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: MIR-875-5P EXERTS TUMOR-PROMOTING FUNCTION VIA DOWN-REGULATION OF CAPZA1 IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Basic reporting
The paper assesses the tumor-promoting function of miR-875-5p via down-regulation of CAPZA1 in esophageal squamous cell carcinoma

It addresses a current and relevant topic since there is a growing interest in miR-875-5p in several malignancies.
Experimental design: The manuscript is quite well written and organized.
Figures and tables are comprehensive and clear.
The introduction explains in a clear and coherent manner the background of this study.
Validity of the findings: Overall, the discussion includes articles of historical importance in this setting. Nevertheless, we recommend the authors to include results and references related to the following recent papers, in order to enrich the discussion:
•	Essadi I, Lalya I, Mansouri H. Esophageal carcinoma. N Engl J Med. 2015 Apr 9;372(15):1470-1. doi: 10.1056/NEJMc1500692. PMID: 25853758
•	Rizzo A, Mollica V, Ricci AD, Maggio I, Massucci M, Rojas Limpe FL, Fabio FD, Ardizzoni A. Third- and later-line treatment in advanced or metastatic gastric cancer: a systematic review and meta-analysis. Future Oncol. 2020 Jan;16(2):4409-4418. doi: 10.2217/fon-2019-0429.
Additional comments: General comments
We believe this article is suitable for publication in the journal although minor revisions are needed.
The main strengths of this paper are that it addresses an interesting and timely question and provides a clear answer, with some limitations.
We suggest linguistic revision.


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: MIR-875-5P EXERTS TUMOR-PROMOTING FUNCTION VIA DOWN-REGULATION OF CAPZA1 IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please have a look at Minor concerns: 1 to 4.
Experimental design: Please have a look at Major concerns: 1 to 7.
Validity of the findings: No comment.
Additional comments: Esophageal cancer is one of the most unknown and deadliest cancers worldwide, mainly because of its extremely aggressive nature and poor survival rate. Despite improvements in the management and treatment of esophageal cancer patients, the general outcome remains very poor for overall 5-year survival rates (∼10%) and 5-year postesophagectomy survival rates (∼15-40%). Esophageal cancer is often diagnosed during its advanced stages, the main reason being the lack of early clinical symptoms and efficient genetic markers for diagnosis and treatment. In this manuscript, Dr. Nan Kang and colleagues investigated the role of miR-875-5p and SNP of CAPZA1 3’UTR (rs373245753) in proliferation and metastasis of ESCC cells in vitro. The study’s conclusion showed that miR-875-5p may function as an oncogene via down-regulation of CAPZA1 expression in ESCC. Although the current study is interesting, there are some major concerns that need to be addressed before consideration of publication.

Major concerns:
1. In general, the authors found that the SNP of CAPZA1 gene 3’URT (rs373245753 T or G allele) could influence the interaction of miR-875-5p and thus have a surprising effect on the malignant phenotype of esophageal cancer. This is a very interesting result, but a key positive control is missing. I strongly recommend the authors to add the CAPZA1-CDS overexpression group as a positive control to observe whether the overexpression of CAPZA1-CDS can directly inhibit the malignant phenotype of esophageal cancer and is not reversed by the minic of miR-875-5p.
2. “Materials & Methods – Statistical analysis” Line 141-145. “Data differences were analyzed by Student’s t-test (two-tailed).” Some of the experiments (eg. Figure 1B) in this study were multi-group comparisons, and t-test was not applicable. The author should choose an appropriate statistical method to analyze the data again and explain it in detail.
3. “Results – miR-875-5p promoted cell proliferation and metastasis of ESCC cells” Line 152-154. “According to the TCGA database, we found that miR-875-5p was amplified 6% and 5% in EAC and ESCC, respectively, while approximately 1% were deleted in EAC and ESCC (Figure 1A).” In the TCGA database, the CNV information only gives the data of Level3, and the CNV of special gene cannot be directly obtained, so further analysis software is needed. The author should present the analytical method or the source of CNV of MIR875. In addition, miR-875-5p is the mature body of miRNA, which is an RNA-level concept, without the presence of CNV, so the authors should present CNV of MIR875 gene.
4. “Results – miR-875-5p targeted the CAPZA1 3’UTR containing the T>G alteration at rs373245753” Line 169-172. “…employed a pair of PCR primers based on…” The author should give the sequence information of primers and probes in the method.
5. “Results – miR-875-5p targeted the CAPZA1 3’UTR containing the T>G alteration at rs373245753” Line 179-181 and Figure 2E. “As shown in Figure 2E, for the pGL3-CAPZA1(T) construct, the miR-875-5p mimic significantly reduced luciferase activity compared with miR-NC…” In Figure 2E, the vertical axis is shown as the relative luciferase activity, so what is the reference for this experiment? The author should be shown on the chart and stated in the results.
6. “Results – CAPZA1(T) was negatively correlated with the proliferation of ESCC cells, while CAPZA1(G) attenuated such a negative effect” Line 188-190. “Cells were stably overexpressed with CAPZA1(T) and CAPZA1(G) by lentiviral infection…” What structures of the CAPZA1 gene are the authors overexpressing here? CZPZA1-5’UTR-CDS-3UTR? CZPZA1-CDS-3URT? Or CZPZA1-3URT? Please explain clearly in the corresponding position.
7. “Results – CAPZA1(T) was negatively correlated with the proliferation of ESCC cells, while CAPZA1(G) attenuated such a negative effect” Line 185-197. If CAPZA1 is a tumor suppressor gene, it is reasonable that its high expression has an effect on the malignant phenotype of esophageal cancer, but why does overexpression of CAPZA1(G) promote the growth of esophageal cancer cells instead (Figure 3A)? Therefore, I think it is necessary to overexpress CZPZA1-CDS alone to observe its effect on esophageal cancer cells.

Minor comments:
1. “Abstract” Line 22-33. In the abstract, the methods and results of this study are too simple. Please modify the abstract according to the requirements of the journal of PeerJ.
2. “Materials & Methods – Stable and transient transfection” Line 90-91. “Stable recombinant plasmids over-expressing CAPZA1(T) and CAPZA1(G) by lentiviral infection and matched control vectors were constructed by Obio Technology Co. Ltd…” The number or name of lentiviral vectos should be presented.
3. “Results – miR-875-5p targeted the CAPZA1 3’UTR containing the T>G alteration at rs373245753” Line 166-167. “We used public databases and predicted that miR-875-5p targeted the CAPZA1 3’UTR containing the T>G alteration at rs373245753 (Figure 2A)…” The author should introduce the predictive software which predicted the binding of miRNA and mRNA 3’UTR used in the method.
4. Figure 2B. The schematic diagram which author given is mRNA of CAPZA1, but "113162075", "113213865" and "113214241" are the physical locations of genomic DNA. Please do not mix them.


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: MIR-875-5P EXERTS TUMOR-PROMOTING FUNCTION VIA DOWN-REGULATION OF CAPZA1 IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: I am afraid to say that, even if the experiments seem well carried out, this article does not rest on any foundation.
1 / Even if the gene is amplified, the authors must prove that this amplification is associated with increased expression, which has never been described in patients. In my opinion, the amlification is not specific and we find an entire region (including miRNAs) amplified, so the effect of miRNAs is an effect among hundreds / thousands of genes.
2 / miRNA is not expressed in any CCLE cell line "upper_aerodigestive" or "esophagus" or "head and neck".
3 / The rs373245753 which is described in the article is only found on pubmed but does not contain any frequency so it has never been described as real in the 1000 genome project.

So I cannot accept this article which for me is only a series of experiences based on artefactual hypotheses.


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: MIR-875-5P EXERTS TUMOR-PROMOTING FUNCTION VIA DOWN-REGULATION OF CAPZA1 IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Improved by revisions.
Nothing to add
Experimental design: nothing to add
Validity of the findings: nothing to add
Additional comments: nothing to add


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

Input:
Title: MIR-875-5P EXERTS TUMOR-PROMOTING FUNCTION VIA DOWN-REGULATION OF CAPZA1 IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Esophageal cancer is one of the most unknown and deadliest cancers worldwide, mainly because of its extremely aggressive nature and poor survival rate. Despite improvements in the management and treatment of esophageal cancer patients, the general outcome remains very poor for overall 5-year survival rates (∼10%) and 5-year postesophagectomy survival rates (∼15-40%). Esophageal cancer is often diagnosed during its advanced stages, the main reason being the lack of early clinical symptoms and efficient genetic markers for diagnosis and treatment. In this manuscript, Dr. Nan Kang and colleagues investigated the role of miR-875-5p and SNP of CAPZA1 3’UTR (rs373245753) in proliferation and metastasis of ESCC cells in vitro. The study’s conclusion showed that miR-875-5p may function as an oncogene via down-regulation of CAPZA1 expression in ESCC. Although the author answered all of the questions, I still have some questions about the responses, which need further answers from the authors.

