interpretation section. The authors rightly point out that this paper should mainly present a genotyping method applicable to non-invasive samples from wild dogs. It would make the main message of this study more clear if the authors would give less details of the population genetics analyses. In this context this is not essential and even more confusing.
Supplementary Figure 2 was successfully revised

I cannot recommend this manuscript for publications as long as the authors do not revise the text according to the above mentioned points. Language editing is still needed.


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: STANDARDIZATION AND VALIDATION OF A PANEL OF CROSS-SPECIES MICROSATELLITES TO INDIVIDUALLY IDENTIFY THE ASIATIC WILD DOG (CUON ALPINUS)
Review round: 4
Reviewer: 1

Output:
Basic reporting: I'm now satisfied with the new version of the manuscript, which is now simpler and more limited in its scope, but also much clearer and more robust
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: LNC-SAMD14-4 CAN REGULATE EXPRESSION OF THE COL1A1 AND COL1A2 IN HUMAN CHONDROCYTES
Review round: 1
Reviewer: 1

Output:
Basic reporting: There is not a structured abstract as peerJ guide recommend. Abstract should organized in background, methods, results and discussion.
Authors have used an English langue editor as a result the manuscript is clear and well writing. However, some sentences in the conclusion are confusing and not well written.
References should be updated if the authors follow this reviewer´s recommendations.
Figures are well labeled with good quality and well described.
Raw data is supplied.
Experimental design: The study might be considered inside the scope of peerJ.
The objectives are not well defined. It is not clear if they want to compare the efficacy of two different microarrays or they really want to discover expression changes in OA patients.
Techniques are well performed and controlled.
There are some gaps in methods that are further commented.
Validity of the findings: Even number of patients is low, statistical analysis is well performed although a an explanation is necessary about the use of ANOVA test

Authors compare their findings with others reported by Fu et al. The authors should extend their comparisons in discussion to other papers related to this topic. Here I suggest some papers that authors might use for this comparison.
Cell Death Dis. 2018 Jun 15;9(7):711. doi: 10.1038/s41419-018-0746-z,
J Cell Biochem. 2018 Jun 12. doi: 10.1002/jcb.27057.
Eur Rev Med Pharmacol Sci. 2018 May;22(10):2966-2972.
Biomed Pharmacother. 2018 Aug;104:825-831. doi: 10.1016/j.biopha.2018.04.124. Epub 2018 Apr 25.10.26355/eurrev_201805_15051.
Biochem Biophys Res Commun. 2018 Jun 7;500(3):658-664. doi: 10.1016/j.bbrc.2018.04.130. Epub 2018 Apr 24.
Cell Physiol Biochem. 2018;45(3):1241-1251. doi: 10.1159/000487455. Epub 2018 Feb 9.
Cell Physiol Biochem. 2018;45(2):832-843. doi: 10.1159/000487175. Epub 2018 Jan 31.

There are few comments in the discussion about the biological importance of the findings and how they might be related to advance in discovering new treatment for OA.

Conclusion is poor and confusing, it needs to be rewritten. It is not clear what the authors conclude and the importance of the study.
Additional comments: Table 1 must be moved to supplementary material

Patient data related to age and sex should be shown. Is it possible that there were any bias in sex or age between OA and non-OA groups?

There are little information about how specimens were treated to perform lncR microarray.

Analysis of qPCR must cited the work of Livat & Schmittgen 2001 (http://www.gene-quantification.net/livak-2001.pdf)

What are the other data analyzed with ANOVA?

A reference is required in this sentence “In previous work, the most enriched GOs targeted by up-regulated mRNAs were anatomical structure development, cell periphery, and lipid binding in biological processes, cellular processes, and molecular function, respectively.


