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: EARLY SKIN-TO-SKIN CONTACT BETWEEN HEALTHY LATE PRETERM INFANTS AND THEIR PARENTS: AN OBSERVATIONAL COHORT STUDY
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 allowing me to review the revised version of this manuscript. The vast majority of the reviewer’s comments were incorporated into the manuscript. There continues to be minor editorial changes needed. Other comments include the following;
Line 159-162 belongs in the introduction and not the methods section
Substitute the word children for infants.
The definitions of the demographic characteristics were defined previously in the methodology section and are redundant in the data analysis section.
Line 249: fetal asphyxia during delivery should be changed to fetal asphyxia. I am assuming that cardiotocography is fetal heart rate monitoring and should be stated as such. I also assume fetal PH is umbilical pH and should be changed to make the terms more easily understood.
Although use of a breast pump, cup, nipple shield were collected, they are not in the results section. Thus should be removed from the methods section.
No need to repeat content found in the table in the text


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

Input:
Title: EARLY SKIN-TO-SKIN CONTACT BETWEEN HEALTHY LATE PRETERM INFANTS AND THEIR PARENTS: AN OBSERVATIONAL COHORT STUDY
Review round: 3
Reviewer: 1

Output:
Basic reporting: generally clear however could be more succinct throughout.
See comments in the document provided
Needs to be edited carefully and consistent use of terms and abbreviations is needed.
Experimental design: see comments in table provided
Validity of the findings: low impact
Additional comments: See table provided for specific 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: EARLY SKIN-TO-SKIN CONTACT BETWEEN HEALTHY LATE PRETERM INFANTS AND THEIR PARENTS: AN OBSERVATIONAL COHORT STUDY
Review round: 3
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: no comment
Validity of the findings: no comment - these have been addressed in the limitation section
Additional comments: Thank you for allowing me to again review this manuscript on a very important topic regarding the late preterm infant who as we know is a high risk of morbidity. Although there are still minor editorial errors that need correcting the authors and adequately and appropriately responded to the reviewers comments and concerns. I just had a few comments/questions
1.	Line 144: was phototherapy an exclusion criteria (would be rare but if the infant had a hemolytic process?)
2.	Line 239: “minus” is confusing
3.	Line 370: ethnicity is also important to rate of breastfeeding


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: ESTIMATING THE POTENTIAL BIODIVERSITY IMPACT OF REDEVELOPING SMALL URBAN SPACES: THE NATURAL HISTORY MUSEUM’S GROUNDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear, unambiguous, professional English language used throughout.

This is an exemplary example of a clearly written, structured and produced piece of work. There are very few typographical errors, errors of omission or of expression/fact. I have annotated comments onto the PDF (attached to this review).

Intro & background to show context. Literature well referenced & relevant.

The introduction has a decent review of the literature but there are a couple of points that require some thought. Firstly, I am not persuaded that by the ‘neglected status’ of urban ecology (p.1, line 44ff). There has been a great deal of recent activity in this field. Perhaps rephrase to say just that. I think the lengthy diversion into the history of the grounds redesign at the NHM has too much detail; it might better sit in the methods of supplementary materials. It needs slimming down to about a third picking up just the salient points. The key point of the paper is that it introduces a means of assessing the impact of site-based developments, which is an area where little robust work exists (aside of classic impact assessments). This is the section that requires emphasising.

Structure conforms to PeerJ standard, discipline norm, or improved for clarity.

This is fine.

Figures are relevant, high quality, well labelled & described.

The Figures are nicely produced but would benefit from some modification:
Fig 1: rotate by 90 degrees
Fig. 2: needs scale and north arrow. Not all data sources are listed (in comparison to Github sources).
Fig. 3: Maybe use a 2nd Y-axis to improve clarity and remove the grey text as you describe what it means in the figure caption.
Fig. 5: A bit small to read easily on the screen - increase figure and font size. It is clear on retina-type displays but not everyone will have access to one of those.

Raw data supplied (See PeerJ policy).

Raw data, R code and latex all available on Github @:  https://github.com/helenphillips/GroundsRenovation. The code indicates possible sources of problems but looks fine. I have not tried to emulate the analyses.
Experimental design: The design is clearly outlined but there are a few areas where the paper would benefit from more methodological detail and support/justification (all minor stuff):
1. unpack what is meant by the dose-response modelling framework - p.4, line 138
2. I understand why going for a Species|Area-type model is sensible given what you are trying to achieve but lots of variables will impact species richness/density (e.g habitat structure, site context ie. situated in areas with lots of similar habitat or isolate, disturbance histories, time and the like) You allude to this at the end of the introduction but I think you need a develop this a little more and revisit to more directly as a critique in the discussion. I was a little puzzled by line 157 where you mention time limitations; what were they? I cannot see that in the discussion either.
3. Data collation: You need to document what data was extracted using ImageJ.
4. GLMM is a sensible approach but it's a small dataset for inference outside the realms of the study. Some justification of that is needed.
5. Your assumptions are sound given the data but why did you carry out sensitivity analysis on only one of the three?
6. Why were both the density and richness models simplified by removing the additive effects? Elsewhere you mention data paucity as a reason why interactions were not undertaken. Is that the reason?
Validity of the findings: The authors have used an appropriate and robust approach to the analysis and they are careful not overextend the reach of their findings. I like the central idea of this paper as it allows an evaluation of outcomes of site developments thus chimes well with UK planning policy which is essentially site-based at the point of decision. The analytical model / approach I believe to be robust enough to be used elsewhere but it may need nuancing for each specific location. Some guidance on what form this might take in the discussion would be helpful. I think also that a fuller review of the issues surrounded a focus on area is needed as a critique in the discussion.
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: ESTIMATING THE POTENTIAL BIODIVERSITY IMPACT OF REDEVELOPING SMALL URBAN SPACES: THE NATURAL HISTORY MUSEUM’S GROUNDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 57-109: This is far too much detail for a study of this type, strongly recommend cutting this down significantly.  You are trying to argue that your study has value beyond being a case study, this much detail on the history and specific challenges of your site does not help make this argument.
Experimental design: Line 190: I think this is a central limiting factor of your research.  You used data from urban areas “when possible”, but we cannot tell when this was, and a huge wealth of literature tells us that relationships between biodiversity and habitat in urban areas are altogether different than in natural landscapes.  This point needs to be made, and strongly.  This is a major assumption that casts some doubt on your findings.

Line 227: When scaling in this matter, you completely ignore edge effects, unless I am missing some part of your methods.  Given how small your habitat patches are, it seems these effects may be extremely important.  And generally, since you are using values from the literature that are collected across much, much wider scales, I don’t think the relationships are likely to scale down to the level of the patches you’re working on.
Validity of the findings: The findings are valid, I think, but the inference space is extraordinarily narrow.  You have applied a simulation modelling exercise to a small urban patch using non-urban data, and you haven’t verified it with any field sampling whatsoever.  Is this a promising approach for other systems?  It might be, though I have some grave concerns about edge effects and scaling as I describe previously.  But to test that you’d need to validate it with field data, which this study fails to do.

So what we have in the end is a simulation study, one that tries to use possibly unrelated data to predict biodiversity outcomes for land management on an extremely small scale, and finds those changes to be minimal for total species richness.  It’s intuitively obvious that by adding new habitats, and high quality habitats, we would expect these values to go up.  Of course, even species richness isn’t usually what we’re interested in, compared to things like diversity of rare species.  The method has promise, but I’d like to see it explored over a much wider area, and with some field data to back it up.

At any rate, I think you need to discuss these limitations more fully in the text—you are way out on a speculative limb, and I think the text needs to work harder to acknowledge that.
Additional comments: Thank you for the opportunity to review this manuscript.  It is very well written, and in general I applaud studies that investigate the relative value of small patches of urban habitat, which are often much more valuable than people realize.  Technically the paper is sound, nothing was done that is misleading or that reaches erroneous conclusions.  So for the purposes of this journal I suspect it is publishable.

Unfortunately, I found the potential benefits of the paper for management or conservation lacking.  This is a fairly simplistic modelling exercise, applied only to a single case site, that makes a variety of unrealistic assumptions to try to make a relatively simple final observation about how wildlife species might use habitat.  I appreciate what the authors are trying to do, but I think the study needs to do at least one or two of the following to be of much use to anyone:

1.	Apply their method to more sites
2.	Validate their predictions with field data
3.	Create a more complex model that incorporates things like edge effects, connectivity, and the like.

I do realize that potential impact is not a determining factor for publication in this journal, so I bring this up largely out of completeness, and in the hopes that the authors would like their work to have a wider impact.  I hope this review is useful to the authors.