Major concerns:
1. “Rebuttal letter – Major Q1” Figure 3E to H. In the two experiments of Ctrl and CDS, CDS and CDS+ Mir-875-5p, are the processing factors of the CDS group the same? In Figure 3F, the clone number of CDS group is about 300. In Figure 3H, the clone formation number of CDS group is about 200. Why is there such a big difference in the number of clones generated in the same experimental treatment? Is there a statistical difference? Is it repeatable? The author must answer this question carefully, or it may give the reader the impression that experimental results in this study are very unstable.
2. “Rebuttal letter – Major Q2” Although the author makes two group comparisons, it is not possible to use the t-test. In the same repeated experiment, if more than two groups (multi-group) are grouped, ANOVA test and post test (eg. Tukey test) should be used for two groups comparison. The author should re-analyze the relevant data according to the statistics tutorial.


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: MIR-875-5P EXERTS TUMOR-PROMOTING FUNCTION VIA DOWN-REGULATION OF CAPZA1 IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Esophageal cancer is one of the most unknown and deadliest cancers worldwide, mainly because of its extremely aggressive nature and poor survival rate. Despite improvements in the management and treatment of esophageal cancer patients, the general outcome remains very poor for overall 5-year survival rates (∼10%) and 5-year postesophagectomy survival rates (∼15-40%). Esophageal cancer is often diagnosed during its advanced stages, the main reason being the lack of early clinical symptoms and efficient genetic markers for diagnosis and treatment. In this manuscript, Dr. Nan Kang and colleagues investigated the role of miR-875-5p and SNP of CAPZA1 3’UTR (rs373245753) in proliferation and metastasis of ESCC cells in vitro. The study’s conclusion showed that miR-875-5p may function as an oncogene via down-regulation of CAPZA1 expression in ESCC. The paper is improved and most concerned raised by the reviewer have been addressed.


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

Input:
Title: MIR-875-5P EXERTS TUMOR-PROMOTING FUNCTION VIA DOWN-REGULATION OF CAPZA1 IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA
Review round: 4
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Esophageal cancer is one of the most unknown and deadliest cancers worldwide, mainly because of its extremely aggressive nature and poor survival rate. Despite improvements in the management and treatment of esophageal cancer patients, the general outcome remains very poor for overall 5-year survival rates (∼10%) and 5-year postesophagectomy survival rates (∼15-40%). Esophageal cancer is often diagnosed during its advanced stages, the main reason being the lack of early clinical symptoms and efficient genetic markers for diagnosis and treatment. In this manuscript, Dr. Nan Kang and colleagues investigated the role of miR-875-5p and SNP of CAPZA1 3’UTR (rs373245753) in proliferation and metastasis of ESCC cells in vitro. The study’s conclusion showed that miR-875-5p may function as an oncogene via down-regulation of CAPZA1 expression in ESCC. The author added to the experiment after his own discovery of the error. Through analysis, I agree with the author to replace with new experimental data, and believe that the conclusion of this study is still well-documented.


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

Input:
Title: THE RELIABILITY AND VALIDITY OF THE WEIGHT-BEARING LUNGE TEST IN A CONGENITAL TALIPES EQUINOVARUS POPULATION (CTEV)
Review round: 1
Reviewer: 1

Output:
Basic reporting: Thank you for the opportunity to review this paper. Overall, I feel this paper is well written and provides new knowledge in the space of home monitoring of CTEV. In particular, providing data which is objective. This paper is well written and clear throughout.

The abstract is well written and the background provides a good context. The structure is clear and the figures and tables are appropriate. The results are relevant to the hypothesis.

Line 100 – the authors refer to a ‘six-week casting process. Consider adding – ‘approximately’ as some take longer.

Line 103 – states ‘in those who do not comply’. Consider reframing to ‘unable to comply’. There is some anectodal thought in this space that compliance may be related to an uncompliant foot rather than an incompliant patient/ family.

Line 107 – states that ‘The primary sign of relapse is a reduction in ankle range of motion’. As this is a deformity which occurs in multiple planes, relapse can also occur in multiple planes. Consider revising to ‘One of the primary signs of relapse is a reduction in ankle range of motion’.

Line 140 – consider changing WBL to WBLT for consistency or define WBL in addition to WBLT.
Experimental design: Line 112 – This is a good paragraph which highlights the need for regular monitoring. Consider adding a sentence around early identification of relapse to minimise the intervention required. This could link nicely to the final sentence around ‘useful in early identification of relapses’.

Sample size was justified well.

Methods – consider stating if participants were blinded to each other’s measures.

Line 172 – Inclusion criteria – Please state if idiopathic cases were only included, or if atypical, those associated with syndromes included.

Line 257 – good justification to use the second measure.

Overall the study was designed well with good rigorous measures such as blinding of the measures until the investigator was happy with the placement. There is sufficient detail to replicate this project.
Validity of the findings: Table 2 presents reliability and validity data. I am wondering if the Mean (SD) column could please be revised for greater clarity. I am unable to ascertain what this data specifically represents.
 I would be interested to know what ranges / averages / median (appropriate measure) were found in this cohort? My query relates to whether, if there was wide variability in the ranges we could be more confident that these tools could measure a wide range of DF scores reliably. If, however, the range of DF was small, we could only be confident in the reliability within that range. It may be possible that the data provided covers this, apologies if this was the data in Table 2 which I misinterpreted.

Conclusions are well stated and within the scope of the project. Overall data is robust and statistically sound.
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: THE RELIABILITY AND VALIDITY OF THE WEIGHT-BEARING LUNGE TEST IN A CONGENITAL TALIPES EQUINOVARUS POPULATION (CTEV)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Basic reporting: The style and clarity is of high standard.  The referencing depth and breadth could be improved upon with regards the statistical tools/rationales as per the PDF comments provided.
The introduction is succinct and effective.  The discussion is also well tethered to the rest of the paper and sets out useful future work that is needed.
The figures seem easy to comprehend and of use.  The raw data is provided and is easy to digest especially considering the small sample size required for such studies.
Experimental design: Experimental design: The selected methods fit the aims of the paper well.  The procedure used could be expanded upon as per PDF comments to enhance further.  The statistical methods used seem sound and logical but enhanced referencing/justification could be added.  Good, honest mention of the challenge of getting inquisitive children to do something repeatedly whilst trying to blind them from the results.
Validity of the findings: Validity of the findings: The impact of the findings should be significant especially in such cases and with remote, vulnerable communities.  If you can provide info regarding the samples community backgrounds that would enhance the impact and the external validity of the claimed benefits.  There needs to be re-wording around the link to clinical relevance as per the PDF comments made.  The limitations are open and honest as well as considerate of the wider picture that is not entirely captured by this single direction measurement.
Additional comments: Your paper is well written and of great interest to such a large population. The methods chosen to investigate seem appropriate and reflective of current telehealth and remote technology use movements/developments.
The paper is of use and with the aforementioned changes should be of great use to a wide audience.  The topic of accurate measurement and enabling patients/families to take responsibility for such methods is of use outside of this pathology group too. The paper flows well and is easy to read and digest.


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

Input:
Title: THE RELIABILITY AND VALIDITY OF THE WEIGHT-BEARING LUNGE TEST IN A CONGENITAL TALIPES EQUINOVARUS POPULATION (CTEV)
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The revisions in this paper have improved the overall work. I congratulate the authors on 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: DETERMINING THE INFLUENCING FACTORS OF PREFERENTIAL FLOW IN GROUND FISSURES FOR COAL MINE DUMP ECO-ENGINEERING
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this manuscript, the Authors investigated the morphological characteristics of preferential flow. The overall language of the manuscript is Ok, proofreading by a native speaker will further improve the quality of the manuscript. Language issues have been observed regarding the correct usage of articles and abbreviations e.g.  “GF” in line 44. In Line 105, from “Water flows” I presume you mean “water flow in soil”. The introduction is well written, however, in the last paragraph adding few studies regarding ground fissures in the dump area will further improve this section. All the Figures and Tables are relevant and well presented.
Experimental design: The topic of the manuscript fits well in the Journal and its objectives are of great interest to readers. The experimental design is easy to follow and the study can be helpful to understand the groundwater movement in mining areas. The study site description needs more details and it must be revised. In the study site description, the location map as shown in Figure 1 does not provide sufficient information regarding the study area it is better to pin this location at the province/country map. The climate conditions have been described in detail but it's not clear what is the main source of this information, how many and which years data have been used to get these averages?. It is better to add a flow chart of your methodology.  There are  61 ground fissures in total why only 3 have been selected? In section 2.2.2 Line 219-221 add some references for the drying method and ring method. Please elaborate on which type of statistical analysis has been performed and how you dealt uncertainties involved during the image processing.
Validity of the findings: The results are well presented and support the conclusions. The discussion is sound and relevant, the literature cited in the introduction is nicely used for discussion. The results will be more meaningful if Authors further elaborate regarding:
The effects of soil macropores on effluent rate and hydraulic conductivity.
The relationship between the dye coverage ratio and plant factors
Additional comments: I enjoyed reading this manuscript which is scientifically sound and the findings are interesting.