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: LNC-SAMD14-4 CAN REGULATE EXPRESSION OF THE COL1A1 AND COL1A2 IN HUMAN CHONDROCYTES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The reporting is overall clear and written in professional English, but I have found quite a few mistakes listed below:
-L43: In genel > is it “in general”?
-L74: informed consent was signed all patients > informed consent was signed by all patients
-L97: according the manual > according to the manual
-L105: were assess > were assessed
-L106: a multiple groups > multiple groups
-L110: “Analysis Differentially expression”: should be rephrased
-L116: “this regulated articular cartilage degradation”: this should be rephrased, as it gives the impression you have confirmed that MMP13 up-regulation had an effect on cartilage degradation.
-L117: “MYOC was minimally expressed”: do you mean “MYOC was the most strongly down-regulated”?
-L119: “abnormal mRNAs”: what do you mean by “abnormal”?
-L133: “it is possible…mRNA”: there is no verb
-L144: was correlates > correlates
-L145: “top mRNA and lncRNA”: what does this mean?
-L146~148: this sentence is also difficult to understand.
-L172: “we identified 23 common …between 2 studies”: this sentence is also difficult to understand.
-L201: their the top > their top

The references seem adequate.

The article is properly structured with all the necessary figures and tables. However, why do you show the volcano plot with the lncRNAs and mRNAs combined, whereas the heatmaps show lncRNAs and mRNAs separately? Furthermore, line 160: “meaning that 244 were common for both studies”: I don’t understand where this figure (244) comes from. This should be better explained either in the main text or with the figure/table legends.
Experimental design: The research presented is within the aims and scope of the journal.

The research question is relevant and meaningful.

The investigation is performed to a high technical standard.

The methods, however, are sometimes confusing, especially regarding the microarrays used: were the chips from Arraystar or Affymetrix? Also, line 170, what is a “specification chip”?
How were the GO and KEGG analyses and the hierarchical clustering carried out?
Also, no data about the purity of the RNA is shown (SDS-PAGE); could you provide these in a supplementary figure?
Validity of the findings: The major strength of this article is, to me, the large number of samples used for the study. However, the results are very different from previously reported studies on microarrays using similar samples, so it is difficult to judge whether the results are robust or not. For instance, line 176~178: “In addition…our work (FC=6.28)”: does this mean there is only one gene that is similarly up-regulated in all three studies (ENST00000426074)? If very little overlapping results can be found between the three studies, how can this be explained? This is not discussed in the discussion. Did the other studies use less samples? Were the samples used treated differently? etc...

Also, PCR was only carried out on 8 genes, which were furthermore the most strongly modulated genes; is this sufficient to show the validity of the microarray results?

Regarding statistics, why was the SEM used instead of SD (standard deviation)?

Regarding the conclusions, only very little is said about the modulated lncRNAs (MMP-13, MMP-9, and TNFAIP6 are mentioned but those are mRNAs). As the paper is originally about lncRNAs in osteoarthritis, could you provide some discussion of the modulated lncRNAs identified in this study?
Additional comments: No additional comments


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

Input:
Title: LNC-SAMD14-4 CAN REGULATE EXPRESSION OF THE COL1A1 AND COL1A2 IN HUMAN CHONDROCYTES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript meets the criteria of the journal
Experimental design: The experimental desing is acceptable.
Validity of the findings: Findings in the manuscript are admisable
Additional comments: The authors have satisfactorily addressed this reviewers 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: LNC-SAMD14-4 CAN REGULATE EXPRESSION OF THE COL1A1 AND COL1A2 IN HUMAN CHONDROCYTES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors say they have used an “English langue editor”; however, many mistakes remain, especially in the parts that have been newly added; I believe the editing has not been carried out properly by a native speaker and strongly recommend this should be done to improve the readability of the article. Here are a few corrections for the abstract:
P7, l31: “Because…not understood”: there is no independent clause
P7, l32: “a new lncRNA-lnc-SAMD14-4 and…” > “a new lncRNA (lnc-SAMD14-4) and…”
P7, l32: “potential ability”: do you mean “potential involvement”?
P7, l34: “The human cartilage tissues” > “Human cartilage tissues”
P7, l36: “related biological modules”: what does this mean? (“module” does not appear again in the text)
P7, l37: “The co-expression analysis … between the lncRNA-mRNA couple associated with PI3K-Akt pathway”: this is not very clear either; do you mean “Co-expression analysis was used to predict interactions between lncRNAs and mRNAs associated with the PI3K-Akt pathway”?
P7, l38: “the expression pattern of lncRNA and mRNA” should probably be “the expression pattern of some modulated lncRNAs and mRNAs”
P7, l39: “to explored” > “to explore”
P7, l42: “total 2042 lncRNAs were detected significantly different expression in OA” > “a total of 2042 lncRNAs were detected with a significantly different expression in OA”
P7, l43: “Total 2011 mRNA was detected significantly different expression in OA” > “A total of 2011 mRNAs were detected with a significantly different expression in OA”
P7, l44: “Go analysis” should probably be “GO analysis”
P7, l44: “all differentially expressed gene on pathogenesis” > “all differentially expressed genes on the pathogenesis”
P7, l44-46: there are 2 verbs in the sentence; maybe “corresponded” should be “corresponding”?
P8, l47: “mediated COL1A1 and COL1A2 expression” should probably be “mediated the COL1A1 and COL1A2 up-regulation”
P8, l49: “mechnisms” > “mechanisms”
In the Methods section,
P10, l84 and l90; “definition”; does that mean “protocol”?
P11, l105: “as previous study” > “as in previous studies”
P13, l155: “including” should probably be “included”