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: MOLECULAR PHYLOGENY AND EVOLUTIONARY HISTORY OF MORICANDIA DC (BRASSICACEAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript explores the phylogenetic relationships of Moricandia species (seven spp.) Sampling seems to be adequate with several specimens per species and nuclear ITS and plastid trnT-F and ndhF sequences. Author also tested the position of the closely related Rytidocarpus moricandioides. Overall, analyses are adequate, and results obtained were interesting, revealing that M. foleyi  is not included in Moricandia. Moricandia s.s. (without M.foleyi) is composed by two geographically structured clades, the African and the Iberian clades. Additionally, Iberian specimens of R. moricandioides are included in the Iberian clade, while African specimens (Rytidocarpus s.s)  were placed sister to Moricandia. Result from divergence time analyses related divergence of Moricandia with the geologic history of the region; however I am not an expert on the geological evolution of this area and cannot give a robust evaluation of these findings.

I think that this manuscript is adequate for its publication in PeerJ after applying minor changes (see comments for the author)
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript explores the phylogenetic relationships of Moricandia species (seven spp.) Sampling seems to be adequate with several specimens per species and nuclear ITS and plastid trnT-F and ndhF sequences. Author also tested the position of the closely related Rytidocarpus moricandioides. Overall, analyses are adequate, and results obtained were interesting, revealing that M. foleyi  is not included in Moricandia. Moricandia s.s. (without M.foleyi) is composed by two geographically structured clades, the African and the Iberian clades. Additionally, Iberian specimens of R. moricandioides are included in the Iberian clade, while African specimens (Rytidocarpus s.s)  were placed sister to Moricandia. Result from divergence time analyses related divergence of Moricandia with the geologic history of the region; however I am not an expert on the geological evolution of this area and cannot give a robust evaluation of these findings.

I think that this manuscript is adequate for its publication in PeerJ after applying minor changes (see comments for the author)

some suggestions/ corrections:

Maybe it will be better if results from individual analyses of ITS and cpDNA are showed before the combined analyses

Authors could indicate on Fig. 4 (the chronogram) the node used for the calibration based on the geological information (uniform 5.39-5.33 mya)

I am not an expert in the tribe Brassiceae, but the authors transferred M. foleyi to the genus Eruca because this species resulted sister of Eruca. However phylogenetic analyses included only four Eruca species, Brassica rapa, and Raphanus sativus. Is this sampling adecuate? Perhaps, in analyses using a broader sampling of Brassiceae M. foleyi could be recovered related to other genus of the tribe.

It would be interesting to analyze this dataset using species tree inference under the multispecies coalescent model

Line 182: GTR+G instead GMT+G


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: MOLECULAR PHYLOGENY AND EVOLUTIONARY HISTORY OF MORICANDIA DC (BRASSICACEAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Francisco Perfectti et al. reported a research to reconstruct the phylogenetic relationships of Moricandia species using the traditionally marker genes of ITS and trnT-trnF. I think this paper is ok and I only have several following concerns.

1. To better present the results, the genus name and clades name of 'African clade' and 'Iberian calde' should be labeled on the Figure 1.

2. I can not understand 'The subspecies M. moricandioides pseudofoetida appeared inside the branch of M. moricandioides but well separated from the other M. moricandioides subspecies' on page 13 line 240-241. Actually, two M. moricandioides pseudofoetida species are sister group of other three M. moricandioides subspecies.

3. on page 13 line 245, the author should specify the exact samples name of the R. moricandioides.

4. The Fig. 4 should be Fig. 3 on page 14 line 271.
Experimental design: 5. Normally, the support value of Bayesian tree pretend to be higher than ML tree. The author should not only label the support value of Bayesian tree, but also label the support value of ML tree on Figure 1 by only presenting the topology obtained by the Bayesian method.

6. The authors only listed the Bayesian trees of separated results on Figure 2. How about the ML tree of separated cpDNA and ITS?
Validity of the findings: 7. The authors claimed that two Rytidocarpus moricandioides subspecies are located into the clade of Moricandia and is indeed a new Moricandia species. But how about the statistical possibilities of other topologies? I suggested the authors to do the AU-test of other potential topologists of Moricandia species using the same dataset as in Figure 1. Unless the result can reject the possibility that the Rytidocarpus moricandioides g01-13 and g01-14 are sister groups of all other species in the pink filled region, I can trust that these two species are actually new Moricandia species.
Additional comments: no comments


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

Input:
Title: MOLECULAR PHYLOGENY AND EVOLUTIONARY HISTORY OF MORICANDIA DC (BRASSICACEAE)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper provides some updates on the evolution and systematics of the genus Moricandia.  Results and conclusions look reliable and the paper is in focus of
the journal. On the other hand, the quality of the text is poor and needs substantial English edition (eg. p 6 line 98. Please replace "their" by "its"). I strongly suggest that you have a native English speaking colleague review your manuscript. Additionally some technical terms including bp (base pair) used differently as "nt" or autor use the terms its1, its2 (page 8 line 140) instad of ITS1, ITS2 (these are commen usage of these regions and also the name of the relevant primers).

Materials used in this study look proper for such kind of phylogenetic studies as well as literature references.
Experimental design: Design of study looks good and proper for filling the gaps regarding phylogeny and systematics of the Moricandia with the exception one analyses. For details, please see comment in "Validity of findings " section.

Regarding model selection, i have never heard the GTM model that autors used in Bayesian analyses. Please check it.
Validity of the findings: Data looks robust but i do not understand why Ancestral Area Recounstruction analyses did not include this comprehensive study. Without these analyses all discussion about origin of the Moricandia species will be speculative.
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: RESPIRATORY SYNCYTIAL VIRUS GENOTYPES NA1, ON1, AND BA9 ARE PREVALENT IN THAILAND, 2012–2015
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript provides additional information on RSV genotypes circulation in Southeast Asia Region. As the RSV molecular epidemiology data from developing countries especially from long term studies are still lacking, this manuscript contributes on the data needed to determine the global RSV circulation and transmission.
.
There are some drawbacks of this manuscript:
1.	There are several grammatical errors and some sentences need to be reframing.
        The flow of idea of the sentences within the paragraphs and between the
         paragraphs are unclear. There are some repetitions of vocabulary used in the
         sentences.

2.	The structure of the manuscript should be revised based on the PeerJ standards.
2.1	The abstract should be revised as it did not follow the PeerJ standard.
2.2   There is no conclusion paragraph. As the PeerJ standard, the author should add
        the conclusion section.
3.	The title of this manuscript is too general. The authors may change it to the more
        specific title which related to the contents of the article.
4.	Figures and tables: Figure  1 needs a revision (left vertical axis ?)

Raw data is not provided. I am unable to verify the RSV sequences obtained from this study from NCBI website as they mentioned that these sequences have not yet been released.
Experimental design: 1.	The topic of this manuscript falls within the scope of the journal
2.	Research questions should be more well-explained, as it seems inconsistent from the
        abstract, introduction and the discussion.
3.	Methodology:
3.1. The author should clarify the methodology regarding the samples as in methods
        section (line 82) the samples were from a retrospective study while in the discussion
        (line 1993) the author mentioned about multi-year surveillance study.
3.2. The author should add the screening diagnostic methods used for the samples as it is
       mentioned in the abstract but it is not well-explained in the methods.
3.3.	Detailed methodology on DNA sequencing should be provided.
Validity of the findings: 1.	The authors do not provide the demography data of the samples.
2.	The discussion on the first and second results should include the comparison with the
        results from the previous RSV studies in Thailand, Southeast Asia countries and
        other countries in tropical region.
3.	The authors should discuss the results of amino acid substitutions (line 161-181) as
        the discussion is limited to N-linked  glycosylation analysis.
4.	The authors should provide more discussions on the third and fourth results. In the
         discussion (line 239-258), it is not clear whether the authors discuss their results or
         the results from the references.
5.	As previously mentioned (point B.2.2), the authors should provide a comprehensive
        conclusion section.
Additional comments: The authors should improve the manuscripts (language, grammar, and the clarity of the presentation) before publication. Furthermore, the authors should provide more detailed discussions on the results presented in the article. Overall, the authors should consider the manuscript to be reviewed and edited by a native English speaker to improve the clarity.


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: RESPIRATORY SYNCYTIAL VIRUS GENOTYPES NA1, ON1, AND BA9 ARE PREVALENT IN THAILAND, 2012–2015
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: RSV infects lower respiratory track of, especially, children worldwide.  In the current study, Thongpan et al. used molecular techniques to describe epidemiological features of RSV Thai strains, detected in several thousands samples, collected over the past couples of years (2013-5). The study is well described, and provides updated data on RSV infection in Thailand. I have some comments, below:

Line 29-30: It will be helpful to give a brief description of 'RSV-A' and 'RSV-B' in the abstract, for readers who are not familiar with these terms.

Line 33: Perhaps the results reported here could describe only in the context of Thailand, not Southeastern Asia.

Line 53: Should it be 13 RSV-A genotypes (not 11)?

Line 65: Any info to describe how the particular strain of virus did spread around the world?

Line 83-91: Were the 3,306 specimens derived from 3,306 subjects/patients? If not, from how many subjects/patients? How many samples were from subjects with flu-like illness and probable RSV infection? How many samples were from King Chulalongkorn Memorial Hospital and from Chum Phae Hospital? Such info should be mentioned in the manuscript.