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: DETERMINING THE INFLUENCING FACTORS OF PREFERENTIAL FLOW IN GROUND FISSURES FOR COAL MINE DUMP ECO-ENGINEERING
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper revealed how preferential flow propagated as affected by natural ground fissures. The data were well analyzed, the contents of this paper are full and complete, the structure is reasonable. But some conclusions drawn from this paper need further consideration.
Experimental design: 1. there were no treatments. The author just selected some experimental points and I wonder how representative these three situ points are.
2. the author just selected one sprinkling intensity, i.e. 50 mm/h. I think this intensity could indirectly affect the preferential regime, including the stained area, strained patterns, infiltration depth, etc. I suggest the author carefully elucidate based on what this intensity was chosen.
Validity of the findings: 1. the scientific problems are not quite clear because of the poor introduction. I think this scientific research is doable but I cannot find the necessity in the introduction section.
2. the thresholding process from color image to binary image determined the infiltration path. I suggest the author explain how you overcome the differences of illumination and photographing time points, homogeneity of staining when the stained images were photographed and binarized.
Additional comments: Line 44: what’s the definition of ‘The preferential flow stained area ratios’. Does it make sense when you chose different size of research area?
Line 54: how can you determine the 3-dimensional water content prior to the experiments?
Line 103~111: the authors did not specify the necessity of research and the scientific problems. The research background was not highly concentrated on the research problem. Such as, until Line 103~111, the author still introduce the research background regarding crack flow, rather than research progress.
Line 112~117: the same problem with Line 103~111, the research progress is not clear, especially on the phenomenon of preferential flow.
The whole introduction failed to introduce recent research progress on fissure preferential flow and did not put forward a clear research problem regarding preferential flow in ground fissures. I think this part should be thoroughly enhanced.
Line 255: the parameter ‘stained area ratio’ is inappropriate here because it is totally determined by the depth of the soil profile which is selected by the authors.
Line 461~476: the discussion on how ground fissures affected preferential flow should be strengthened, such as the whole hydrology process from sprinkling to the end of preferential flow; why the top 5 cm showed a matrix flow regime; the specific process of how preferential flow were “above wide and below narrow”, etc.
Line 487~489: I still cannot understand why the preferential flow will be promoted with the increase in the contact area between roots and soil. Please further clarify.


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: IMPROVING INSECT CONSERVATION ACROSS HETEROGENEOUS LANDSCAPES USING SPECIES–HABITAT NETWORKS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is very well written, clear to follow, and very interesting. There are a few minor typographic errors or missing words, so I suggest the authors check the manuscript thoroughly before publication.

I have one comment: as described in the methods, the study site is one protected area that contains four Natura 2000 sites. For most of the study, the authors refer to the one protected area as the overall habitat. I'm not 100% familiar with the Natura 2000 system, so I did find this a little confusing - for example, in Australia there are different types of protected areas, but they exist as independent areas..we don't generally have one type embedded within another type. Perhaps this could be clarified for readers not familiar with the system.
Experimental design: The study is an interesting application of an emerging method for understanding more complex relationships between species and their habitats. I am familiar with this method and found the experimental design here rigorous and well defined.

As someone who is familiar with this method, I was interested about the choice to use unipartite analysis for some aspects (L147). Could the authors perhaps include more detail to explain the reasoning for this and why unipartite was considered more useful here than bipartite? I think this would be helpful for others working on this emerging method of research.

L149: were the edges/links 'number of shared butterfly species'? I'm not clear how species information was included in the edges information.

Table 1: I think it would also be helpful for readers to include in this table some detail on the scale and meaning of the metric to assist interpretation, e.g. what do high vs low levels of these metrics mean? etc.
Validity of the findings: I think the results are valid and robust based on the aims and methods used. The authors have interpreted conclusions thoughtfully.
Additional comments: Nice 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: IMPROVING INSECT CONSERVATION ACROSS HETEROGENEOUS LANDSCAPES USING SPECIES–HABITAT NETWORKS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Manuscript is very well written in clear and concise English. I have made a few suggestions to reduce ambiguity (in the general comments section below), but none of these require major changes.
Experimental design: Nothing to add here. The experimental design is robust and methodology is rigorous and appropriate to address the research questions posed.
Validity of the findings: Nothing to add here. Results are clear and robust. The discussion and conclusions are appropriate for the results.
Additional comments: I thoroughly enjoyed reviewing this manuscript by Cappellari and Marini – I found it to be excellently written, with robust experimental design and clear results. The manuscript is very polished, with some complex ideas and methodologies that are presented in a simple and clear manner. The manuscript structure is concise, and the analyses used are robust and elegant. I must emphasise that this is a very timely piece of work following Marini et al 2019 and provides an elegant solution to an often complex and difficult problem in conservation ecology. Overall, I do not have any major criticisms of this work. My comments below are minor, and mainly concern clarity in the writing. Also, because this work is likely to be applicable to a general audience, the authors may need to provide more frequent and thorough explanations of the network jargon.

Minor comments:
Title: might be confusing for those who aren’t familiar with “Natura 2000 protected areas”. Perhaps rephrase or remove?
Line 18: you need to explain what “Natura 2000 protected areas” are here.
Line 21: give examples of the traditional diversity measures that you used here.
Line 22: again, you need to explain what unipartite and bipartite networks are for your more general/lay audience.
Lines 23-24: “key habitats for butterfly conservation” is a little vague. What do you mean by “key habitats” and how did you define these? Are they habitats/patches with exceptionally high conversation value, for example?
Lines 29-31: just because species were observed in a particular habitat type doesn’t mean they were using resources in those habitats. Or were they? Can you explain this a little more?
Line 33: “…providing key implications for conservation” is too vague. You need to elaborate on what these implications for conservation are.
Line 39: could you provide examples of the most common habitat types here?
Lines 55-72: would it be worth discussing “Hackett et al. 2019. Reshaping our understanding of the roles of species in landscape-scale networks. Ecology Letters. 22,1367-1377”. This paper uses a different network approach to your study but essentially seeks to address the same problem in a somewhat similar way.
Line 75: can you provide a little more explanation as to why butterflies are an excellent indicator of habitat quality?
Line 77: which life history traits? Can you give examples?
Lines 84-85: as indicated in the abstract, what do you mean by “key habitats” and how did you define these? You just need to give a little more context.
Lines 125-126: it’s not clear why you selected these two diversity indexes, especially considering that there are so many indexes available. I’m not saying that you should have looked at more, but you need to give a strong justification for using these two.
Line 150: you need to give a more detailed explanation of what centrality is, especially for those who aren’t familiar.
Lines 168-181: species names need to be italicised throughout.
Lines 197-198: do you mean that these indices were different from each other among habitat types?
Lines 222-223: again, it’s not clear whether these butterflies were using resources within the habitats that they were observed in, or if they were simply passing through. Can you clarify/expand on this?
234: weak compared to what? You need to provide some more context here.
Lines 240-241: some discussion around network stability might be useful here too?
Line 242: Differences in what?
Lines 250 -253: this all makes sense, but then what would be the directive/recommendation to conservation decision makers? I think you need to go beyond this broad statement and provide an example of what you would recommend based on your findings (i.e., are you saying that we allocate resources to conserve a certain subset of patches or that we need to spread resources to conserve/manage all patches?).
lines 260-262: this sentence is a little confusing. Over what time scale was this “evolution”? Do you have a relevant citation(s)?
Line 263: central in terms of the network structure or in a spatial sense?
Lines 270-271: so, is the key driver here low level/frequency of disturbance? Maybe you could explore that concept a little more.
Lines 283-284: again, I wonder if butterflies in some of these patches were simply passing through (perhaps due to the patch’s location relative to other more important/resource-rich patches) rather than using resources within the patch they were observed in? Does spatial location of a particular patch relative to other patches (and their importance) play a role in determining the importance of that patch for butterfly species? I guess this becomes quite circular.
Lines 290-291: I absolutely agree but feel this statement is too vague and not really that helpful in a practical sense. Can you take the discussion in this paragraph one step further and explain how your findings could be applied by a conservation practitioner (i.e., by giving an explicit example)?
Line 296: needs to be written in present tense.
Line 303: I’m not sure that “ineffective” is the right word here. Protecting only one or a subset of habitats might be suboptimal, but not necessarily “ineffective”.
Figure 1: what do the codes within each coloured node represent? Each figure needs to be stand-alone.
Figure 3: I wonder if you could indicate on this graph which habitats were statistically different from each other, as determined by your linear models? Package emmeans will do this for you (using contrasts/comparisons).
Figure 4: what do the different colours represent? This need to be explained in your figure caption.