The references seem, overall, adequate. However, P16, l208-209: “COL1A1… which are associated with cartilage biology and OA”: one or two references should be given here.

The article is properly structured with all the necessary figures and tables. The raw data has been shared on the GEO database.
Regarding the figures, however,
- in Figure 3 legend: H and I should probably be A and B.
- legends of Fig6 and Fig7 should clearly state what A and B are.
- fig4 and P13, l156-157: one mRNA does seem to correlate with many lncRNAs but most lncRNAs only seem to correlate with one mRNA, so this sentence should be modified accordingly or better explained. Furthermore, the legend of Fig4 does not explain why the nodes have different sizes.
- finally, P10, l84: the contents of Supplement (or Supplementary?) figure 1 should be at least briefly mentioned
Experimental design: The research presented is within the aims and scope of the journal.

The research question is relevant and meaningful.

The investigation is performed to a high technical standard.

The methods, however, are still somewhat lacking:
- The authors mention that OA samples were taken from 19 patients and normal tissue from 11; however, the list of patients contains 53 patients; could this be explained?
- P14, l164: the main text states that 53 clinical specimens were used for PCR, but the legend of Fig5 mentions n=3; why is that?
- P11, l114-116: some more details regarding the transfection experiments should be given: how was the expression vector pcDNA3.1+-lnc-SAMD14-4 obtained? How many cells were seeded or transfected? With how much vector? etc
Validity of the findings: The previous version of the manuscript did not discuss the modulated lncRNAs identified in this study, but the revised version focuses on lncSAMD14-4 and its possible role in modulating COL1A1 and COL1A2, which has brought some improvement to the manuscript.

However, one of my main concerns, which is also completely lacking from the discussion, is the age of the OA and control groups: the mean age of the 5 OA patients used for microarray is 63 years; the mean age of the 5 control patients used for microarray is 28.8 years; are the differences in lncRNA and mRNA expression detected by microarray the result of OA or just old age? As the age difference could account for some or most of the differences observed (Loeser et al., Arthritis Rheum, 2012; Peffers et al., Arthritis Res Ther, 2013; Adapala et al., Gene, 2017), this should be discussed.

Furthermore, some conclusions seem to lack supporting evidence:
- P15, l189-190: it isn’t clear whether lnc-SAMD14-4 overexpression induces a significant increase in COL1A1 and COL1A2 expression in IL-1b-treated cells; the * only shows a difference compared to the control group; is there a significant difference between the IL-1b and IL-1b + lnc-SAMD14-4 group? Furthermore, how much was the lnc-SAMD14-4 expression increased in the transfected cells?
- P15, l193: the effect of the knock-down of lnc-SAMD14-4 by si-lnc-SAMD14-4(2#) should be shown (it generally is). As mentioned in the previous comment, the * only shows a difference compared to the control group; is there a significant difference between the IL-1b and IL-1b + si-lnc-SAMD14-4 group? What about between the siRNA (I suppose it’s the control siRNA group?) and si-lnc-SAMD14-4 group?
- P15, l195: “These data…osteoarthritis”: the only clear difference in COL1A1-2 expression is between the vector group and the lnc-SAMD14-4 group (unless further statistical analysis shows other significant differences); furthermore, the link between osteoarthritis and COL1A1 and COL1A2 expression has not been supported (by experimental results or references to other studies), so this sentence is, I believe, an overstatement.