Line 108: There were 278 positive samples. I wonder whether a total of 212 strains (118 RSV-A and 94 RSV-B) were identified from all these positive samples, or there were just some positive samples that failed to identify the strains?

Line 112: What are the genotypes of the RSV reference strains B1 and A2?

Line 171 and 176: duplication of which gene or region?

Line 211: It will be more interesting to see if there is any info about the percentages of co-infection by both RSV subgroups.

Figure 1: Is it possible to adjust the scale of the Y-axis to 0 - 100%, to clearer describe % positive samples, in relation to the number of samples, at any time points?

Figure 2 and 3: Can the Figures 2 and 3 be combined into one tree, to see overall phylogenetic relationships of all RSV Thai strains? In the legends, adding some info regarding about number of samples, where they were derived, how many samples in each year, and indicators of which are outgroup/reference strains, would be helpful to readers.

Figure 4 and 5: the symbols '.' and '~' and any others, marked in the figure, should be given a short description in the legend. Can these two figures be combined into one sequence alignment of both RSV subgroups?


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: IMMUNOFLUORESCENCE CHARACTERIZATION OF SPINAL CORD DORSAL HORN MICROGLIA AND ASTROCYTES IN HORSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a reasonably clearly written manuscript.Overall the English is quite good, and only occasional sentences are unclear (as pointed out below). References are appropriate and reasonably up-to-date, and the manuscript overall comply with guidelines regarding structure etc.
Experimental design: In this manuscript, the authors (Meneses et al) describe their studies of distribution, cell sizes and other characteristics of spinal cord dorsal horn microglial cells and astrocytes in horses. The studies are based on material from (only) 5 supposedly healthy, adult horses. However, the selection criteria for the horses are a bit vague and, while that may be due to the “opportunistic” sampling at an abattoir, it is still a concern. Information should, as a minimum, be provided about the estimated age of the horses, their sex and breed. The description of the morphometric assessments (lines 216-226) is somewhat vague and could be greatly aided by some illustrations of the approach (this could be provided in the supplementary file). Moreover, as detailed below, the illustrations of some of the features described cannot be appreciated in the figures provided, notably Figure 2.
Validity of the findings: The authors have a good point about the way the literature regarding shape of microglial cells, as a reflection of activation state, is largely based on dogma originating in some of the early description of these cells, and that the amoeboid shape tends to be the “resting state” in many species rather than the active or reactive state, as originally described. This is certainly the case for species as disparate as mouse, rabbit, ruminants and horses (e.g., J Virol 82: 755; Microb Pathog 103:71). In contrast, the statement that GFAP is not present throughout the astrocyte cytoplasm (line 310-11) is not correct for all species either. It is actually present in such species as mouse and rabbit. What can be concluded from all this is that generalizations cannot be made regarding either of these cell types and that for each species under study appropriate controls must always be applied.
The authors discuss at length the size differences of astroglia between horses and humans (lines 317-25), but do not take into account regions/locations (e.g., the Oberheim paper cited describes neocortical astrocytes). Moreover, differences could be influenced by fixation method used (type of fixative, time of fixation etc) as well as other preparatory methods that could influence tissue shrinkage. This aspect should be commented on.
Additional comments: The manuscript could be further improved by addressing the following points:
•	It is misleading to call the approach “immunohistochemistry”, since a chromogen was not used for visualization of the binding of antibodies. Rather fluorochromes were used and the read-out was immunofluorescence, whether by conventional fluorescent microscopy or confocal. This should be corrected throughout the manuscript, starting with the Abstract (line 31). Furthermore, sections are not “stained” (e.g., line 206), but immune-labeled. Please correct this throughout the manuscript.
•	Line 160: what do the authors mean by “mounted in xylene embedded slides”? Please clarify.
•	Lines 176 & 178: it is very confusing when the authors say that the primary antibodies were “conjugated” with an [fluorocrome] goat anti-rabbit or donkey anti-goat secondary antibody. The use of the word ‘conjugate’ is misleading in this context, as the term is normally used to describe the joining of two or more chemical compounds, notably the conjugation of an enzyme or a fluorochrome to an antibody. Thus, the secondary antibodies are conjugated with fluorochromes, and the secondary antibodies are used to visualize the binding of the primary antibodies by binding the former to the latter. This should be corrected in the text.
•	Figure 2: some of the panels are mis-labeled, i.e., there is no panel D, causing some confusion when reading the results section. In general panels E, F and G (which should be D, E, and F) and in particular panel G (i.e., F) are difficult to interpret. It is not possible to appreciate “a tight connection between end-point processes and capillary walls” (line 268) in the figures provided.
•	Lines 41: correct to “amoeboid or spherical”
•	Line 58: correct to “found in rodents, with …….”
•	Line 108: correct to ‘major’.
•	Line 114-115: what do the author mean when saying “that highly coupled these cells”?
•	Line 122: correct to “activation at the dorsal horn level”
•	Line 128: correct to “under either normal conditions or during painful….”
•	Line 130: correct author name to “Bielefeldt-Ohmann” as in the reference list.
•	Line 134: replace “disposition” with ‘distribution’.
•	Line 141: delete “has”.
•	Line 281: delete the second “in”.
•	Line 284: replace “stain” with ‘label’
•	Line 294: correct to “resting and active states”
•	Line 297: replace “o” with ‘and’.
•	Line 308: correct “immunostain” to “immunolabel”
END


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: IMMUNOFLUORESCENCE CHARACTERIZATION OF SPINAL CORD DORSAL HORN MICROGLIA AND ASTROCYTES IN HORSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors described the morphological studies of microglia and astrocytes in horse spinal dorsal horn with immunohistochemistry in this manuscript titled 'Immunohistochemical characterization of spinal dorsal horn microglia and astrocytes in horses'. Since the whole study was based on Iba1 and GFAP antibodies, therefore I would recommend authors to use the best well-known antibodies for Iba1 (WAKO) and GFAP (DAKO) instead of the antibodies from Santa Cruz. And the staining for glia cells in the manuscript was not good. As we know that the fixation could also affect the glia cell staining, where Iba1 and GFAP antibodies show perfect staining with mild fixed tissues. And the authors used around 10% PFA for immersion fixation tor two days, which would definitely reduce the staining quality (usually 4% PFA for 2 hrs of immersion fixation).


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: IMMUNOFLUORESCENCE CHARACTERIZATION OF SPINAL CORD DORSAL HORN MICROGLIA AND ASTROCYTES IN HORSES
Review round: 1
Reviewer: 3

Output:
Basic reporting: English is fine and there is adequate background literature presented to justify studying the topic, but that material leads the reader to think a somewhat different study will be done.  A bit more background is needed for the specific work that was actually done and a bit more justification is needed for what actually was done.  The general structure of the article is fine.  There is no concrete hypothesis stated.  See General Comments section below for suggested improvements.
Experimental design: The research appears to be original within the Aims and Scope of the journal.  However, the research question needs to be more well defined as does the research gap being filled.  Portions of the Methods need to be described in more sufficient detail for replication.  See General Comments section below for suggested improvements.
Validity of the findings: Clearer description of some of the Methods would make the data more robust and validity easier to evaluate.  The conclusions could be more tightly linked to the major issue presented, which is the substrates of pathological pain states.  See General Comments section below for suggested improvements.
Additional comments: INTRODUCTION

A significant issue with the Introduction is that it leads the reader to believe that the authors will be doing a study that examines the interaction of microglia and astrocytes underlying pain syndromes when in reality they are simply looking at the normal morphology of microglia and astrocytes in horse.  In essence, the last paragraph could serve as the principal portion of the Introduction if the authors just talked a bit about the glial markers in that particular paragraph, talked a bit more about the existing work in the horse, and provided a more detailed explanation of how this normal data will be useful in future pain studies.

Some more specific comments regarding the Introduction:
In line 68, the concept of the term “static” is a bit vague.

For the sentences that begin on lines 73 and 74, in-text examples would be useful (aside from simple referencing).

Perhaps “differential functionality” might be better at the beginning of line 77.

Line 78: maybe “duration” instead of “time”.

Line 79: “Glial-derived” might be better.

In the sentence beginning on line 83, it’s not clear whether the authors are suggesting pain syndromes cause the microglial response or whether the response underlies the syndrome.  So maybe the beginning should read “Painful syndromes involve the triggering of”.  Between this sentence and the following one (beginning on line 86), there is unnecessary repetition regarding Iba-1.

Line 89:  This sentence needs some clarification.

Line 100:  what do the authors mean by housekeeping function?  This sentence seems to have some circular reasoning (e.g. resting astrocytes express basal levels of GFAP, but how do you know they’re basal levels unless you first know they’re in the resting state?  By GFAP levels?  See what I mean?).

Lines 101/102: Please clarify “external stimulation”.

Line 103:  What do the authors mean by “pre-synaptic space”?

Lines 102-106:  The sequence of interaction between neuron and astrocyte could be clarified here.

Line 106:  Perhaps change “at the spinal level” to “at the spinal cord”.

Line 108:  “major” instead of “mayor”.