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

Input:
Title: BACTERIAL META-ANALYSIS OF CHICKEN CECAL MICROBIOTA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is written in a good English with the sufficient amount of background information. It has the professional structure and self-contained.
Experimental design: The research question is well defined. The paper is providing a meta-analysis of taxonomic diversity of cecal bacterial taxonomy diversity in chicken. Methods are well-described and appropriate expect some minor issues listed below.
Validity of the findings: The findings look valid and meaningful. Although the papers with raw data are provided it would be good to add to the Table S1 SRA IDs of the raw read datasets and PubMed IDs of the papers to make the search easier. Additionally, it would be beneficial for reproducibility to provide exact commands for Trimmomatic and QIIME2 used for the data processing.
Additional comments: Line 135: The title should be “Core microbiome”
Line 189 and below: “Salmonella Enteriditis” Usually species name of a bacteria is written in italics and lower case.
Line 199: The text states that Kruskal Wallis test was implemented, but Figure 4C shows Wilcoxon test.
Line 207: “(p-value = 0.001)” QIIMEII should give you q-value which is more appropriate for pairwise PERMANOVA than unadjusted p-value. Was p-value the same for all comparisons?
Line 437: Please provide explanation of the two-step exclusion criteria on the Figure 1 as in the methods section they are described as a one step with 3 (i-iii) criteria. It would be good to provide more explanation of the steps in this flow chart.
Line 444: It is impossible to associate index numbers to the study from the table 1 as according to the table S1 the numbers are associated to studies even not in alphabetical order. Please add indexes to the table 1.
Line 455: Please clarify meaning of asterisks. Also is not clear what base mean stands for. Kruskal Wallis test shows if different groups are drawn from the same distribution or not. Was the test implemented as a pairwise against some artificial mean group? What does a different number of asterisks mean? Maybe authors should consider remove the asterisks as I don’t see if they were discussed.


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: BACTERIAL META-ANALYSIS OF CHICKEN CECAL MICROBIOTA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please see detailed comments below.
Experimental design: Please see detailed comments below.
Validity of the findings: Please see detailed comments below.
Additional comments: The ms by Cardenas et al. “Bacterial meta-analysis of chicken cecal microbiota” synthesizes and reanalyzes 16S rRNA sequence data from nine previous studies with a goal to identify a core set of microbial taxa in the chicken cecal microbiome.  While this is an interesting objective, the ms needs to be strengthened considerably before it would be suitable for publication in my opinion.  Some general and specific comments are below with some suggestions for improvement.

General comments:
Similar work has recently been performed that the authors do not mention.  Please see and discuss:  NPJ Biofilms Microbiomes 4, 27 (2018).

Searching for a universal set of core taxa in the chicken cecal microbiome is a valid and important question.  The main result of five ‘core’ genera is interesting but needs to be strengthened in several ways.  This result is such that it can only be defined by loosening criteria to allow only 50% membership.  This is not surprising given that the data analyzed comes from such different contexts, arguably undermining this result.

As Figure 5 shows, there are significant differences among communities according to breed, study, experimental group, etc.  These results are interesting and have been shown previously.  One additional parameter not shown here that is also well known to have a significant effect on the cecal/GI microbiome is the age of the birds.  Given the heterogeneous population of birds collected from the nine studies in question, it’s no wonder that finding a core set of taxa requires such non-stringent criteria.  I agree with the value of searching for a core microbiome, but the ms as it currently stands is trying to force a square peg into a round hole.  Recognizing that things are different and then trying to show how they are the same is somewhat illogical but rather than being a fatal flaw in this ms, this situation presents some interesting opportunities that can easily improve the ms significantly.

Because of the community-wide differences among studies (many of which are significant as the authors show with permanova), it would be more logical and informative to stratify the data by study, grouping into categories as appropriate.  Identifying sets of core taxa for each study/condition would be more meaningful and biologically relevant.  These core sets of taxa could then be compared to identify commonalities AND differences.  This type of approach would significantly strengthen the work and increase its value to researchers working in this field.

Finally, the number and types of ASVs within each core genus should be reported.  Collapsing the ASV classifications to the genus level is valid, but since this information exists, it should be reported.  These data would be helpful to other researchers and should be reported for full disclosure as the authors advocate.  Reporting these in a phylogenetic context (i.e. tree) would be most helpful.

Some additional specific comments:

L105-107:  unconventional reporting and not meaningful description of roles, rubrics, decision-criteria, etc.

L109: review

L119-120 – Seems like a kind of crude approach.  How do you know this was sufficient?  Many barcode/adapter combinations are up much longer than this and are of variable length.  Either search for specific sequences and/or identify sequences that were removed.  Since ASVs are being derived, these trimming procedures can have important effects on the results.

L129:  taxa

L131:  Please note Greengenes does not appear to have been updated since 2013.
Also see BMC Genomics volume 18, 114 (2017) for comparisons of 16S rRNA databases.

L229:  Need specific citation for reference here.
L231-2:  This argument needs to be refined.  Please cite evidence that 99% or 97% OTUs can merge genera.  Additionally, this argument doesn’t seem to be valid since ASVs were only used to identify genera.


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: BACTERIAL META-ANALYSIS OF CHICKEN CECAL MICROBIOTA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors improved manuscript sufficiently for publication.
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: LEAD TRANSFER IN THE SOIL-ROOT-PLANT SYSTEM IN A HIGHLY CONTAMINATED ANDEAN AREA
Review round: 1
Reviewer: 1

Output:
Basic reporting: I consider this manuscript of interest because it contributes to current knowledge on Pb transfer in soil-root-shoot systems in the Andean region, a seldom explored topic in this part of the world.
I strongly suggest the authors to find editorial assistance. The style and writing needs a major review and more care and prolixity to avoid grammatical and concordance mistakes, which already makes the article difficult to read in some sections. In addition, the article has very long sentences (e.g. L6-L11), which makes it difficult to follow the ideas.
Although the article has a good grasp of relevant and recent literature, the authors fail to support statements with appropriate references, and in some cases they do not use them correctly.
The structure of the article is disorganized, there is no clear hypothesis, information is repeated in different parts, and tables and figures contain the same information.
Experimental design: This article meets the aims and scopes of the journal.
The research question is not well defined, although the topic is important and deserves attention given the lack of information on metal soil polluiton and translocation to grass tissues in the Andean region.
Authors should explain why they compare between natural and cultivated pastures, and  two seasons in the year. There is no hypothesis to support this experimental design.
Methods do not describe pH and organic matter analysis, and do not mention brand/model of equipment used.
Validity of the findings: All data have been provided.
A strong concern is that results are not compared to reference or background Pb soil levels in Peru, nor to widely used international standards.
Although conclusions are limited to supporting results, they do not report on the dynamics of Pb transfer in the soil-root-shoot system as proposed in the research question.
Additional comments: I have the following observations and suggestions:

INTRODUCTION
L31. Toxic for what biological system?
L33. Compared to what standard or reference scale: wildlife, ecosystems, human health? Methylmercury, mercury, chromium VI, cadmium, arsenic, organochloride compounds, among others, are much more toxic and dangerous.
L51-53. I recommend the authors Chang Kee et al 2018 (Accumulation of heavy metals in native Andean plants: potential tools for soil phytoremediation in Ancash (Peru). Environ Sci Poll Res 25(34), 33957-33966. (10.1007/s11356-018-3325-z))
L60. Says 1992. Should say 1922.

METHODS
L93. Avoid Latitude and Longitude
L 93. Include m “above sea level”, and then only refer as “m a.s.l.”
L96. Repeats information (1922) given in the introduction. Avoid.
L100. Repeats information given in the introduction about the degree of pollution. Avoid.
L108-109. Should be included under Laboratory Analysis.
L114-L118. Should be included in Figure 1 legend.
L125. Reduce number or references.
L133. How was pH and organic matter analysed? Give methods and brand/model of equipment used. Also for FLAA.
L149. Authors must refer also to the National Soil Environmental Quality Standard. Why using a Standard from Finland as an international reference? There are others commonly used, such as US EPA, the Canadian Environmental Protection Act Registry, and the Dutch RIVM Standards.
L155-L164. The authors use the transfer factor incorrectly. Hu et al., 2017 mention, “this factor represents the potential capability of heavy metals’ transmission from soil to the edible parts of vegetable”. In the present work, this would only be represented by TBss = [Pb shoot]/[Pb soil]. However, the authors also use it to describe the translocation root/soil and shoot/root. In this case, I recommend the application of the bioconcentration factor (BCF) (Yoon et al. 2006), and translocation factor (TF) (Zu et al. 2005), which can be calculated using the following formulas:
BCF = metal contents in root tissue/metal contents in soil
TF = metal concentration in shoot tissue/metal concentration in root tissue
References
Yoon J, Cao X, Zhou Q, Ma LQ (2006) Accumulation of Pb, Cu, and Zn in native plants growing on a contaminated Florida site. Sci Total Environ 368:456–464. https://doi.org/10.1016/j.scitotenv.2006.01.
Zu YQ, Li Y, Chen JJ, Chen HY, Qin L, Christian S (2005) Hyperaccumulation of Pb, Zn and Cd in herbaceous plants grown on lead–zinc mining area in Yunnan, China. Environ Int 31:755–762. https://doi.org/10.1016/j.envint.2005.02.004
L166. If analyses include the effect of type of pasture and sampling period, two-way ANOVA should be performed.

L173. RESULTS
Authors should develop in more extent this section and not merely refer to tables and figures.
The information in Tables 2 and 3 is the same as in Figures 2 and 3, respectively. This should be avoided. I suggest to use Tables.
There are no results for soil composition.
L 196. This is part of the Discussion section.