Finally, the study does not compare its findings with similar reports on lncRNA expression in OA. On p16, l205-206: “Because…problematic”: although the platforms are different, I believe a comparison of the lists of modulated lncRNAs should be possible; if not, why could this not been done? If a comparison is possible, are there any overlaps between the current and previous studies and what are the main differences between the current and previous studies (especially Fu et al. and Liu et al.)?
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: LNC-SAMD14-4 CAN REGULATE EXPRESSION OF THE COL1A1 AND COL1A2 IN HUMAN CHONDROCYTES
Review round: 4
Reviewer: 1

Output:
Basic reporting: The reporting is overall clear and written mostly in professional English, but I have still found quite a few mistakes. I appreciate that my English corrections for the abstract were taken into account, but the whole manuscript should have been checked by a native English speaker or a professional editing service before submitting the manuscript (as is usually the case).

The references seem adequate.

The article is properly structured with all the necessary figures and tables.

The article is self-contained with relevant results to hypotheses.
Experimental design: The research presented is within the aims and scope of the journal.

The research question is relevant and meaningful.

The investigation is performed to a high technical standard.

The methods are described with sufficient detail.
Validity of the findings: My main concern regarding the age difference between the control and OA groups has been mentioned in the discussion. I understand that getting age-matched controls for such a study is very challenging, so, as long as this limitation is clearly mentioned in the discussion, I can accept the results (knowing that I will have to be cautious with the data).

All underlying data have been provided. Controls have been added to figures 7 and 8 as required.

Conclusions are well stated, although, on L243-245: “most dysregulated… were OA-related”: this is probably an overstatement: just because a couple of strongly regulated genes are OA-related does not mean that “many of the dysregulated lncRNAs and mRNAs” are also OA-related; I suggest instead “… which suggests that the differences observed in our arrays were at least in part a consequence of OA”. (Though the authors mention that they “have contribute other paper about common dysregulated lncRNA between our Affymetrix chips work with Fu’s group Arraystar work”, a proper comparison between the current microarray data and previously published data would have been helpful to confirm that the gene modulations observed here were indeed OA-related.)
Additional comments: I appreciate that most of my previous concerns have been addressed.
I still have a few comments and questions:

The whole manuscript will have to be checked again by a native English speaker or a professional editing service before submitting the manuscript (as is usually the case...); there are still too many mistakes for me to point every one out (e.g. l112: "All study were" should be "All studies were", etc.).

L243-245: “most dysregulated… were OA-related”: this is probably an overstatement: just because a couple of strongly regulated genes are OA-related does not mean that “many of the dysregulated lncRNAs and mRNAs” are also OA-related; I suggest “… which suggests that the differences observed in our arrays were at least in part a consequence of OA”. (Though the authors mention that they “have contribute other paper about common dysregulated lncRNA between our Affymetrix chips work with Fu’s group Arraystar work”, a proper comparison between the current microarray data and previously published data would have been helpful to confirm that the gene modulations observed here were indeed OA-related.).

There are too many significant digits (in the main text and in tables). For all the fold changes, 2 decimals seem more appropriate (for example: for lnc-SAMD14-4, 86.11 seems more appropriate than 86.11047, etc...).

Figure 1: why do you show the volcano plot with the lncRNAs and mRNAs combined, whereas the heatmaps show lncRNAs and mRNAs separately?

Figure 3 legend in the pdf file: H and I should probably be A and B (the word document has been correctly modified).

L204: “Fu’s group… and Liu’s group…compared with controls”: could you insert the references at the end of this sentence?