Line 112:  Might add “with GFAP antibody” after “astrocyte”.

Line 120-122:  This sentence needs to be clarified.

Lines 125-126:  This sentence sounds like the goal of this study, given the preceding Introduction material.

MATERIALS AND METHODS

The description of the sectioning and staining are fairly straightforward.  However, the description of the image collection and analysis is much less clear/detailed and can use significant improvement, particularly how the samples from a given horse were assigned to different staining and analysis procedures, and the description of the various morphological measures.  More specific comments follow:

For animal selection, indicators of pain, which would be important in such a study, were of the most general nature.

Why were either cervical or lumbar samples used from a given horse and not both?

There are no references for the tissue fixation and staining.  How did the authors derive those procedures?

How were the sections parsed for the different staining procedures?

The logic for the double antibody staining should be explained more clearly.

Lines 154/155:  Transverse sections?  70%, 80% 90%, etc. or 70%, 75%, 80%, 85%, etc.?

Line 159:  Do the authors really mean sagittal?  If so, why?

Line 160:  Model of Jung microtome?  What is meant by xylene embedded slides?

Line 162:  Please see query for lines 154/155.

Line 163:  What type of microwave and what were the settings?

Lines 164/165:  Was there some kind of serum used along with the TCT buffer for blocking?

Line 176 and 178:  Do authors mean “incubated” instead of “conjugated”.

Line 179:  Was there any wash after the secondary antibody incubation before DAPI staining?

Line 180:  What form of DAPI was used?  Aren’t there a few different types?

Line 188:  Regarding “initially processed as previously described”, up to what point?

Line 189:  It’s not clear what “incubated separately” means regarding the primary antibody double-staining.

Line 203:  Should probably add, “and then counterstained with DAPI”.

Line 210:  Might add “was performed” after “laser scanning”.

Line 212:  Are the 15 sections all the horses combined, or 15 sections from each horse?

Line 213:  It’s not clear what the authors mean by “the entire spinal cord”.

Line 214:  Please clarify what is meant by “completely integrated dorsal horns”.

Lines 216-226:  The description of the morphological parameters that were measured should be explained more clearly and with more detail.  For example, how was shape determined?  How was the decision made to classify a cell as spherical in form or hyper-ramified?  Was the length of all processes of a cell measured?  Did diameter of hyper-ramified cells include the processes?  A figure would be helpful showing the various morphometric measures that are described.  When measuring process length, was bending of the process taken into account at all?

Line 224:  Should the word “cells” be “processes”?

Line 227-234:  Please elaborate on what is meant by “distribution” in line 227.  How was the dorsal horn and its laminae identified in these fluorescence sections?  What was the criterion for an immunopositive cell at 10X magnification?  Was it any stained piece that was on the perimeter of the defined square?  How thick was the perimeter line?

There is no mention of background staining in the omission controls.  How was background staining dealt with?

RESULTS

It is difficult to assess the validity of the data without clarifying some of the issues raised above for the Materials and Methods, particularly with respect to how tissue slices were assigned to the various staining procedures for each animal.

A table summarizing the results would be useful.

Is there a way to know whether the microglia are all amoeboid/spherical, or whether processes just can’t be detected by the staining method?

When talking about differences between any measures (e.g. between numbers of cells in dorsal horn regions), the authors should note they are simply numerical differences as opposed to statistically significant differences, since no statistical comparisons were made.

Is there a reason why the microglial/astrocyte branch contacts weren’t quantified?  Perhaps they differ between dorsal horn regions.

How were primary and secondary branches defined?

Many of the descriptions are quite qualitative and it’s hard to evaluate how representative they really are.

Lines 252-254:  The authors should clarify what they mean when they say, “GFAP was mainly found in astrocytic primary processes”.  Compared to what?  Small cell bodies relative to what?  How were “long branches” defined when the authors say “extended 2-4 long branches”?

Lines 254-257:  So primary astrocyte processes then give off 2-4 long branches (why weren’t descriptive statistics calculated here?), which each give off 2 small thin branches?  Correct?  How were the blood vessels and neurons that the authors mention identified?

Lines 258-259:  How did double fluorescence make it better for defining astrocyte cell bodies and extensions of processes?

Lines 259-261:  The authors should explain why it was necessary to use diameter measurements for cell body size in astrocytes but area for cell body size in microglia.  How was “main process” defined?

Lines 261-263:  The authors should clarify why they needed to see the whole cell size for the astrocyte density measurement, and how the Cx-43 staining help with that.

DISCUSSION

The Discussion makes some interesting points regarding differences with other species and with humans.  However, the authors do not really explain how these particular findings are relevant to deciphering the substrates of pathological pain states.  Also, no limitations of the study are discussed and there is no mention of effects of background staining.

Lines 286-287:  Microglial cells were also found in the other dorsal horn regions examined.  They were just numerically less dense.

Lines 288-289:  Maybe no processes were observed using Iba-1 because of staining quality.

Lines 299-302:  The data you talk about in this paragraph actually do seem to support a difference in microglial morphology between healthy and (encephalitic) diseased horses.

Why did the authors not discuss the findings on dorsal horn laminar distribution of microglia?

Lines 317-325:  The horse, rat human comparisons are interesting, but what does it mean for deciphering mechanisms of pathological pain states?  Same question for the laminar distribution?

Line 326:  This statement should be toned down given that no comparative statistics were performed.

Lines 330-333:  It is hard to see what the authors describe here in Figure 2F, and the statement about “stretch connection” is not clear.

Lines 333-336:  The authors need to elaborate a bit here in order to clarify the statement.

CONCLUSIONS

Lines 344-348:  Perhaps the authors might say that exploring other species might reveal glial organization that is closer to human than is rodent, and might represent better models for investigating glial substrates of pathological pain.

Lines 353-355:  So are you saying you feel horse is a better model for human pathological pain states than rodent?


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: IMMUNOFLUORESCENCE CHARACTERIZATION OF SPINAL CORD DORSAL HORN MICROGLIA AND ASTROCYTES IN HORSES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Only minor spelling and syntax issues still need to be corrected. Otherwise much imporved.
Experimental design: No further comments
Validity of the findings: No further comments
Additional comments: The Authors should be commended for their attention to the reviewers' comments and criticisms.The manuscript is now notably improved, and only minor issues remain to be dealt with:

line 53: correct to 'immuno-label'
line 202: correct to 'three'
line 216: suggest modify the sentence to the following "GFAP primary antibody binding was visualized by incubation with Alexa Fluor 488 conjugated donkey anti-goat secondary antibody (1:500, Invitrogen, Camarillo, CA, USA)...."

line 339: delete "to obtain"
line 394: replace "o" with "and"
line 397: change "evidencing" to "suggesting"
line 406: correct to "used for study of the morphology......"
line 507: correct to "focus on exploring...."
line 530: change to "immunofluorescence analysis".

END


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: IMMUNOFLUORESCENCE CHARACTERIZATION OF SPINAL CORD DORSAL HORN MICROGLIA AND ASTROCYTES IN HORSES
Review round: 2
Reviewer: 2

Output:
Basic reporting: Please note that this is a re-review of the original submission.  The basic reporting is now satisfactory and no longer misleads the reader to think that a different type of study will be done.
Experimental design: The descriptive nature of the study has now been made clearer and many of the methodological details lacking in the original have been added or clarified.  However, there are still a few details that could use clarification that are noted below.
Validity of the findings: As noted, several methodological details have been clarified, improving the perceived validity of the findings.  The authors have added a few theoretical connections between their findings and pathological pain states, but the discussion of limitations of the study is still minimal.
Additional comments: The authors have addressed the vast majority of the comments made by this reviewer on the original submission.

INTRODUCTION:
The Introduction now seems adequate.

MATERIALS AND METHODS:
The reference that the authors cite in their responses, for the Bouin’s fixation procedure, should be added near the end of paragraph 1 of the Methods.

The author’s response describing the 3 adjacent sections for the 3 antibodies and the total of 15 sections per horse should be added to the Methods text.

In the Methods section it still needs to be made clearer why there seems to be a set of single labeled tissue produced, and then another set of double labelled tissue produced, despite the fact that the authors feel it’s clear.

Line 167: The authors should further clarify what “separately” means (is it really sequentially, with first primary antibody on Day 1 and second primary antibody on Day 2?).

Line 198: How was a hyper-ramified cell defined?

Line 207: When the authors say GFAP and Cx-43, do they mean double labelled, while the Iba-1 is single labelled tissue?  Irrespective, this should be clarified in text.

Lines 212-215:  The authors say in their response letter that dorsal horn laminae were not identified, so how did they make the laminar distinctions mentioned in these lines and how were they able to visualize dorsal horn regions?

It’s still not clear how the authors can have confidence in their numbers without a background estimate/correction.

For Supplemental File A: the numbers in Part A should be explained.  Line 12 should read “using 30 square perimeters”.

RESULTS
For the qualitative descriptions in lines 225-227, and 234-235, some idea of how representative this is should be presented.  Otherwise, it’s hard to interpret the validity of the observations.

