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I am glad to accept your excellent manuscript for publication. Thank you for choosing PeerJ as the venue for this interesting research.
[# PeerJ Staff Note - this decision was reviewed and approved by Konstantinos Kormas, a PeerJ Section Editor covering this Section #]
Please address the remaining minor points. I also found a typo: you state "It is important to note that the significant positive correlation of males of P. siculus was reversed when the smallest sized individual (0.59 mm) was removed from the dataset (R = -0.62, p = 0.04)." I believe you mean "59 mm" instead of "0.59 mm"
This manuscript is well written and contextualized within the existing literature. Only a few small typographical/figure issues remain:
Perhaps re-order figure four to group males and females together, for ease of comparison?
Point size on Figure 5 could be increased for legibility, and to better distinguish data points from outliers of the boxplots. Currently it looks like there are open points and filled points with no legend to explain them.
There is still code in lines 251-255, this is unnecessary as the script is included in supplementary materials.
The experimental question is clearly stated and the methods well explained and suited to answer this question.
I appreciate the authors adding the additional scripts to their supplemental info. The data are well analyzed and the conclusions the authors draw are supported and reasonable. My only concern is the low number of females for P. lusitanicus (n = 2), I would hesitate to draw any conclusions of sex differences (or lack thereof) based on such a low sample size.
The authors have addressed my major concerns or given an acceptable reason why they will not be addressed. This is a well written and interesting manuscript that will be a valuable addition to the field of microbial ecology, for reptiles in particular.
We have received high-quality reviews. Every one of them is very supportive of your paper, but they include many requests for clarification of (usually) minor points. Please address them all. In spite of their number, I do not expect any of them to offer any challenges.
Vasconcelos et al., in the manuscript entitled “Factors shaping the gut microbiome of five species of lizards from different habitats”, present an interesting, well written, descriptive study on the effects of environment on the gut microbiota. They provide an important contribution to the field by focusing on reptiles, and present an informative study by comparing multiple species from very divergent sites.While enough figures and tables are provided, one aspect that has to be addressed is the presentation of the supplementary tables (proper naming and legend, see my comments below),
The project has a clear question and was properly designed for the question. As the authors mention, it would have benefited from sampling the same species from different environments, but this was likely a budget constrain and does not invalidate the discoveries from the authors in any way.
The authors present well supported data to support their claims. Their attention to details in methods description allows for the study to be verified and/or repeated.
Line 33: Please add the Oxford comma throughout the manuscript.
Line 45: This might be a pdf conversion error, but the space between “environments” and “having” is highlighted.
Line 50: The “P.” from Podarcis is not italicized.
Line 73: Square bracket should be curve. Additionally, add a comma after “e.g.”
Line 102: There seems to be a double space before “Individuals”.
Line 103-105: Only fully spell the full genus name on its first instance in a section. Replace all other “Podarcis” by P..
Line 109-112: This should be two sentences. Otherwise it may be interpreted by the readers that the diet is also part of the sexual dimorphism.
Line 111: Please replace “Teira” by T.. This is not the first instance in which the genus name is introduced.
Line 115: I believe the authors mean “to datE”.
Line 116-117: Only fully spell the full genus name on its first instance in a section. Replace all “Podarcis” by P..
Line 134-135: Only fully spell the full genus name on its first instance in a section. Replace all other “Podarcis” by P..
Line 136: Please refer to supplementary table 1 in here. And replace the sheet names in the supplementary excel as supplementary table 1 and supplementary table 2. Additionally, I can’t find the legends for these tables, I recommend creating a README sheet on the supplementary excel where the legends for both tables can be found. Finally. I do not agree with the name of this excel, the information provided on supplementary table 2 is not the raw data, it has been processed. I suggest calling the file supplementarytables.
Line 150: Two sites are shown on figure 1, but there are only GPS coordinates for one of them. Please add the second set of GPS coordinates.
Line 162: Please replace “Teira” by T.. This is not the first instance in which the genus name is introduced.
Line 163: In the previous instances the figure 1 was introduced with the coordinates. Please add it here to maintain the structure of the text.
Line 175: Swabs of different materials have different efficiencies, please indicate the type of swab used.
Line 185-186: Did the authors mean “… along with the extraction blanks, [AND] PCR controls….”?
Line 190: Add a comma after “e.g.”
Line 195: R is not properly cited. It should be R Core Team, 2020, R Core is not the “first name”.
Line 206: Please add the thousand separator, it is impossible to easily read this number as it is. Also, add the thousand separator throughout the rest of the manuscript.
Line 208: Appendix 1 does not tell the reader which table to look at (the excel has 2 sheets). Additionally appendix is not the term endorsed by the journal. See my comment to line 136.
Line 237: Unclear what the authors are referring to, please replace for Supplementary table, see my previous comments.