DISCUSSION
L230-231. This quote is something the authors do not know or have not proven for the case of La Oroya. I suggest they mention it as a possibility but not as a fact, unless they provide evidence of this biomagnification process.
L232. This relationship between Pb in the air and the accumulation chain proposed is not clear.
L234. This is obvious and is not a finding of this work. Polluted soils will always have more metal concentration than plant tissues.
L237-238. An explanation and reference are needed to support this statement.
L241-243. A reference is needed for 25 mg/kg as the world average concentration. Authors should compare Pb soil concentrations found in this study with polluted sites around the world to state that soils of the central Peruvian highlands are at the upper end on a global scale.
L255-258. It is crucial that the authors mention soil Pb background levels for Peru in order to assure the extent of impact of the metallurgic activities in La Oroya on soil quality and its potential negative effect on environmental and human health.
L266. Are considered toxic for what biological system: plants, animals, humans, the environment?
L268. Morera et al., 2013 report on suspended sediment yield and hydrology of Andean rivers, not in soil pollution. Authors must be careful when using others’ results to support their findings.
L286. Indicate Pb concentrations under which these mechanisms to attenuate the absorption and transfer to the aerial parts were observed. In addition, mention phytostabilizing strategies in plant roots based on exudates and polymers.
L320. Indicate Pb levels in other regions mentioned.
L364-368. Should be moved to “Implications for central Peru”.
FIGURE 1.
The legend is not clear about the significance of shapes (square, circle) and colours regarding period (April/September) and origin (cultivated/natural).
Avoid picture of the city. It does not provide useful information. Replace image for sampling sites with a black/white map showing distance from La Oroya city.


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: LEAD TRANSFER IN THE SOIL-ROOT-PLANT SYSTEM IN A HIGHLY CONTAMINATED ANDEAN AREA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: - Line 134 : What the authors mean by normal temperature? the usual procedure for plant material drying is 60-80°C for 48h or till a constant weight was obtained. This is supported by many published works for example: Maryland, 1968, Agron. J. 60, 658-9; Sharkey 1970 Grass Forage Sci. 25, 289-294; Ramsumair et al., 2014.Trop Agric. 91,179-86; Cone et al., 1996. J Ag Sci. 126, 7-14).

- For laboratory analysis, the authors wash the roots and shoots with deionized water. This washing is poorly appropriate and does not eliminate the metals adsorbed on the surfaces as well as the Pb deposits on the shoots that can increase the Pb level recorded in the aboveground part of the plant. In addition to washing plant material with water, many authors rince roots and shoots using cold 0.2 mM CaSO 4 solution (auguy et al 2013). this makes it possible to eliminate the Pb adsorbed on the surface of the organs and makes it possible to really estimate the metal absorbed by the plant
Validity of the findings: - Line 372 : “Our findings are important and impressive and demonstrate that the grasslands in the central highlands of Peru would have high concentrations of heavy metals compared to other parts of the country and the world…….”. There is no data in this work that support such confirmation. This study is focused on lead and it is difficult to generalize to other heavy metals.

- In this work, it would be knowledgeable if the authors provided informations supporting that the Pb analyzed in the aerial part of the plants comes from the soil and that the Pb from the air do not overestimate the results.
Additional comments: Lead is a highly toxic heavy metal that affect plants growth and yield and impact grass based forage quality in many natural and cultivated pastures areas. This paper provides new data and interesting point of view on this topic.
There still some questions and points to clarify

- Figure 1 : The study site and sampling location are 20 kilometers from the lead extraction site. To better estimate the impact of La Oroya Mining-Metallurgical complex on the site contamination, I propose to indicate the orientation of the sampling site and the direction of the wind.
On the map, I suggest to eliminate the arrows and to put a square indicating the studied area.
The images also show a river that may contribute to the transport of metal contaminants. If it is the same river, indicate the direction of the water flow.

- Line 59 and 60: it seems there is an error in the date. Put “The polymetallic mining-metallurgical center located in the central sierra, which has been in operation since 1922” (as indicated in line 96) instade of “The polymetallic mining-metallurgical center located in the central sierra, which has been in operation since 1992 ”

- It appears that the study area is subject to emissions of particulate matter that contaminate the surrounding ecosystems. This source of Pb is described in the introduction and the authors are well aware of it (lines 61, 62 and 63). However, they have not integrated this aspect into their experimental approach and did not take this into account in the analysis and discussion of the results.

- In discussion of lead concentration in the soil, the authors give no reference to mobile and mobilizable fractions of Pb. This is important because the Pb is poorly soluble and a very small part of the metal is really bioavailable to plants. In practice, such fractions can be determined by quantification of Pb in CaCl2 and EDTA extractable fractions (Auguy et al 2013).

- Line 290 and 291 : this sentence is not clear

- Line 319 : the authors claim that “In the current study, no differences were found between Pb concentrations in natural and cultivated pastures”. I think this is mostly due to Pb deposit from air emission Pb particulate matter.

- Line 337 : please put 26.8 mg/kg instead of 26.8 mg/g

- Line 340 : “In practice, accumulation of Pb in forage roots instead of shoot are desirable… “ will be more appropriate for this sentence

- Lines 355 to 357: BF also reflect the capacity of plants to mobilize and uptake heavy metals.


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: LEAD TRANSFER IN THE SOIL-ROOT-PLANT SYSTEM IN A HIGHLY CONTAMINATED ANDEAN AREA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Although there has been some improvement, I’m afraid the manuscript still does not meet the standards of a scientific article in terms of writing and English style. I think the information is worth considering, especially because it covers a seldom explored topic in the Andean region. However, it needs to be presented in a more organized way, in particular in the Introduction and Discussion. These sections need to be restructured. Ideas do not follow a consistent path, from general to specific topics, which makes it difficult to read. Also, the manuscript still has very long sentences (e.g. L62-L68, L79-L85, L86-L92), which is not helpful to follow.
Experimental design: Still is not clear for me what is the hypothesis behind sampling native and cultivated grasses. Also, it would have been very interesting if the authors analysed metals in each grass species. This may provide important information on bioaccumulation profiles in edible parts, and hence, the potential risk of each grass species to animal (and human) health.
L102. I meant to avoid the terms “Latitude” and “Longitude”, not the information on coordinates.
L102. Include m “above sea level” for the first time, and then only refer as “m a.s.l.”
L116-118. Should be included under Laboratory Analysis. This was noted and suggested in the first review.
L149. Include reference to the Walkley-Black method and equipment used.
L189. If analyses include the effect of plant species and sampling period, a two-way ANOVA should be performed to check for interactions between these two factors.
Validity of the findings: There are no results for soil composition although calculation of percentages of sand, silt and clay are mentioned under Laboratory analysis. I noted this in my first review.
- Discussion
L258. This quote should explicit that this is a possibility. I suggest “which would be deposited in the soil and most likely assimilated by plants and animals…”
L260. Include Kryshna and Mohan, 2016 in the list of references.
L266. A brief explanation to this barrier mechanism should be included.
- Figure 1.
The images are not clear enough for a publication. Replace image of sampling sites with a black/white map showing distance from La Oroya city.
Additional comments: Comments are already included in the above sections.


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: LEAD TRANSFER IN THE SOIL-ROOT-PLANT SYSTEM IN A HIGHLY CONTAMINATED ANDEAN AREA
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The revised version of the article is consistent with the observations and comments. Several sections of the manuscript have been really improved.
The maps in Figure 1 can be improved. The position of the studied site is still not clear and the the writing in the maps is very small.


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: GRAZER DENSITY AND SONGBIRD COUNTS IN A RESTORED CONSERVATION AREA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written and quickly gets to the point of the study, there are only two minor suggestions on the background information and writing:

Lines 36-38: A more balanced introduction could mention when negative effects of grazing have been observed on bird communities (e.g. in cases of over-grazing) or be more specific to what types of grazing that may be beneficial to bird communities.

Line 52:  “For example, (Batary et al. 2007) found that...” should read “For example, Batary et al. (2007) found that...”.
Experimental design: The study is carried out in a systematic and purposeful way. My main point here is that the analysis is very limited in relation to the data that has been collected and the questions being asked. Please explain why the response variables were limited to counts of each species across the whole season in the manuscript, or consider expanding the analysis according to the suggestions below. Another important point is how the purpose of the study and its method is described, see comments below.

Firstly, it would also be useful to know what time of the year the birds benefit from presence of grazers, and differences to proximity of horses or cattle. There is an emphasis on the strength in carrying out the study for a long time on lines 219-224. Although this is a strength, the opportunity in describing when the birds are most dependent on grazer presence is missed. The manuscript would be strengthened by giving further detail to how the grazer-bird relationship changes over time. This would, for example, provide useful knowledge to nature reserve managers where grazing is part of the conservation measures during parts of or the whole year. (See also the comment regarding lines 253-255 below.)

Moreover, it would also be interesting to know how the overall abundance of birds and species richness of songbirds was related to grazer presence. Given the data that has been collected, these variables should be easy to incorporate in the analysis or analyse separately. If there is a reason for not including these measures in the analysis, this reason should be stated in the manuscript.