Line 232: Why is the number of primary processes presented as a range and not a mean?

Line 242: Cx-43 alone?

The HE staining comment in the author’s response regarding tissue integrity and blood vessels should be added to the Methods.

DISCUSSION
The author’s response regarding lines 299-302 of the original submission should be placed in the Discussion around line 275.

Lines 278-279 need to be toned down because the very limited data available in horse, although inconclusive due to small numbers, seem to indicate there might be a relation between disease state and cell form.

Line 306:  Should say, “…the highest number of astrocytes were counted in laminae I, II and III…”

Line 313:  Should say, “…we showed some evidence that astrocytes…”


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: GENOME-WIDE IDENTIFICATION AND EXPRESSION PROFILE ANALYSIS OF CCH GENE FAMILY IN POPULUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Figure 6 (RT-qPCR results) should include the raw data or bar plots and the statistical significance of the difference should be discusses. It is not easy to identify the level of difference from heatmaps. In addition, because untreated and treated samples are in separate plots, they cannot be directly compared due to separate normalization.
Experimental design: The article will fall within the scope of PeerJ. The analyses are routinely used and are well done. There are some issues however. See specific comments related to experimental design are below.

1. The definition or identity of CCH gene family is not clear and objective. Is the MXCXXC motif and the secondary structure pattern not present in any other gene family? Is there a minimum sequence identity that authors looked for. A single criteria of BLASTp with an E-value seems arbitrary. It should be clearly discussed what makes a CCH family unique, and use that criteria.

2. Is the induction of expression level differences upon Cu stress statistically significant? It is not clear from the heatmaps. If it is not significant, it should be clearly stated and discussed.

3. It is not clear how the A. thaliana, and O. sativa CCH genes were identified. Will a similar sequence search conducted for CCH in these genomes. The criteria should ideally be same for all genomes.

4. It is known that a single query may not be enough to cover all the genes in a gene family and the choice of query also affects the family coverage (see e.g. Kaushik et al. PLOS ONE 2013). It can be tested by a sequence search using all A. thaliana CCH if any new CCH proteins are identified in any of the 3 organisms.

5. Are the functions of the predicted motifs known? And based on the motifs, can a new functions of CCH in Populus be identified? Domain combination analysis can also be used to identify novel functions of CCH if any.

6. Has the lineage leading to the evolution of Populus experienced multiple Whole Genome Duplications? How does genomic distribution of CCH relate to other WGD events in the past?

7. Is there any specific property in ohnolog PtCCH and the ones that are retained from SSDs? It will be interesting to discuss.

8. Has the expression pattern diverged between the leaves in Pt and PnCCH? What could be the reasons and implications for such a divergence.
Validity of the findings: The finding would appeal to limited audience interested in CCH family in plants. Most of the article is very descriptive. If the results are put in a broader context, it will make the manuscript more appealing to broad audience.
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: GENOME-WIDE IDENTIFICATION AND EXPRESSION PROFILE ANALYSIS OF CCH GENE FAMILY IN POPULUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Xu et al. describes the identification of genes encoding copper chaperone (CCH) family proteins in Populus. Based on the known P. trichocarpa genomic sequence, they perform a bioinformatic analysis and identify 21 proteins with sequence and structural similarity to Arabidopsis CCH, analyze the chromosomal location of the corresponding genes, and perform a phylogenetic analysis including proteins from rice and Arabidopsis. They also analyze the expression of several of the Populus genes in different organs under different conditions (control, copper deficiency, copper excess). Their main conclusion is that the CCH family is expanded in Populus and they suggest that this may be related to the ability to grow on contaminated areas and accumulate metals.

The manuscript is correctly written, with references to the relevant literature. The bioinfomatic and experimental analysis was properly performed, although some corrections and clarifications are required, as described below.

Figures and figure legends:
-Fig. 2 legend: Please, explain the meaning of the different boxes connecting the chromosomes.
-Fig. 3C: References to motifs are too small and difficult to read. Please, refer in the legend that the sequences of the motifs are listed in Table S4. Also, are these motifs similar to any known motif, thus giving clues to their possible functions, or are they just conserved sequences between CCH proteins?
-Fig. 3C legend: explain the meaning of S1 to S3.
-Please, add bootstrap values to the trees, or an indication of how significant the different branches are. Related to this, do the trees shown in Figs. 3 and 4 have the same structure? If not, how do the authors explain this? How do clades S1 to S3 in Fig. 3 relate to clades SI-SIII observed in Fig. 4? Ideally, the labeling of clades should be the same (S1-S3 or SI-SIII).
-Fig.  4: I guess there is a mistake in the labeling of some groups (blue and green in the upper left part should be SIII instead of SII).
-Fig. 5: The labels in the bar graphs are difficult to see (too small). The legend should be more descriptive, so that the reader can understand the figure without referring to the text. This applies to all figures. Please revise this.
-Fig. 6: Relative expression is shown. Relative to what? Untreated controls? Please, clarify this. Also, give a hint of the statistical significance of the results (for example, include an asterisk in those rectangles where changes in expression are statistically significant).

Discussion:
Lines 425-434, explaining primer design and why the expression of some genes was not measured, should be removed from here and included under Results.

Conclusions:
The Conclusions are not properly written. English is poor and there is repetition of concepts already mentioned in other sections. Re-write or, preferably, remove this section, since it does not add much to the paper.
Experimental design: I find that a basic question that is not correctly addressed in the manuscript is that of the function of members of the CCH family. The authors refer ambiguously to the CCH gene from Arabidopsis, which has been functionally characterized, and then identify 13 CCH genes in this species. Are there any studies on these other CCH genes? Which one of these 13 is the “real” CCH? (I guess it is AtCCH6). These matters should be discussed in the manuscript. Also, I suggest that the authors perform complementation studies in yeast to analyze if representative Populus CCH proteins from different clades are functionally equivalent to Arabidopsis CCH. This would add a functional significance to the expansion of the family.
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: NEW CERNOTINA CADDISFLIES FROM THE ECUADORIAN AMAZON (TRICHOPTERA: POLYCENTROPODIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: I  have made my comments regarding terminology, nomenclatural conventions, and typos or missing commas.
Experimental design: The description of these new species is important, especially given the environmental threats and possible extinction of species and habitat.
Validity of the findings: These are the first new species of this genus discovered from the region.
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: NEW CERNOTINA CADDISFLIES FROM THE ECUADORIAN AMAZON (TRICHOPTERA: POLYCENTROPODIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: Line 70; Dudgeon et al, 2005 is cited as 2006 in the literature, in this same line, Finlay 2010 has ae extra comma and a D before the )
                Line 81; mentions a supplementary file 1 which I didn’t see
                Line 89; should be a comma after  For examination and description
                Line 134;  ..thumb-like, ca. as long as – what is ca., suggest authors write out word which ca. is describing, nearly??
                Line 140, 165, 167; same comment about use of ca.
                Line 187; Cernotina hastilis Flint 1996, Flint 1996 is not in literature cited, nor is Botosaneanu 2002, in the next line (line 189)
                Line 201; the reference Paprocki et al. 2004 is not in the literature cited
                Line 212, 213; the reference Paprocki and Franca 2015 is not in the literature cited


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: SKIN AND FUR BACTERIAL DIVERSITY AND COMMUNITY STRUCTURE ON AMERICAN SOUTHWESTERN BATS: EFFECTS OF HABITAT, GEOGRAPHY AND BAT TRAITS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper describes the bacterial communities found in the fur and skin of 14 species of insectivorous bats (in a total of 186 animals) in 5 caves in the USA states of Arizona and New Mexico. Bats were caught either directly in the caves or outside of them, from 2011 to 2014.  Using 454 massive sequencing, the authors analyzed 16S rRNA.
	The sampling appears to be good (but we are never informed of the total samples per bat species per cave, for instance), and in general, the molecular and statistical methods seem competent.
	The main result is that, according to statistical analyses, cave- caught vs. surface-netted was the most important factor in determining the differences in the bacterial communities, and the second more important factor was the eco-region were the animals were sampled.
	In relation to these two main findings, I have my most important concerns of the study.
	1) Why do the bacterial communities associated to the bats change so much in a cave and outside? If they change so drastically, perhaps most of the bacteria the authors are finding are just transient, just acquired from the environment, but actually not living and interacting in the skin and hair of the bat, and perhaps they are not even active, since 16S rDNA does not distinguish if they are dormant or even dead.
	It would be critical to design a method to actually evaluate the active and resident bacteria in a bat: the bacteria that are alive, interacting with other microorganisms, both bacteria and fungi, and with the other organisms in the skin (ectoparasites) and also organisms conducting a functional role in the external biome of the bat.
	While we could devise molecular methods for disentangling this (to know which bacterial are the active vs. dormant or dead bacteria), such as analyzing the transcriptome of the bacterial RNA in the skins and hair of the bat (and should be discussed!), at least the authors could compare the bacterial communities that were found in a given bat species in a given time, both inside and outside the cave. These shared OTUs between the biomes inside and outside the cave may represent the true resident bacteria.