Line 255: Please add the degrees of freedom to the F values here and throughout the rest of the results.
Line 265: Please remove the . before “(Fig. 3)”.
Line 287: In order to keep consistency throughout the text replace “about” by ~.
Line 302: This might be a pdf conversion error, but the space between “diet” and “items” is highlighted.
Line 305: I don’t see a value in adding personal observation to the reference when there is literature supporting it. I would suggest to remove it.
Line 309: Add a comma after “e.g.”
Line 350: I recommend the authors to remove “personal observations” if they did not actually measure the distance between lizard pairs to be able to say it with confidence.
Line 366: Please replace “Podarcis” by P.. This is not the first instance in which the genus name is introduced.
Line 390-393: For manuscripts, only the first word should be capitalized.
Line 410-411: For manuscripts, only the first word should be capitalized.
Line 445-449: This paper is not cited on the manuscript, please remove.
Line 456: Journal name not italicized.
Line 481-486: For manuscripts, only the first word should be capitalized.
Line 507-510: This paper is not cited on the manuscript, please remove.
Line 515: Journal name not italicized.
Line 576-578: This paper is not cited on the manuscript, please remove.
Line 579-581: For manuscripts, only the first word should be capitalized.
Line 598-601: For manuscripts, only the first word should be capitalized.
Figure 4: Parenthesis not closed on the last instance of the legend.
Table 2: Please add the degrees of freedom.
I have only a few suggestions in this area. The manuscript is excellent in structure and clarity.
Line 80 – I would suggest adding references to some recent broad-scale studies of gut microbiota variation across vertebrates, including Youngblut et al. 2019 and Song et al. 2020.
Figure 1: A map with field sites indicated and a scale bar would be helpful.
Again, I have only minor revisions.
Line 154 – could you add the native range of P. siculus and approximate time since their introduction?
Line 168 – are your three collection locations similar in temperature and rainfall, with the main source of variation being human disturbance? Or are there other climate variables that differentiate them?
Line 177 – did you use a buffer or ethanol to preserve swabs, or were they stored dry? What was the maximum length of time between taking the sample and storage in the -80?
Line 209 – did any reads appear in the negative control (ie through index hopping)? If so, what was the maximum read depth for any OTU that appeared?
All findings are well-stated and follow logically from the results.
This is an excellent and well thought out paper addressing interesting questions in invasion biology and lizard gut microbiome composition. The methods section is comprehensive and clearly written. I would like to hear the authors’ thoughts on the implications of their results for broader evolutionary patterns in the introduction and conclusion, but this suggestion should not impact the manuscript’s acceptance.
The authors are clear and concise in their reporting, and the background for their study is well explained. Authors could improve their literature review, and make small but meaningful changes to most figures. However, the caption for Figure 1 contradicts the description of habitats in the manuscript, and this could call into question the major outcomes of the paper, depending on which one is correct. See below.
Figure 1 does not seem to match the habitat descriptions in the manuscript – Panel A shows coastal farmland but is noted as the habitat for P. siculus, which should be one of the urban species. Panel D is described as the habitat for the two rural species, but appears to be an urban parking lot (based on the train track this should be T. dugesii?). This is an important correction, since the rural/urban comparison is integral to the manuscript, but I assume that this is a typographical error for the figure, rather than a problem with the manuscript.
The literature review in the introduction could be expanded. In several places, the authors have a long list of factors, but cite only 1-2 papers that do not cover all of the things they list (eg line 78.) While they are correct the microbiome research on lizard species is limited, they overlook several relevant papers (Hong et al, 2011, Montoya-Ciriaco et al, 2020, Zhang et al, 2017, etc)
The authors could break their methods up into sections to make it more readable.
Small typos throughout the Introduction, particularly in references associated with species descriptions which are missing from the Literature Cited.
The code for the mixed effects models (line 219-223) would be better included in the supplemental R script, which currently includes the processing of raw data but not the statistical analyses. Please provide your script for analyses.
In the Discussion, common taxa found in this study should be compared to what has been found (by cloacal swab) in other (lizard) species. Also, the discussion of the differentially abundant taxa could be expanded, referencing the potential impact of the specific taxa which varied, as well as reasons why they may vary.
Authors also briefly mention an increase in diversity by size in one lizard species. This is a common finding, and could be better placed in context with more discussion and references. However, as noted in the experimental design section, the finding seems to be strongly influenced by the one 59mm individual and should be considered more cautiously.
The sentence beginning on line 206 is difficult to understand as written.
Please provide your script for analyses.
Increase point size and axis font size on Figure 2, 3 and 5, and consider using a colorblind friendly palette, or one that can be distinguished in black and white (for all figures).
In figure 2, it would be helpful to include to location of each species, and which are invasive or native, for ease of comparison. Similarly, in Figure 4, the sex of the animals could be included.
