Turtle species and ecology drive carapace microbiome diversity in three seasonally interconnected wetland habitats
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Different species of freshwater turtles exhibit primary behaviours ranging from aerial basking to benthic bottom-walking, cycle between wet and dry conditions at different time intervals, and undertake short-distance overland movements between aquatic habitats. These behaviours in turn may impact the accumulation of microbes on external shell surfaces of turtles and provide novel niches for differentiation of microbial communities. We assessed microbial diversity using 16S and 18S rRNA metabarcoding on carapace surfaces of six species of freshwater turtles residing in three adjacent and seasonally interconnected wetland habitats in southeast Oklahoma (United States). Communities were highly diverse, with nearly 4200 prokaryotic and 500 micro-eukaryotic amplicon sequence variants recovered, and included taxa previously reported as common or differentially abundant on turtle shells. The 16S rRNA alpha diversity tended to be highest for two species of benthic turtles, while 18S rRNA alpha diversity was highest for two basking and one shallow-water benthic species. Beta diversity of communities was more strongly differentiated by turtle species than by collection site, and ordination patterns were largely reflective of turtle species’ primary habits (i.e. benthic, basking, or benthic-basking). Our data support that freshwater turtles could play a role in microbial ecology and evolution in freshwater habitats and warrant additional exploration including in areas with high native turtle diversity and inter-habitat turtle movements.
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Thank you for your detailed and clear response to the points raised by the reviewers which you have addressed carefully and with detailed explanations
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This is a study that would be of interest to the field and community. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments. Both reviewers have identified several areas that need addressing. Please make sure that any raw data is made available as this specified in the platforms open access policy (https://www.microbiologyresearch.org/open-data) Furthermore several aspects were raised for giving more detailed context of the study and as well as amendments in the presentation of the results.
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Comments to Author
ACMI-D-23-00155 Turtle species and ecology drive carapace microbiome diversity in three seasonally interconnected wetland habitats SYNOPSIS AND MAJOR COMMENTS The manuscript entitled "Turtle species and ecology drive carapace microbiome diversity in three seasonally interconnected wetland habitats" focuses on the characterization of the microbial communities inhabiting the carapaces of six turtle species in four different wetlands locations in Southeast Okhaloma (USA). The manuscript is broadly well-written, with the major results being presented in a straightforward manner and mostly comprehensible based on the performed metataxonomic survey. All the bioinformatic analyses were run accordingly and the data produced is reproducible and provided for transparency means in the Microbiology Society …
Comments to Author
ACMI-D-23-00155 Turtle species and ecology drive carapace microbiome diversity in three seasonally interconnected wetland habitats SYNOPSIS AND MAJOR COMMENTS The manuscript entitled "Turtle species and ecology drive carapace microbiome diversity in three seasonally interconnected wetland habitats" focuses on the characterization of the microbial communities inhabiting the carapaces of six turtle species in four different wetlands locations in Southeast Okhaloma (USA). The manuscript is broadly well-written, with the major results being presented in a straightforward manner and mostly comprehensible based on the performed metataxonomic survey. All the bioinformatic analyses were run accordingly and the data produced is reproducible and provided for transparency means in the Microbiology Society Figshare platform. The work is pioneering in assessing turtle microbiomes from a holistic perspective, targeting both prokaryotic and eukaryotic communities, by targeting an interesting microcosm in a well-structured experimental design and sampling approaches that could be easily replicated in future studies. The derived insights on the key results were totally coherent with what has been already published in the literature and the authors discuss elegantly how these turtle-associated microbiomes might be intertwined with microbial dispersion and ecological roles in their original ecosystems. A number of minor issues that deserve attention includes: (1) writing in the full form "16S rRNA gene" and "18S rRNA gene" throughout the manuscript; (2) making clearer in Material and Methods the purpose of some of the statistical approaches adopted in beta-diversity analyses; and (3) updating and changing some of the figures in a way they are more informative and comprehensible. The Impact Statement section needs full rewriting. Finally, the authors should check if the raw metataxonomic data has been deposited in SRA/NCBI. DETAILED REVIEW Abstract Line 20 Replace "16S" and "18S" with "16S rRNA" and "18S rRNA". Line 29 Replace "16S and 18S diversity" with "16S rRNA and 18S rRNA gene-based diversity survey". Line 32 Correct: "4,200". Lines 32-33 Avoid using "16S and 18S" in the sole form. This is wrong. Rewrite specifying the gene, such as "16S and 18S rRNA gene". In this particular stretch, just remove both of them. Lines 34-35 Maybe replace "16S" and "18S" here with "prokaryotic" and "microeukaryotic", respectively. Impact Statement Line 48 The authors might consider replacing the too-wordy stretch "using a DNA sequencing strategy called metabarcoding" with "by metataxonomics". Line 49 Correct: "4,200". Lines 50-58 This is the abstract all over again. The authors might consider rewriting this whole part of the impact statement in the revised manuscript version. What the work will contribute to a better understanding of turtle microbiomes and the interplay between turtle-associated microbial communities and ecosystem functioning? How might the current work have revealed novel microbial indicators or key taxa related to turtle behavioural habitats? How that could be extended to other not-yet-investigated animal species living in these freshwater environments? Data Summary Line 62 The referred BioProject ID Number returns no result after searching on the NCBI BioProject database. The authors should ascertain that the data has been deposited or if the ID is correctly provided in the manuscript text. Introduction Line 78 Replace "antibiotics" with "antimicrobials". Lines 111-112 Rephrase the beginning of this sentence, perhaps: "(...) At least, ten species of freshwater turtles are present in these wetlands, (...)". Line 114 There is no transition between the end of the theoretical background and the aim of the study. Try to rewrite this sentence providing a link between these two sentences. Methods Lines 124, 143 Indicate in the manuscript text: "(Figure 1A)", (Figure 1B)" and "(Figure 1C)", not just as Figure 1. Line 174 Add "in the present study" just by the end of the first sentence of this Table legend. Line 180 Correct: "V3-V4 hypervariable regions 16S rRNA gene and the V8-V9 hypervariable regions of the 18S rRNA gene". Line 187 Replace "16S and 18S" with "both biomarkers". Lines 191-193 Again, correct "16S rRNA gene and 18S rRNA gene". Additionally, correct that all throughout the manuscript, especially in this Material and Methods subsection. Line 193 Avoid abbreviations like "ca." Just write circa. Line 203 Avoid the abbreviation "ver." for "version". Write it fully or just as "v." Line 205 Correct "16S rRNA gene and 18S rRNA gene". Also, correct "DADA2" (it's written in capital letters). Line 211 What does that mean "99%" for the SILVA version? Also, the "ver." abbreviation, avoid it. Line 220 Capitalize "QIIME 2". Lines 225, 231, 233, 236, 239, 253, 256, 259 Correct the "ver." abbreviation again. Line 259 Capitalise ANOSIM. Results Line 268 Correct: "16S rRNA gene". Line 269 Correct: "18S rRNA gene". Line 271 Correct: "16S and 18S rRNA gene". Line 273 Correct: "16S and 18S rRNA gene" in the Table legend and also in the first lines of both Table columns. Lines 276-277 Not clear what authors stated about the results being redundant. Is it possible to clarify that better? Do the results shown in the manuscript refer to the non-redundant ones? Line 284 It might be worth reviewing the size of the stacked barplots for each relative abundance graph, it is quite hard to see any of it in the current version of the submitted manuscript. Line 291 Avoid using the abbreviations (e.g., "STOD" and "TRSC") throughout the manuscript text. Instead, keep these abbreviations for figures and tables, but for the manuscript text is more consistent to adopt the turtle species names so it's easier for the reader's comprehension. Line 327 Even though it is understandable that the authors tried to represent systematically in one figure the pairwise comparisons of alpha diversity results, Figure 4 is really hard to comprehend and does not make evident to the reader what are the key insights on the microbial diversity data derived in the current work. The recommendation is just to keep the graphs and present numerical results as included in [1]. Line 342 Figure 5 could be plotted in another manner, not as a heatmap. Additionally, it can not be derived any substantial result on the distribution of microbial communities without the ASV taxonomy. Line 362 Add the stress values for each ordination plot. Line 400 Add "diversity" after "beta". Lines 402-405 "t" and "p" in "t-test" and "p-values" should be italicized. Line 422 Avoid "ca.", write "approximately" or "circa". Lines 419, 428 Write ANOSIM in capital letters. Line 461 Instead of writing "(16S)" or "(18S)", the authors could consider particularizing such as "(prokaryotic)" or "(eukaryotic)". Lines 484-485 It could be considered to specify in the Material and Methods section the impotence of analysing both beta dispersion and centroid locations in surveying the structure of microbial communities inhabiting turtles' carapaces. Line 525 Correct "does not". Line 552 Do the authors refer to "antimicrobial resistance" and not "antimicrobial properties"? Or does the microorganisms associated with these turtle species have been detected as potential producers of antimicrobial substances?