	2) The sampling scheme is never clear. In particular, only 5 caves were analyzed, but in Figure 5 there are 8 environments. A table indicating caves, the external sites, sampling dates and bat species would help understand the final sampling design and to evaluate how well balanced is the study and which other possible analyses could be conducted with confidence. Also a clarification indicating if the bats in the caves are the same as the ones in the nets, or if the caves represent another population -- that may explain some of the results-- would be important.
Experimental design: Collecting Methods: Lemieux-Labonté et al. 2016, used as a negative control a humidified sterile swab, page 4, that sounds like a good idea, as they detected evidences of contamination, page 5.  Apparently in the manuscript there were not similar controls; this cannot be changed if it is the case, but this point at least should be discussed.
	Also, environmental samples of both the cave and the outside samples would have been interesting to check for the identity of the bacteria, instead of using the global or arbitrary data bases you used. Discuss.
Validity of the findings: I think the main findings are adequate, but the presentation of them should be improved and better interpreted. See the other sections for comments on the validity.
Additional comments: Abstract:
Lines 28: Briefly, explain how and why the microbiomes from day to night can be so different! At first glance, this does not seem to make any sense.
Line 31: Explain what is WNS.

Introduction:
In general, I feel that more emphasis should be given to the bacterial communities in the skin and hair in the Introduction.
	The skin and hair of the bats should represent complex environments, as the bats are notorious because the high number of ectoparasites found, including fleas, different mites, and other arthropods, and their immune system seems to be very developed. Clearly the environment also includes fungi, as the one responsible of the white-nose disease.
	The authors should make an effort to discuss better bacterial and the communities of other organisms from the skin (and hair in mammals or feathers in birds) in vertebrates.
	An important reference that should be reviewed in the Introduction and more carefully discussed is the study of Lemieux-Labonté et al. 2016. PeerJ. The introduction of that paper cites several relevant references to understand the skin bacterial ecology that should be explained in the ms. Also, apparently this is the only similar analysis of culture free bacterial communities in bats, and for this reason it should be carefully described in the Introduction, and compared in the Discussion. The paper is indeed mentioned in the Discussion in line 299, but is not mentioned in the Introduction and the paper is missing in the References section.

Line 46: “in the Southwest”: Make it more general, so people outside North America will understand that you are talking about a specific part of the South of USA.

Line 49: I am not sure what “syntopic” means, explain.

Around line 66: Improve the review of the skin and hair communities in animals, in particular for mammals and bats. You mention the white-nose fungus, but as I explained above, many other organisms live there, and there are some interesting recent papers included and reviewed in Lemieux-Labonté et al. 2016.
	Also, a critical issue is the functional role of the bacteria in this environment, as it can include transient (that may be interesting but irrelevant), and resident bacteria, and methods to disentangle both.

Lines 91 to 93: And if fungi communities are so important, Why not conduct a parallel study using ITS for the fungi? Not in this paper, but here you could discuss this (perhaps some bacteria defend from fungi in general) and you could use the DNA already extracted for this paper.

Methods:
As I mentioned above, the sampling is not clear, as there are 5 caves, but 8 eco-regions in Fig. 5.
A table stating the sampled caves, eco-regions, dates, and number of bats per species will help the reader to understand the total sampling, and how unbalanced was the sampling, and if it is possible to compare microbiomes of a bat species and a given date with samples in and outside the cave, to explore for the existence of resident and transient species.

Lines 111 to 122: As I mentioned above, some controls for contamination seem to be missing, and environmental samples would have been useful to interpret the data.
Also, how do you decide the number and species of bats in each cave and sampling? How many animals were sampled at each sampling date?

Lines 150 to 151 and 219 to 228: It is not clear if you rarefied or not the data, or rarefied in some analyses and not in other.
	I am not sure if McMurdie and Holmes 2014 is actually right, but I am sure that using different sample sizes would bias the estimates of diversity.

Line 201: What is the meaning and interpretation of the VDS. I am not familiar at all with this statistic.

Results and Discussion:
In general, I do not like to make a joint section of Results and Discussion, as I feel the authors tend to confuse what they found and what the wanted to find. Separating both sections in clearer for the reader.
	I strongly recommend to separate both sections, and to carefully rewrite the Discussion, making clearer comparisons with other studies of external microbiome in animals, in particular to Lemieux-Labonté et al. 2016 paper, as I mentioned above.

Fig. 1: It is very attractive, but make it more general:  a) Mark the sampled caves. b) As these caves are more related to the southern populations, and as the national borders are meaningless for the biology, also include (North of?) Mexico (as Canada is included). This would make the map and the paper more useful and even more attractive.

Line 247: The number of reads among samples is very variable, 843 to 20,515 and thus I would be very careful of comparisons between samples if they are not rarefied. What was the average number of reads?

Lines 252: An easy to read and compare figure of the dominant bacterial groups in each sample, and perhaps in other categories, in particular of the bat species, would be important.
	For instance, you could show this data in a simple pie figure, as in Figure 1 of Lemieux-Labonté, et al. 2016. If you use the same order and colors, this figure could be directly compared and discussed.
	In contrast, your Figure S1 is completely useless. It is impossible to read or to make any sense of it.

Lines 255 to 264: The Random Forest results apparently are the main finding of the paper. I am not familiar with the method, but I think that if it is so important, these results should be shown in a figure or table, to give more emphasis, but also include the associate statistics to evaluate the probability or reliability of this analysis.
	On the other hand, I am not so sure of the results of the Random Forest, given the values shown in Figure 7 for some other variables as soil PH, the values for some environments and some bat species, or sampling dates, that seem to be very important. I suggest to restructure the paper and give the Random Forest and the MDS analyses together, to try to disentangle what is happening and which are the more important variables that determinate the bacterial communities.

Lines 265 to 278:  Above, in lines 252 to 254, there is a brief description of the communities. Put both sections together, to support the suggested above pie figure.

Figure 3 is interesting, but obscure, as the Y-axis is a complicated and non-intuitive variable (Log-relative normalized). Adding simple (additional?) pie figures or something like that may help understand the abundance of the bacterial groups. Also, explain better the estimate of the number in the Y axis, I guess a higher number means a higher number of that taxa, but it is not obvious.

Line 270: Supplementary figure S5 is cited here, but apparently the figures (or tables) S2 to S4 have not been cited yet. S5 figure is very complex, and I am sure there should be an easier way to visualize this information. Improve, please.

Lines 299: Extend and improve discussion of Lemieux-Labont et al. 2016. Also include reference.

Fig. 5: I do not see any gray line. May be just a problem of my computer, but it is a good idea to make sure the figure can be opened OK in any software.

Figure 6: As this analysis explains only 4% of the variance, I would send it to supplementary material, and just mention the statistic values in the text, lines.

Line 317 and Figure 7: What is the meaning of the VDS corrected richness? How you interpret this statistic?

Lines 342 to 359: While I think this analysis is interesting, I am not sure how reliable it is, as the comparison with the data sets are very heterogeneous: Barberán et al. 2015 seems to be for the complete continent, Newton et al. 2011 for all the available lakes in the world, and for the caves it seems very arbitrary, lines 213 to 218.
	I am not confident at all of this analysis. It would have been better to take as controls environmental samples of each sampling locality. Please, consider sending this to supplementary material and carefully discuss its limitations.

Line 372: The “C” of cave is missing.

Line 375: Indicate the dominant phyla.

In general all supplementary materials will need a careful evaluation and edition, and a list of these materials was missing in the paper (and one of them I could not open). Some of the materials are very difficult to understand what they are or their meaning and the text is some case is too difficult or impossible to read, as was the case of S1.


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: SKIN AND FUR BACTERIAL DIVERSITY AND COMMUNITY STRUCTURE ON AMERICAN SOUTHWESTERN BATS: EFFECTS OF HABITAT, GEOGRAPHY AND BAT TRAITS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Winter et al. analyze the external surface microbiota of 14 species of bats across the Southwestern United States. This is a relevant study that addresses important questions about the factors influencing bat microbiomes. Unfortunately, I consider that the manuscript has several structural and technical flaws that prevent it from its publication in PeerJ the way it is. At the end of this report I describe general comments and suggestions to correct and improve the manuscript. Should the authors address the corrections and improvements suggested, I will be happy to review a new version of the manuscript. Specifically, the article fails in explaining in a clear way their main findings. The manuscript needs a better structuring of the information. The introduction requires a better background on skin microbiota and some of the figures can be improved.
Experimental design: Overall, this study is correct in terms of the experimental design except for one major aspect that fails: my major concern is the decision of swabbing the entire skin and fur surfaces. Based on previous studies in other mammals, skin microbiota differs across skin regions so the decision of swabbing everything will obscure some important patterns. Also, the authors emphasize the importance of this study in the context of an emerging disease (WNS) however their swabbing technique neither replicates previous studies like Hoyt et al., 2015 nor it is focused on the regions infected by P. destructans (wings and uropatagium). Another important aspect, is that swabbing all external surfaces might also catch a high proportion of transient bacteria which are not specifically associated to bat skin, therefore making the interpretation of the results difficult. The authors need to clearly explain why they made this decision and how this would influence their results.
Validity of the findings: Overall, this study is correct in terms of the validity of the findings. However at the end I have several suggestions that will improve this section.
Additional comments: General comments
Overall, I consider that the methods, results and discussion are confusing due to the way the manuscript is structured. It is not clear which are the main findings and it is hard to follow the main line of the manuscript. For example, in the abstract it is clear that ecoregion and location are the main factors influencing “external surface” microbiota, however in the results (using different methods) it is mentioned that species, sampling site, seasonality, NPP, soil pH, Conifer Woodlands and Savannas, and Chihuahuan Basins and Playas are also influencing bacterial diversity.  The authors need to determine which are the main findings and streamline their results and discussion. This includes, evaluating which methods explain better their results.