Lastly, if all grazers are marked with individual GPS collars, it should be possible to test whether the birds preferred grazing by one grazer more than the other (i.e. horses or cattle). This information is, for example, useful when selecting the type of livestock/grazers for conservation management.

Lines 253-255: This sentence makes the reader wonder why this was not investigated in the current study as the data seem to be available. I’m referring to one point in the reviewing criteria: “Coherent bodies of work should not be inappropriately subdivided merely to increase publication count.” If more data is needed, please state (briefly) what more information that is required to carry out this analysis. I also suggest making this statement more relevant to the reader by encouraging more studies on this topic from researchers in general, rather than discussing what the authors can do in the future.

Lines: 55-59: It is not clearly stated what new information the correlation of grazer GPS points and bird counts bring, in comparison to the more common method described above (measuring bird density/community under a specific herbivore density). This study would have a higher relevance to conservation management if the reason for the method used was clearer, especially when the differences in study methods is used to motivate the study. For example, is there a problem with the more common method, does the method used here provide a more robust understanding of how dependent different bird species are on recent grazing by livestock, or does the method used provide results that can confirm results obtained with another method? Moreover, adding a sentence or two after the first sentence in this paragraph (i.e. on line 56) about the ecological/conservation management implications of the knowledge obtained from this study would significantly improve this section.

Lines 218-219: Again, what new information does this approach bring to the field? If using the difference in methods as a justification for carrying out the study, it needs to be clear why another method is important.

Lines 101-102: It would be useful, if possible, to say how accurate/precise the GPS points are. Can the actual position of the livestock be assumed to fall within a certain range of the reported position? This would help in interpreting the reliability of the results.

Lines 195-196: “The only species that certainly bred on the 196 grazed area were the Skylark and the Red-Backed Shrike.“ - How was this confirmed, by observations of nests and/or displays of territorial behavior such as singing? For the species where breeding status within the survey area was uncertain, which observations were done? Providing these details would back up the statements about breeding status.
Validity of the findings: Lines 255-257: I commend the authors for being clear with the limitations in demonstrating causal effects from correlational studies. By also providing an example of the kind of study that would be required to demonstrate a causal effect, (e.g. a randomised and replicated grazer experiment) and encouraging such studies to be carried out by others, this statement gets more meaning and is more useful to readers in the same research field.
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: GRAZER DENSITY AND SONGBIRD COUNTS IN A RESTORED CONSERVATION AREA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This paper deals with the important issue of the effects of grazing on the abundance of birds, in this example in a grassland habitat. Although other studies have already addressed this issue, this experiment brings the novelty of using grazers marked with GPS allowing for a continuous analysis of grazing pressure. Although I think this study has interesting information, I have some questions and comments that I list below, expecting those can help improve the paper.

Introduction

Regarding the introduction I think that, although much information is given, there are some aspects that were not mention and could help to frame the work. Here are some examples:

Lines 42-44: the consequences of shorter vegetation can relate not only with food availability but also to food accessibility

Line 44: It is said that other birds “may be impeded by the effect of trampling”, but why? What are the effects of trampling that can affect birds?
Although some examples are given, I miss a deeper discussion here about the direct and indirect consequences of grazing for birds, namely through the effects on vegetation height and composition, soil compaction, etc...

Line 50: The authors focused mainly on the consequences for birds during the breeding season, but grazing can have further consequences in other parts of the year. I suggest this is mentioned here.

Lines 55-56: This sentence is difficult to read

Line 60: By counts you mean abundance? Total number of individuals?

Lines 60-69: I think that this paragraph needs a revision because it is a mixture of introduction with methods (for example it is not necessary to say here how many horses and cows were marked). Maybe it would be better if you start by a small description of management/grazing regime in your study area, then explain why is your work important and what is the novelty of your study and finally present the objectives

Line 66-69: This last sentence is very similar to the 1st in this paragraph. Also, I suggest that you improved the description of your objectives, maybe by using hypotheses. What were you expecting to obtain?

Materials & Methods

Lines 76-77: Is there any reference that you can add here? I suggest you rewrite the sentences that follows so it is clear that those were actions taken as part of that restoration project

Line 92: I think it is more correct to say cows or bulls...

Line 99: Do you know what is the estimated error (in meters)?

Line 117: Is it possible to include a map of the study area? I think it will help the reader to get a better notion of the study area, habitats, transects used for data collection, etc.

Line 119: What was the distance between parallel transects?

Line 122: I understand why you considered skylarks in song flights in your analysis, but depending on the scale used to make an association between grazers and birds use of the area, it may be difficult to make a strong connection...

Line 133: Again, here by count you mean total of observations during the sampling period?

Lines 144-146: I think this needs to be better explained. As far as I understood you had hourly data for the position of the animal, for 24h. So as a measure of grazing pressure you used the sum of animals present in the cell, every hour, for 30 days prior to bird count?

Line 161: Can you please provide a Reference to sustain these criteria?

Results

Lines 194-195: I suggest adding a table in Supp Material with a list of the total species detected and number of individuals/species

Lines 197-98: Did you collect data to confirm this information? If not, this is not a result of you study but an observation (although I understand that this information will be probably important to discuss some of your findings)

Lines 200-201: Please also include information in terms of nr of grazers/ha so these results are comparable with those of other studies in terms of grazing pressure.

Line 201: In figure 1 I think it would help if you could had information about months or the separation/limits of the three seasons that you considered in the analysis (instead of just having in xx axis the number of days from 1st January)

Discussion

Lines 217-218: I think this paper has good data to add information about the effects of grazing in bird abundance, however I think that it could go further and gain extra importance and strength if the author had data regarding other variables that could help explain the patterns found (namely regarding food abundance and/or availability; changes in vegetation caused by grazing etc.). As far as I understood this data are not available, so I suggest that at least these aspects are mentioned in the Discussion.

Lines 225-236: In this paragraph you present some possible explanations for the patterns found in terms of relationship of birds with grazing intensity. One think that I feel important to know is if those patterns for each species response were maintained thorough all sampling period, or was it more evident in some of the seasons?

I did not understand if you tested the possible influence of season, but, as you mentioned, the fact that you collected data in more than one season is one relevant aspect of you work.

Line 237: I agree that this work has the novelty of analysing this effect on a continuous scale but, on the other hand, it seems to me that this sampling design makes it more difficult to attribute a causal-effect relationship between grazers density and bird abundance.

Line 245-252: Don´t you think that some of these patterns detected for species as White Wagtail and Blackbird can be influenced by the low number of birds detected?

Finally, I suggest a careful revision to all the text since I detect some typos errors.


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: GRAZER DENSITY AND SONGBIRD COUNTS IN A RESTORED CONSERVATION AREA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I appreciate that the authors have considered all comments and suggestions from myself and the other reviewer. The inclusion of the new measures: total bird abundance, bird species richness and separate analyses of horse and cattle correlations to bird abundances have particularly improved the study.

Although it is good to avoid drawing conclusions of causal effects from correlative studies, I would say it is possible to speculate into what the results from a correlative study may suggest in a wider ecological context, while still making it clear that no causal effects can be confirmed. However, this is a matter of taste and the authors should be free in limiting their conclusions and speculations to what they feel is appropriate. The authors have given enough context to their study and enough motivation and explanation to their choices for the study to be accepted, in my opinion.


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: EXPRESSION OF VITAMIN D 1Α-HYDROXYLASE IN HUMAN GINGIVAL FIBROBLASTS IN VIVO
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is clearly written, providing sufficient literature references for background, methodology and discussion. The structure of the article is appropriate with figures and tables properly designed.

One issue is that the data in figures 1 and 2 are presented as relative mRNA expression, apparently each is relative to b-actin levels. However, figure 2 is simply a reshowing of figure 1. Is this just the controls or the periodontitis? Regardless, it would be much more appropriate to make the control set to 1.0 for all cases in figure 1, with the periodontitis shown relative to that amount. Then, it would make sense to show the data in figure 2, setting either one as the 1.0. As it stands, there is no reason for figure 2, and the data are hard to understand in figure 1.
Experimental design: The authors have previously shown that CYP27B1 is expressed in vitro, so it is important to show this expression in vivo, especially where it is difficult to distinguish cell type (fibroblast vs. epithelium). Thus, the immunohistochemistry data are important. The results are clearly shown and support the in vitro data. All of the methods are sufficiently detailed. That said, the ultimate significance of the fact that CYP27B1 is expressed in these tissues is not well elaborated. It would strengthen the manuscript to explain to the reader why it is important to know A) that CYP27B1 is expressed in both (or either) cell type, and B) that CYP27B1 expression apparently differs in hGF vs. hGE in periodontal disease.
Validity of the findings: The data appear to be fairly well presented. It would strengthen the rigor of the study to present the PCR data with each data point (either as a box plot or to keep the column graph, but to indicate each data point).  The conclusions are presented, but some speculation on the further utility of this information would truly strengthen the manuscript.
Additional comments: Overall, this is a straightforward study demonstrating the expression of  CYP27B1 in gingival tissue in vivo. It is important to the field to know this information, but the overall significance of the findings should be elaborated upon.