Include units on x-axis for Figure 5
Typos in captions for Figures 4 and 5.
The sampling design of this study is well thought out and detailed enough to answer questions the authors propose. While most of the major analyses are sufficient to answer the proposed questions, I have some concerns regarding a few of the secondary analyses.
Lines 276-278: Authors should provide all statistical output and not just the one significant relationship. Also, I am suspect of that significant relationship and the (apparently) very strong influence of the one 59mm individual. If this one sample is removed, does the pattern remain? What can you tell us about that one individual? If it was a juvenile, for instance, your effect may not be related to body size so much as it is to life stage. The pattern was only found in one of your five species… By chance, did the other species have a narrower range of SVLs sampled?
Lines 279-288: It is unclear to me how the FEAST analysis can differentiate between microbes that are being transferred between species from those that are merely present in both species, especially without including environmental or diet samples as a source which could also be contributing to either species’ microbiota. Based on the referenced paper, it does not seem that the analysis is meant to make that type of conclusion. Are these two species pairs physically interacting in a way that could transfer microbes? Particularly P. siculus and P. virescens, which the authors note are rarely observed within 50 meters of one another? Authors should clarify how this works both in the statistical analysis and the behavior and ecology of the lizards, or at the very least include a caveat about these results.
With the current set up of the manuscript, I am not sure of the value of including the Tiera dugesii species; it does not have a paired species for comparison, and is not mentioned specifically in either the Results or Discussion, beyond a general summary sentence. If the authors wish to include this species, it should be more carefully worked into the analyses. For example, the authors mention briefly the effect of host phylogeny on gut microbiota in the Discussion, and this more distantly related species with similar environmental pressures could help to parse some of those questions, particularly based on Figure 3, which shows T. dugesii grouping separately from all other species.
In Figure 1, three of the locations appear to be near a water source (panel A includes a beach), and this was not mentioned in the description of the habitats (except that T. dugesii was located near a port). The authors should explore the potential marine influence on diet and microbes for the relevant species, and note how this would affect comparisons between locations.
A test of dispersion should be included with all PERAMOVA analyses, as this is an assumption of the test.
Controls were mentioned for extractions and PCR, but how were these utilized? How were contaminants identified and removed?
Please include more detail about the mock community results, perhaps expected vs. recovered ratios of species? Was the mock community used to determine the parameters in line 198, or only checked after the fact?
Consider using a differential abundance test designed for microbial data (deseq2, corncob, LEFse, etc), that examines all taxa and not just the most abundant ones, as high abundance does not necessarily mean high importance.
The ratio of males and females is extremely skewed for basically all species. How could this have affected the results around variation by sex?
Line 137 – instead of a range, provide specific mean snout-vent length for each species (even if it is just the animal measured for this study).
Also include mean SVL of the groups that were used in the analysis for each species on line 244-245, since individuals were chosen based on matching size.
The major conclusions of this paper, based around the impact of urban vs rural habitats, are well supported and the analyses are sound. However, the questions raised regarding some of the analyses used for secondary comparisons (in the experimental design section), as well as the Figure 1 caption error, need to be addressed to assess whether those findings are valid and the authors’ conclusions correct.
This manuscript explored that variation in microbiomes between several lizard species, both invasive and native, in urban and rural environments. For the most part, the data are well analyzed and the authors draw appropriate conclusions. My biggest concerns are with the interpretation of the FEAST analysis, and the discrepancies between habitat descriptions and their photographs in Figure 1. The authors should also expand their discussion of the specific taxa they found in the cloaca, and better situate these findings with the existing literature.
The authors have conducted a fine study on the diversity of gut microbes (bacteria) found in 5 lizard species of Podacris lizards. Overall the study is well reasoned and thorough, and the methodology for inferring transmission was elegant. While the study is straightforward and the findings generally unsurprising, there are a few areas the manuscript needs clarification or improvement.
1) The methods for sample collection are insufficient as written. Details on whether sterile technique was used, if exterior or cloaca was sterilized (see Colston et al 2015 PLoSOne) time samples were handled until being frozen etc all need to be thoroughly documented.
2) The authors do not indicate whether data were rarified prior to calculating diversity statistics. This is necessary (or justify why it is not) , particularly when dealing with amplicon data such as 16S that has high PCR bias.
3) To insure transparency and repeatability processed data (biom files, alignments etc) should also be made available in addition to the RAW reads and scripts provided.
4) Figures need improvement and should be checked for accessibility to color-vision impaired readers. The symbol size used for localities in Figure 3 is not legible.
See above regarding methods for sample collection and rarefaction.
Overall, I feel the results support the conclusions drawn. The authors could expand on the underlying impact in their findings regarding differences in weighted/unweighted analyses (i.e. community structure vs membership).
This was a pleasure to read and will add to the growing body of literature on factors structuring gut microbiomes in reptiles.
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