I have some concerns with some of the methods and analyses. Specifically, I don’t think the mantel test is strong enough to mention that geographic distance is playing a role. Even though the regression is significant the R value is extremely low. I would not include this test. Also, I am not convinced by the comparison of specific bacterial taxa with environmental sources. Bacterial communities in nature are highly diverse and therefore if the authors want to compare environmental microbes with the microbes obtained from bats they should have collected samples from the exact site where the bats were sampled. In my opinion, this is the correct way of comparing environmental microbes with potentially symbiotic ones and also to determine the proportion of transient bacteria present on the bat surfaces.

Since the authors analyzed 14 different species it would be interesting to analyze if there were any patterns associated to the bats habits. Do these bats eat the same food? Do they have similar life histories? What about a phylogenetic component?

Specific comments

Introduction

The authors need to expand the background on skin microbiota and their role to prevent emerging diseases in bats and in other non-human systems that have been studied with more detail.

Line 42. Eliminate “second to rodents at 2277 species”.

Lines 55-64. I am not sure how this information is relevant to this study, since all of these aspects are not analyzed later in the text.

Lines 77-78. You need to be more specific here, since this is the only study about antifungal bacteria in bats. Also, I think this study needs to be discussed later in the text in the context of your results.

Lines 79-85. Need more references of skin bacteria in bats such as Lemieux-Labonte et al. 2016.

Methods

The methods are a little confusing and they are disorganized. A better structure would help the reader follow the text better. For example, Sampling also includes molecular techniques like PCR. Normalization is mentioned twice. Diversity estimates are divided in different sections (alpha and beta diversity, NMDS plots etc) and they should be included in one.

Line 118. Explain what is Ringer’s solution

Line 151. Why did you use a depth of 1500? Please explain this sentence better.

Line 191. Eliminate the last sentence that starts with “NMDS…”

Line 203-209. There is no need to include the R script that you used here, unless you decide to include all the other scripts used for this study. If so, I would send all the scripts to supplementary figures.

Lines 219-234. Explain with more detail how DESeq2 and CSS work. Also, explain better how the data obtained with these methods was correlated with rarefied data. If these were highly correlated with the rarefied data, why not use rarefaction? Maybe I am missing something here since I am not familiar with DESeq2 and CSS.

Results and discussion

The results in general are difficult to follow. The authors describe patterns of diversity associated to cave caught and surface netted and then explain pattern associated to ecoregions and then go back to describing cave caught etc. I would reorganize the results in subsections that reflect the main findings.

Line 249: By looking at the supplementary figures, the authors need to explain why there was such a large proportion of unassigned reads.

Line 252-254: This sentence seems out of place. I think it should go later in the results.

Line 255: Change “The data were tested using a random forest model to see if the data…” to “A random forest model was used to see if the data…”

Line 256-259: You mentioned in the methods that a reasonable ratio of the estimated
generalization error compared to the baseline error should be two or greater, i.e. the random forests classifier does at least twice as well as random guessing for an unlabeled community. So why do you say the method was minimally successful if a ratio of 2 or greater seems enough? Please explain

Line 274-277: To determine the effect of ecoregions and habitat with random forest the authors should only include regions where both bats from caves and from surfaces were collected. Otherwise it is not possible to distinguish the effect of the ecoregion versus location.

Line 285: Why chloroplasts? Please explain.

Line 285-288: This needs to be explained better. I would think the opposite, that cave caught bats would be dominated by bacteria adapted to cave environments and that surface netted would have a higher diversity of bacteria because they are in contact with many environments, soil, air, plants etc.

Lines 289-299: There needs to be a stronger discussion section comparing with previous findings. The studies mentioned here were done in different species of bats with different habitats and the samples were obtained from different origins (guano, guts or ocular surfaces). There needs to be a more thorough explanation of why the authors find differences or similarities with these studies. Why did you not find differences among host species? were the samples taken differently that what Lemieux-Labonte et al. did?

Lines 300-301: The authors need to discuss the fact that transient bacteria may be present in their samples. How do you take this factor into account?

Lines 302-304: This was mentioned in the previous section too.

Lines 337-338: What are the implications of these differences? Overall, throughout the text the presence of specific groups of bacteria need to be explained better: What are these bacteria characterized by? What does it mean if a specific group is enriched in cave caught or in surface-netted bats etc?

Lines 339-341: I don’t think these sentences are relevant for the discussion

Conclusion

Lines 362: Why does comparing with one soil study (Ma et al., 2016) is relevant? Please explain. I would suggest to focus on specifying which are the factors that mostly contribute to explain your data instead of comparing with other unrelated studies.

Lines 363: “Internal” means “gut”?

Lines 372: What does “.Ave” mean?

Lines 374-387: I think there needs to be a clearer conclusion. What are the main findings? Which factors determine the microbiome in bats? Which factors will require more studies in order to determine their relevance?

Figures and tables

Figure 1: I don’t think this figure is relevant in the context of this study.

Figure 2: This maps need a scale. If you decide to keep the mantel test you could make a figure with the map and the graph together? This is just a suggestion.

Figure 7: To make the interpretation easier I would highlight with some color the variables associated to each of the NMDS and richness.


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: SKIN AND FUR BACTERIAL DIVERSITY AND COMMUNITY STRUCTURE ON AMERICAN SOUTHWESTERN BATS: EFFECTS OF HABITAT, GEOGRAPHY AND BAT TRAITS
Review round: 2
Reviewer: 1

Output:
Basic reporting: For this new version of the manuscript the authors changed the figures and answered some of my concerns.

Nevertheless, there are still some issues, in particular about the figures that the authors will need to correct in the next version of the paper.
Experimental design: See below.
Validity of the findings: Somewhere in the Discussion or in the Conclusions, the limitation of the study should be clearly discussed in order to try to improve similar research in the future. From my original review I copy some of points that are still needed for this:
1) “It would be critical to design a method to actually evaluate the active and resident bacteria in a bat”, and explain how you could solve this.
2) “Lemieux-Labonté et al. 2016, used as a negative control a humidified sterile swab, page 4, that sounds like a good idea, as they detected evidences of contamination, page 5. Apparently in the manuscript there were not similar controls; this cannot be changed if it is the case, but this point at least should be discussed.”
3) “Also, environmental samples of both the cave and the outside samples would have been interesting to check for the identity of the bacteria, instead of using the global or arbitrary data bases you used”.
Additional comments: Line 137:  It should say “Figure 1”, as the figures were changed.

Line 147: You should mention Figure 2a around here.

Line 161: Figure 2b should be cited here.

Line 197: Explain better. Apparently all the samples belonging to a bat species were removed. If this is the case, mention this bat species here.

Lines 207 and 294: Explain what is BLM.

Lines 276 to 286 need work. Move lines 276 and 277 to lines 195, were this explanation seems to belong.
And move lines 279 to 286 to Sampling section, around line 162.
Lines 286, remove an extra dot at the end.

Figure 3: Include the units in the figure: 0 to 60 of what? And check the species of the bats and include the name of the “M” species that is missing in the figure caption.  Also, put in italics the Latin names in the caption.

Lines 360: Remove an extra dot at the end.

Figure 5: Indicate what is the meaning of the colors of the dots in the graph.  Change bat name to “Myotis velifer”

Figure 6: Indicate the axis of the figure: latitude and richness, and also indicat the meaning of the two colors, red and blue of the dots.

Figure 7: Indicate the meaning of the colors of the dots.

Line 522: “likely behavior”?  Maybe it is flying or feeding behavior, as in the graph. The figure should be 5, not 6. Correct.


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: SKIN AND FUR BACTERIAL DIVERSITY AND COMMUNITY STRUCTURE ON AMERICAN SOUTHWESTERN BATS: EFFECTS OF HABITAT, GEOGRAPHY AND BAT TRAITS
Review round: 2
Reviewer: 2

Output:
Basic reporting: I consider that the manuscript has improved from the last version. The introduction and the results are clearer now. However, I still consider the manuscript requires some improvements before being considered for publication. In particular, I consider that the authors didn´t properly address some of my concerns regarding the effect that transient bacteria may have on their results and also, they need to correct a few sections and figures. My comments are shown below (general comments to the author).
Experimental design: I have no further comments regarding the experimental design.
Validity of the findings: I have some comments here but I will present everything below(general comments to the author).
Additional comments: I don’t think the term “external microbiota” is the most appropriate term: First I think it´s too vague, second, if external means skin and internal means gut I would argue that actually the gut is also external since it´s not INSIDE the host. I would modify these terms (external and internal) and be more specific: skin and gut microbiota maybe?.