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: NUTRIENT AVAILABILITY INDUCES COMMUNITY SHIFTS IN SEAGRASS MEADOWS GRAZED BY TURTLES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper is very well-written in clear, unambiguous professional English. The introduction does a great job motivating the study, with appropriate literature from both marine and terrestrial literature. The paper generally follows the PeerJ standards as outline in the instructions for authors, with the exception that the manuscript has no Conclusions section. This does not trouble me as a reviewer and reader, but it may require reformatting to end with a conclusions section if directed by the editors. The figures as they appear in the paper are high-quality and relevant, but in the comments I have a suggestion for ether modifying or replacing Fig 2. And, although the authors did provide the near-raw data in a link in their submission materials, there is no link to the raw data nor instructions how to find them in the manuscript. Also, the data are lacking the metadata that will allow future readers to decipher the meaning of the data columns.
Experimental design: This research is within the scope of PeerJ. The paper describes mechanisms responsible for species shifts in plant communities under heavy grazing pressure. The work has special pertinence for seagrass meadows of the tropics, given recent reports of overgrazing and loss of these important ecosystems as sea turtle populations rebound. This paper is the first to point to mechanisms leading to change in species dominance under grazing in seagrasses. The investigators were rigorous and designed experiments well to test their hypotheses. As far as I can tell from the manuscript, the authors have a high ethical standard. I do have to take their word for it that they obtained all necessary permits from the Mexican authorities, but I have no reason to doubt them nor do I have enough expertise with local laws to know if they got all of the permissions they were required to have. The methods are well-described.
Validity of the findings: I found the work to be statistically sound and based on theoretical predictions. This paper links the seagrass grazing literature to more basic literature on the competitive interactions among plants to shed light on the mechanisms for changing species dominance under grazing. Interestingly, these data show how grazing of all species without preference can still lead to changes in competitive hierarchy and therefore species composition of the grazed plant community compare to ungrazed plots.
Additional comments: I like the paper very much. Below I list my specific comments and questions that arose as I was reading the paper.
Line 75 – Rotational grazing was proposed long before the Molina-Hernandez and van Tussenbroek 2014 paper, by, among others, Bjorndal, Susan Williams, Gordon Thayer and Jay Zieman.
Line 98 – be more clear about the nature of light available to seagrasses. In clear waters, light availability at the top of the canopy can be high – but the canopy itself can produce light limitation in dense meadows. This would put the statement about light attenuation of the canopies on line 100 into context.
Line 141. Please revisit the terminology used for the different grazing treatments. Both what are now described as short- and medium-term grazing treatments were done over the same time interval, so there is no difference in the term of them. Rather, the grazing interval was different among the these treatment. Perhaps call them short term frequent and short-term, infrequent grazing treatments?
Line 148. Shears d not really mimic the size of a turtle bite; rather they leave behind ungrazed shoot bases that mimic those following turtle grazing.
Line 182-end of paragraph. Please state the limits of detection of the chemical analysis methods. And, did the authors check for sulfide interference in these colorimetric analyses? My experience has been that sulfides need to be purged before porewater analyses because they poison colorimetric determinations of ammonium and phosphate.
Line 190. Technically, quadrats have to be squares (hence the name QUADrat). Another name for “circular quadrat”?
Line 263 and throughout. Do the alanlyses really justify reporting sulfide (and other parameters) to 2 decimal places? And, at 380 uM, sulfides would likely cause interferences in the phosphate ad ammonium determinations.
Line 290 and throughout. I’d rather see “N concentrations” rather than “N%” or the redundant “N% concentrations” since % is just the unit of expressing N concentration.
Line 305. “species composition” rather than “specific composition” since the adjective “specific” could mean the composition at a particular time or place.
Line 322. C:N ratio, even at the latitude of this study, is seasonal, with nutrient limitation signals generally only evident during times of peak growth rate. Did the authors consider seasonality of the seagrass stoichiometry when designing the study?
Line 329. I believe that almost all studies of the change in N content of seagrasses in response to grazing find no decrease in N content driven by grazing (examples, the Moran and Bjorndal papers, Fourqure et al 2010, others). The speculation in the Thayer et al 1984 paper that depleted N in tissues should serve as a patch abandonment cue was made with no supporting data, but this somehow became part of the paradigm, despite decades of experimental work that shows N content does not decrease in the face of grazing, simulated or natural.
Line 366. Awkward to start a sentence with “But”, I suggest linking this sentence to the previous one with ‘…), however, …”
Line 381, and figure 2. One logical problem in the data is that ungrazed plots have higher biomass of the early successional species that should be out-competed by the slow growing climax species than in clipped plots. How can this be? Perhaps it would be good to calculate the relative contribution of early sucessional species to total biomass ((Halodule+Syringodium+Calcareous Algae)/ (Halodule+Syringodium+Calcareous Algae+Thalassia). Shouldn’t this relative contribution to total biomass be the real critical response to changing nutrient competition?
Literature cited -  did not check all references, but I note that there was no Heithaus et al 2014 in the literature cited section. Please check to make sure all cited papers are in the literature cited section.