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: EXPRESSION OF VITAMIN D 1Α-HYDROXYLASE IN HUMAN GINGIVAL FIBROBLASTS IN VIVO
Review round: 1
Reviewer: 2

Output:
Basic reporting: Literature analysis is incomplete, own works are over cited.
Some figures need to be improved.
Experimental design: There are some questions regarding patient selection and tissue sampling.
Validity of the findings: The number of replicates is not mentioned for some quantitative data.
Additional comments: In the present study, the expression of CYP27B1 in the gingival soft tissue of periodontitis patients and healthy controls is compared. Vitamin D3 seems to play an essential role in periodontitis. Several previous studies of this group and other groups show that vitamin D3 could be locally converted into 25(OH)D3 and subsequently into bioactive 1,25(OH)2D3 by several cells of periodontal tissue.

The primary concern is the sampling of gingival tissue. In periodontitis patients, samples were taken from “part of the wall of periodontal pocket”, whereas, in the healthy subjects, the gingiva resected during crown lengthening procedure was used. First, how these anatomically different tissues could be compared? Second, the wall of the periodontal pocket could be the alveolar bone.

The second critical point is the control of serum vitamin D level or at least vitamin D supplementation by the study participants.

Further comments
Lines. 58-61. The conversion of vitamin D3 into 25(OH)D3 by the liver and subsequently into 1,25(OH)2D3 by the kidney is well known. However, the studies mentioned here (Liu et al. 2012a,b) refers only to the vitamin D3 conversion by gingival fibroblasts/periodontal ligament cells.

Line 62. 1-alpha hydroxylase CYP27B1 expression in the kidney was first detected before the study of Nykjaer et al.

Line 85. No analysis of CYP27B1 expression in endothelial cells was performed. Endothelial cells should be counterstained with CD31 or von Willebrand factor. Periodontal inflammation is characterized by increased vascularization and, therefore, the increased number of endothelial cells. Thus, the possibility that the increased CYP27B1 expression is due to higher endothelial cell numbers should be excluded.

The number of replicates should be shown for each experiment.
Gene expression analysis was done only for some 17 periodontitis patients and 12 healthy controls. How were these patients selected? What were the demographic and clinical data of these patients?
Immunohistochemistry, Figure 3. The expression of CYP27B1 should be shown by arrows so that inexperienced readers can recognize it.
Figure 3. Scale bars should be used instead of providing the magnifications because the pictures could be scaled during the publication process.
Figure 4. Any scale bar is absent.
Fig 5A. What kind of plot is it? Is it a box-plot? In this case, the median and quartiles should be visible. Otherwise, some graphical presentations, for example, histogram, should be used.

Discussion; lines 246-255. How could the increased level of 25(OH)D3 in the gingival crevicular fluid be related to the CYP27B1 expression? CYP 27B1 converts 25(OH)D3 into biologically active 1,25(OH)2D3 but is hardly connected to the local 25(OH)D3 levels.

Most existing literature suggests that vitamin D deficiency is associated with an increased risk of periodontal disease. This fact should be discussed in connection with the obtained data.


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: EXPRESSION OF VITAMIN D 1Α-HYDROXYLASE IN HUMAN GINGIVAL FIBROBLASTS IN VIVO
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have appropriately responded to the initial review.


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: EXPRESSION OF VITAMIN D 1Α-HYDROXYLASE IN HUMAN GINGIVAL FIBROBLASTS IN VIVO
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Thank you for considering the criticism.


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: LIMITED EFFECT OF A HIGHWAY BARRIER ON THE GENETIC STRUCTURE OF A GYPSUM SOIL SPECIALIST
Review round: 1
Reviewer: 1

Output:
Basic reporting: In my opinion there is no need for revision of the English (but also not my mother tongue). The manuscript provided sufficient field background with good article structure. On the other hand, several figures and table are still a lot of room for improvement.
Experimental design: I believe that the questions investigated here are very relevant to understand plant edaphic species adaptation and conservation in general. Thereby, the study is of broad interest for PeerJ readership. Although analysis methods described with sufficient detailed and information, I fee that additional analyses need for this study.
Validity of the findings: The conclusions are speculative because of weak evidence based on only 7 (or 6) loci. Furthermore, several interpretations for the results of analyses were wrong.
Additional comments: Martín-Rodríguez and colleagues investigated the genetic status of a soil endemic Lepidium species occurring in the fragmented landscape due to a linear infrastructure (i.e., highway). Using analyses of nuclear microsatellite as well as statistical analyses, the authors evaluated genetic diversity, structure, gene flow, the effects of landscape factors. The authors found a high level of genetic diversity and complex spatial genetic structure in the species with the presence of frequent gene flow among patches. They concluded that fragmentation effects due to agriculture and infrastructure seem to be negligible for this species and do not limit species viability.
I believe that the questions investigated here are very relevant to understand plant edaphic species adaptation and conservation in general. The study is of broad interest for PeerJ readership. I think one main challenge to its publication of this study is clarity – as is, I had a hard time following many aspects of the manuscript. I am particularly concerned with the “gene flow” and “genetic structure” results section for which I was not able to interpret the same findings as the authors. The authors have limited the estimation of gene flow among populations as well as the genetic spatial structure. In my opinion, simulation-based gene flow inferences can provide valuable insights into the barrier effects of highway. I would like to see more robust analyses here of contemporary and historical migration. Perhaps use BAYESASS and MIGRATE.
Overall, although I feel that the aim of this study is interesting, there is still a lot of room for improvement. In my opinion there is no need for revision of the English (but also not my mother tongue).

Detailed Comments:
Abstract
•	Background is too long. On the other hand, Methods and Results are shallow. It should be more concretely.
•	Line 54-55: IBD was not fully validated in this study. I believe that even GLM would not suffice.
•	Line 56-57: Population’s genetic diversity and structure is usually the result of biological, demographic and ecological features. Thus, no need to mention again.
•	Line 57-58: I feel that the interpretation is biased. Is the genetic diversity is an only indicator for displaying the barrier effects of highway?
•	Line 58-59: I cannot follow the logic.

Introduction
•	Line 71-31: This sentence is irrelevant to the conclusion and aims of this study.
•	Line 111-113: This sentence is ambiguous. I think this explanation (i.e., evolutionary history) is too brief so it will be hard for readers to understand why soil specialists are resilient against to fragmentation due to human activities. Please be more specific.
•	Line 114: It may lack the term “gene” or “pollen” before “flow”.
•	Line 117-124: The former (L117-121) and later (L. 123-124) are disconnected logically. Previous studies should have been indicated that the species exhibits high levels of genetic connectivity between the fragmented patches. Is this understanding of mine correct? If correct, these previous works suggest that there is no barrier preventing gene flow and/or migration in this area. So I cannot fully understand the argument for your hypothesis.

Materials and methods
•	Line 135-143: Is there any information available about the generation time of the species? It may be important for this study.
•	Line 145: “... corner of Madrid (central Spain).”, better “... corner of Madrid, central Spain.”
•	Line 145-153: The position information (i.e., latitude and longitude) concerning “the area” should be provided in the text or figure 1.
•	Line 179-181: Only seven marker names were listed in the text.
•	Line 189-191: “... the samples were amplified with an ABI 3730XL” is wrong. “the amplified fragments were analyzed with an ABI 3730XL” is better.
•	Line 203-206: Parameters for TPM model should be exhibited.
•	Line 211-213: Please provide package and function name used here.
•	Line 225-227: Why did you use DAPC among several model-free methods? I recommend to substitute a spatially explicit multivariate method principal component analysis (sPCA, Jombart et al. 2008) for DAPC. This can allow to test fine scale genetic structure, and to estimate the strength of spatial structure.

Results
•	Overall, STRUCTURE analyses would not be affected by null alleles, but other analyses might. I would suggest doing a null-allele correction on FST.
•	Line 247-253: The statistics related to number of alleles (e.g., allelic richness) should be described as one digit after the decimal point (also in Table1 and S1).
•	Line 254-256: “Expected heterozygosity was higher than...”, better “A relatively high level of heterozygosity observed in all populations”.
•	Line 263-263: “discarded” is wrong, “detected” is correct.
•	Line 268-270: I think that the level of genetic differentiation is low. And also, readers may want to the values of pairwise FST between populations but not within each population.
•	Line 296: You should provide the delta K value or the log likelihood values for each K.
•	Line 299-303: Several results discussed here were not presented graphically so it will be hard to understand. It can be informative to visualize lower and upper hierarchical genetic structures for relevant K (e.g. K = 3, 7, 8) in addition to the main observed genetic structure (K 4).
•	Line 303-305: This sentence should move to Discussion section.
•	Figure 2 (A): “Geneland” is not explained in Materials and Methods section. Is this a result of STRUCTURE analysis? If so, I wonder that all individuals within a population were assigned by single cluster despite the low genetic differentiation and frequent migrants were observed in the studied populations.
•	Figure 2 (C): Are there reasons for order of populations in the figure? If there is not it, populations should be rearranged in coordinate longitude or population code order.