The authors expanded the methods section and explained why the sampled the fur in addition to the skin. However, I still think that this approach will be likely catching transient bacteria from the environment. In fact, there is a hole section in the discussion (lines 509-519) where the authors discuss how the differences they found between bats from different habitats may be related to changes in the environmental bacteria present in caves in contrast to surface environments. One interpretation of these results is that the sampling scheme is promoting the capture of transient bacteria and therefore the microbial communities on the skin are just a reflection of the environment. Therefore, I strongly think that the authors need to address the main caveat of the study, which is that they didn´t took environmental samples to compare with and therefore they can't discard the possibility of transient bacteria being sampled.

Specific comments

L42-44: I suggest adding more references on microbiomes in other animals (i.e amphibians hydra) in addition to the whale paper.

L122-124: Maybe rephrase this first question. Something like: does the daily routine of bats (spending time outside or inside a cave) influences their skin bacterial composition?

L197-200: This should be part of the section Sequence procesing.

L236-237: Phylum? I guess you mean Class? Alpha, beta and gammaproteobacteria are classes.

L262-265: I don’t understand the difference between the first and the second sentence.

L276-277. This section should be included in the introduction.

L279-286: This section should go in the “sampling section”.

L299-306: I would add this paragraph with the next section and eliminate the first sentence which is unnecessary.

L308: Change “Our study stands apart from culture-based studies and other next generation sequencing studies by focusing on the diversity of the external bacteria from...” to “Our study focused on describing the diversity of the external bacteria of 163 wild-caught bats from…”

L314-316: Be careful here: you are confusing classes with phyla. Choose either one but fix the mistake here and throughout the manuscript and figures.

L334. Change the title of this section to something like: The effect of habitat, ecoregion and species type on skin microbiota

L357-360: I suggest eliminating this paragraph. I don’t think is necessary.

L362: I don’t think you need so many subtitles for the results section. I suggest eliminating this one.

L387-388: Eliminate this first sentence and add this paragraph as a continuation of the previous paragraph. You can start L379 with: “A bayesian hierarchical model was fit to explain the amount each predictor contributed to the community dissimilarity on the NMDS1 and NMDS 2 axis. In the case of NMDS1…. In the case of NMDS 2…

L410: eliminate: (defined by QIIME’s alpha_diversity.py)

Figures 6: This graph needs more work: What are the axis? Explain the top part of the figure, what are the colors of the dots? The same with figure 7. They both need to be clearer.

L424: I am not familiar with these bayesian hierarchical models; thus, I really don't understand the values described below. I strongly suggest explaining this part more clearly. Specifically, the meaning of the values and the sign of the value need to be explained for a general audience: What does a negative or positive value mean? The larger the value the greater the effect?

L436-440: This section to me is telling the readers that habitat and latitude are confounding factors and that maybe is not latitude but habitat what is explaining these patterns. I would reconsider presenting these data as it is.

L487-498: I don’t think this paragraph of the discussion is relevant for this study. The comparison with other animal system at the bacterial phylum level is, in my opinion, not informative. The bacterial phyla that the authors describe here are pretty much present, not only on the skin of different organism but also on any environment.

Correct all the “cofounding” by “confounding”.


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: INFERRING MICROEVOLUTION FROM MUSEUM COLLECTIONS AND RESAMPLING: LESSONS LEARNED FROM CEPAEA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a clearly presented and interesting account of changes in recorded morph frequencies in the snail Cepaea nemoralis over time. The animal is an important model for the study of changes, rapid or longer term, in response to changing environment. The evidence for rapid response and short-term fluctuation is an area of current interest and this paper is a worthy addition to it.

I do, however, think that the authors need to think more fully about the conclusions from the data and make a much more forceful case for their interpretation if they wish to stand by it.
Experimental design: Experimental design is clearly stated and appropriate to the objectives of the analysis.
Validity of the findings: The data are robust and statistically sound and the analysis appropriate to meet the objectives set out. I have some issues with the interpretation, which will be set out in the next box.
Additional comments: Comments from Laurence Cook.

From my own experience of the subject of your paper I have the following comments to make on the conclusions.

Line 55. ff. The authors will know that I have reservations about the treatment of eU as a genetic category. Although Cain and Sheppard (line 206. particularly Cain, who emphasised the fact that this was a visual phenotype) noted the predictive value of eU in relation to habitat, analysis of further data suggests that it is no improvement of the category unbanded itself (Cameron & Cook. 2012. Folia Malacologia 20, 255-263). It would be safer to refer to the change as adaptive rather than genetic.

Line 61. ff. Another important long-term study, which appears to show stability and also considers movement and one of the selective agents, is by Charles Goodhart ( 1956 Proc. Linnean Society of London 167, 50-67; 1956 Proc. Linnean Society of London 169, 163-167.

Line 127. ff. The sample sizes for Lobith are colossal, surely much larger than a possible effective population? I have not gone back to Wolda’s paper, but is there a possibility that some frequencies quoted are ensemble values whereas with the later smaller densities you would have been examining sections? I think a note on this possibility should be added.

Line 153. Does that imply that some morph or morphs entered the samples after the first sampling?

Line 218. ff. Surely the caution should be applied to the short-term states not the long-term states (see below)?

Line 250. ff. But patterns can be perceived, and are very much dependent on one’s confidence that the appropriate populations are being sampled at the different times (Cameron & Cook. 2012. Folia Malacologia 20, 255-263; Cook. 2013. J. Moll. Stud. 80, 43-46).

General. I think it is important to investigate and interpret the results you describe. Nevertheless, the problem I have with the interpretation is in your sentence beginning line 202. If short term fluctuations characteristically exceed longer term change, apparently sometimes by large amounts, the implication must be that some kind of stabilizing force also operates. Otherwise fluctuations would accumulate and lead to monomorphism or extinction. Why does this not happen? We carried out a very detailed analysis of the data looked at by Bell (Cain et al., 1990) and in the end concluded that there was no evidence of forces shifting frequencies on a yearly basis. The estimations of ingress and egress did not show up age classes suffering from high mortality or radical difference in contribution of morph types to a particular recruitment. The alternative would have to be that, for whatever reason, the methods or circumstances of sampling sometimes introduce biases. At Point of Air (Cook & Pettitt, 1998. Biol. J. Linn. Soc. 64, 137-150) there is no evidence of selection introduced by climate etc. but plenty of reason to suppose that extensive movement of individuals took place that could account for what would look like a selective change if sampling was restricted to a limited part. In another sampling (Cook 2003. J. Conchology 38, 73-78) I also noted a change in frequency in the middle of a three-sample (in fact, a four-sample as one of yours) sequence which later appeared to be cancelled. A possible reason discussed could have been that when density was low relative visibility of morphs changed. I think that at least you need to discuss these problems more fully before concluding that frequencies do in fact change radically on a very short-term (sometimes apparently within-generation) basis.


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: INFERRING MICROEVOLUTION FROM MUSEUM COLLECTIONS AND RESAMPLING: LESSONS LEARNED FROM CEPAEA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This short manuscript is right to the point with an accurate and relevant message. However, I found the message so trivial that it should not be necessary to point out these things. So, what I should like of the authors is that they give more meat on the bones when referring to the older literature and give very detailed examples of when authors has drawn conclusions of long-term trends from just two data points (one historical and one contemporary).
Moreover, it would be interesting to learn if this is the case for colour-variation in Cepaea snails, usually, or if similar inferences have been drawn for other traits that are tentatively under selection. Examples would be welcome.
Finally, I would suggest yet another possible way of assessing interannual variation in order to separate this from long-term trends if only one historical sample is available: Instead of using only one year of contemporary sampling, repeat sampling over 2-3 contemporary years to assess interannual variation.
Experimental design: The experimental design is straight forward and only basic statistics is used.
Validity of the findings: I wish I could conclude that this information is of very little value because conclusions of long-term trends are never drawn from only two data points. But, if this is so (and as suggested above, I think the authors must do more to show that this is actually the case) then this study has some value. In particular, if the authors (as is partly done) suggest approaches to solve the problem.
However, if the authors fail to find good example of studies in which conclusions are based on only one historical and one contemporary sample, then I think this study has only very minor value, as this should be rather obvious to anyone.
Additional comments: My main point is that the authors should put more emphasize on examples of studies in which this has been a problem. It would also be good to focus the study more on how the problem can be solved. There is some discussion about this but this part can be more of a focus in both Abstract, Introduction and aim of the study. How do you deal with a situation when there is one historical data point available....
Such a focus would be much more of a contribution to the scientific society than only pointing to an obvious problem.