Discussion
•	Line 310-341: The two paragraphs are redundant. It can be shortened.
•	Line 358-360: Your interpretation about the Bottleneck test is wrong. I think that the results of Bottleneck suggest the presence of recent bottlenecks in the all populations. Cornuet and Luikart (1996) said that “If P-value is lower than 0.05, the null hypothesis is rejected in favor of the hypothesis of an overall heterozygosity excess and a recent genetic bottleneck” (Genetics 144, pp. 2005).
•	Line 359-360: I guess so, but you should pay more attention to a high variance among loci. The interpretations are speculative because of weak evidence based on only 7 (or 6) loci. The high FIS within populations may be attributable to the presence of null alleles.
•	Line 369: Which genetic parameter suggests the weak effects of highway?
•	Line 375-377: Why do you think that only SW population is suffering in a barrier effect? There is no anthropogenic barrier within the SW side (population). I believe that FST values were not clearly different between both side.
•	Line 381-383: A complex spatial genetic structure does not always mean a high level of genetic diversity.
•	Line 394-396: A distance from highway may be candidate to explain high private allele, if the highway functions as an effective gene flow barrier.
•	Line 404-406: OK. When moving, do these livestock use the highway (or side road)? If they use, the high way may play important role in seed flow among populations.


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: LIMITED EFFECT OF A HIGHWAY BARRIER ON THE GENETIC STRUCTURE OF A GYPSUM SOIL SPECIALIST
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article title “Limited effect of a highway barrier on the genetic structure of a gypsum soil specialist” addresses an engaging question on the effect of fragmentation on the genetic structure of a plant. I consider that the manuscript is in general well structured, with some points to improve. The authors show a broad knowledge in the study system, but they might improve the descritpion of their study species (e.g. seed dispersal distances, longevity,…). The proposed hypothesis is interesting and well justified and they have designed a proper methodological framework to test it. I miss some interesting points in the Discussion (e.g. the role of time in delaying the impact of fragmentation on genetic structure) as I specify below. These are my specific comments:

Abstract (line 50-51). “Here, the effect of "a" highway with heavy traffic on the genetic flow and diversity of Lepidiuma dominant shrubby gypsophyte, was evaluated.” I think that this sentence needs to be rephrased.

Abstract (line 55). “….exploring in detail the effect of "a" highway….”

Introduction (line 67-73). The first paragraph is very important. Readers need to see the importance of the issue that will be studied in the manuscript and how your research is going to shed light in the field. You start the intro talking about landscape genetics, this is the methodological framework you are going to use to answer your question, but this is not the ecological problem you are addressing. Your manuscript is about fragmentation, so the first sentence must talk about how fragmentation threaten biodiversity worldwide. Then, you can talk about how landscape genetics approaches are suitable to address these kind of questions and how your study will contribute to increase the knowledge in the field. Please change the structure of this paragraph.

Introduction (line 86). “…profoundly affects the "gene" flow between…”

Introduction (line 102). Change dispersion for dispersal.

Introduction (line 117-120). You say that your plant is a perfect model species to study the effect of linear infrastructure because its known capability of maintaining gene flow in highly fragmented landscapes. I do not see that as a positive point for your study.  If you select a species which tolerates high levels of fragmentation, you will not probably detect any effect of this perturbation and you are probably overlooking the effect of this perturbance in other species of this habitat which have less ability to cope with fragmentation. I think that you must change this part. You can say that your species has the ability of maintaining gene flow, but this does not justify that your species is a good model for this study. Maybe you might say that it is a good model because there is a lot of previous information, there are molecular markers available, it is a widespread species in the community, etc.

Introduction (line 124). “…sides of the highway but also among the fragments in each side”. I do not understand how your experimental design research on the effect of the highway among the fragments in each side, what is the effect of the highway in the connectivity among fragments in each side?

Study species (line 134). What do we know on the role of animals in seed dispersal of the species? Could you include information on this issue? As is covered by a mucilage, might it be dispersed by birds?

Study area (line 157-159). You say that you collected 20 individuals where possible. I could not find the number of individuals collected in each population in any of the tables or files, could you please provide this information?

Study area (line 161-165). How did you estimate fragment area, patch perimeter….? I suppose that you used a GIS tool. Please include this information.

DNA extraction, amplification and sequencing. Did you amplify the microsatellites in individual pcr or in multiplex? Please add this information.

Statistical analysis. You did not calculate the frequency of null alleles. This is an important parameter to verify that your genetic estimates are accurate. You have to check that. I think it could be also interesting to check if your populations are in Hardy-Weinberg equilibrium as this parameter might impact some of your results (e.g. STRUCTURE).

Statistical analysis (line 221-227). You apply 2 clustering methods. Please justify that. You might read this reference for this part: Janes, J. K., Miller, J. M., Dupuis, J. R., Malenfant, R. M., Gorrell, J. C., Cullingham, C. I., & Andrew, R. L. (2017). The K= 2 conundrum. Molecular Ecology, 26(14), 3594– 3602.

Statistical analysis (line 230). Which software did you use to perform the AMOVA? Please specify that.

Results (line 305-307). I suppose that you did not find any relationship among genetic descriptors and the remaining environmental variables, but please say it.

Discussion (general comment). I think that time can be someting important to consider in your Discussion. This could be a critical question for your study. Fragmentation can need time to be detectable. It is quite common to find a delay in the impact of fragmentation on genetic descriptors. How long can the individuals of your plant live? Is there any information about how long can seeds remain in the soil seed bank? The highway was built 35 years ago, maybe this is not enough time how to see genetic effect in your species. Please consider to include this aspect in your Discussion.

Discussion (general comment). Is there any information on seed dispersal distances or pollen dispersal distances in this species? It might be something important to comment

Discussion (line 404). Change dispersion for dispersal

Conclusion (line 427). Eliminate "has been". The final sentece "As described....."

Figure 1. How did you elaborate the map? Please say it

Figure 2. You used STRUCTURE not GENELAND, right?
Experimental design: The experiemental design is suitable for the proposed question. Authors are cautious about their results since the spatial scale is restricted, but it is an interesting case study to report. Some points of the methology need to be improved (specified in Basic reporting)
Validity of the findings: Conclusions are supported by the results. However, some points of the methodology can be improved to strengthen the accuracy of the results
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: LIMITED EFFECT OF A HIGHWAY BARRIER ON THE GENETIC STRUCTURE OF A GYPSUM SOIL SPECIALIST
Review round: 2
Reviewer: 1

Output:
Basic reporting: included the general comments.
Experimental design: included the general comments.
Validity of the findings: included the general comments.
Additional comments: This is my second time reviewing this manuscript, and I found that the authors have done a good job in addressing the reviewer’s comments. I have no further comments, but I picked on several minor things:
•	Abstract: It is kind for readers to include DNA maker information, such as the number of loci and type (i.e., nuclear or chloroplast).
•	Line 149: A period is need in these geographic coordinates (e.g., 44.38624).
•	Line 213: Again, parameters for T.P.M. model should be exhibited.
•	Line 224: STRUCTURE’s “K” is better in Italic.
•	Line 225: Why authors conducted STRUCTURE analysis ranging from K = 27? I think K = 1 to 24 (= the number of focal populations) is enough for this study.
•	Line 300: “Meanwhile”, better “Namely”
•	Line 301-303: This sentence is too long and complex, so should be simply.
•	Line 318-342: Also, this paragraph is long, so divide two paragraphs.
•	Figure 2: For readers, the name of population group (i.e., NE or SW) should be in the bar plots. In addition, in the delta K methods, K = 2 was relatively high. I’m not only one who wants to see the results of K = 2 assignment.
•	Figure 3: The labels on these axes are too small and hard to see.
•	Figure 4: “p” is better in Italic.
•	“All” supplemental files: Very hard to see without a caption on the tables. Such irresponsible jobs can lead to doubts about the quality of this paper. That was unfortunate.


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: LIMITED EFFECT OF A HIGHWAY BARRIER ON THE GENETIC STRUCTURE OF A GYPSUM SOIL SPECIALIST
Review round: 2
Reviewer: 2

Output:
Basic reporting: Well-written.

Enough background information.

Relevant results.
Experimental design: Good design.

Methods are suitable to address the proposed hypotheses.
Validity of the findings: Findings are valuable. Conclusions are relevant in the field. However, the spatial scale is quite small, and hence, it is difficult to do general assumptions with these results.

Anyway, a valuable case study.
Additional comments: The manuscript has been improved. Authors applied most of the suggested changes and I consider that the manuscript can be now accepted.

I only have two suggestions. It is a little bit strange to me the way that you report null alleles in Table1. From my point of view, you shoul report a frequency by marker and/or population. Maybe you have null alleles but the frequency is low, this can not be evaluated in the current version.

You said that you collected 20 individuals  where possible. However, you only got 20 individuals in 7 populations. You should give a range (between 8 and 20 individuals, average XXX+-XXX). This would be more informative.