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
The manuscript titled "Turtle species and ecology drive carapace microbiome diversity in three seasonally interconnected wetland habitats" uses great language and the authors stuck to a concise description of their results and line of reasoning and understandable wording. The authors could explain persuasively why their research project is relevant for their field of study and that it focuses on under-researched aspects of turtle carapace microbiomes in this specific habitat what brings novelty to the scientific subject. In general, I think the authors have a great experimental design and their methodology of data acquisition did not show obvious weaknesses. Nevertheless, I would suggest a thorough makeover of large aspects of the introduction and the first half of the discussion, since I think it …
Comments to Author
The manuscript titled "Turtle species and ecology drive carapace microbiome diversity in three seasonally interconnected wetland habitats" uses great language and the authors stuck to a concise description of their results and line of reasoning and understandable wording. The authors could explain persuasively why their research project is relevant for their field of study and that it focuses on under-researched aspects of turtle carapace microbiomes in this specific habitat what brings novelty to the scientific subject. In general, I think the authors have a great experimental design and their methodology of data acquisition did not show obvious weaknesses. Nevertheless, I would suggest a thorough makeover of large aspects of the introduction and the first half of the discussion, since I think it lacks substantial information about data regarding the microbiome in the tested ecosystem and its role on ecological functions as well as partly references to known studies from comparable research projects. Further, some of the figures seems to be in a very "default" state regardless of subjective mediocre esthetics and readability, although the data itself look very interesting, here I would like to see optimization. A general suggestion would also be to distinguish more between different groups of the eukaryotic community and the function of the found taxa like algae and others, as this might help to understand how different turtle lifestyles could be influential for this. The same applies for the ecological relevance of the prokaryotic taxa. On this, the authors lost a lot of information and potential explanations for their findings that I would consider interesting for the community. I admit, that I am not aware of the fundamental principles of the concept of beta diversity dispersal, therefore I could not comment on the correctness of the conclusions made on this topic. I would just recommend to elaborate this more to increase understanding. In the following there are some more detailed suggestions and questions that should help you to improve the quality of your manuscript. L 23 In my browser, I could not find the BioProject indicated as PRNJA975189 when searching in NCBI, but that might be caused by my system, nevertheless please doublecheck to ensure accessibility upon publication. l. 20 Would suggest to rename the keywords 16S and 18S in "16S rRNA gene amplicon sequencing" Further, I know from other journals, that they recommend to do not use keywords that are included in the title. Line 29: 16S and 18S microbial diversity does sound to much like jargon language. Would suggest rephrasing. l. 32-34 s. comment above. l. 34 Think about rephrasing "alpha diversity […] in two species". Since you refer to the carapace this sounds a bit inelegant. l. 39 After going through the manuscript, I think that the statement regarding "ecology and evolution" is a bit exaggerating considering the data you present. l. 57 Not sure if your data could provide information enable you to distinguish between habitat/lifestyle-influenced and species-influenced microbial assemblages. l. 72 Its not my subject of expertise, but I would have expected, that especially amphibious animals encounter strong physical and chemical parameter changes due to their changing of aquatic and terrestrial habitats. l. 83 would recommend to add 1-2 other references before the starting with the "Knight et al. reference" to underline your statement here. l. 86 "distance" can be left out or further explained what is meant with this. l. 89. Would include both citations, when referring to "both cases" l. 89 "robust" is in my opinion not a common description for a bacterial taxon l. 90 What are "potentially extremophiles"? I would either go for known extremophilic properties or properties which are relevant for your study or tested habitats. l. 90 If you italize phylum names, you should italize all phylogenetic names throughout the manuscript to stick to consistency. Section in ll. 80-91 I would add some more detailed microbial taxa information for instance what are keystone taxa and maybe add some more reference if applicable. l. 93 Would be interesting, in case there are information available, how the carapace microbiome is influenced/ changing from the birth of a turtle throughout its life. l.101 Would implement more details and references on deterministic and stochastic processes during community assembly. l. 110 Please explain what do you mean with "interconnected wetlands" l. 116 S. above regarding 16S, 18S It would be beneficial for your line of reasoning if you there would be a more hypothesis-based approach on the study project. Indeed, you nicely explain why especially the Southeast of the US is a relevant field for study, but it misses a bit what is known of the influence of these microbial communities on the ecology in this area and if there are known changes. Are there studies of invading turtle species and how their microbiome changes the microbial conditions in the invaded habitat? l. 124 If available, please add GPS data at least for one reference site or at least add names of lakes/ ponds, rivers in figure 1 to facilitate the identification of sampling spots. What is the meaning of "seasonally interconnected" l. 125, 126 Is there a certain reason for the site descriptors as S1 and S4 or why not included S2 and S3 if they exists? l. 129 If applicable, please add information on human-made changes to the vegetation, like mowing or other changes that might influence turtle ecology/ fitness l. 143 Refer to Figure 1B maybe. Does the catch distribution roughly resembles the expected abundance of turtles in this field or was this just based on hunting luck? Figure 1 Personally, I prefer more capital letters in legend captions, but do as you wish. The dashed white circles are hard to see and the colour contrast to the river-blue is not ideal. Please increase contrast/ visibility. ll.161-173 Avoid empty lines Table 1 Is there a caption phrase missing for "#" or why you added this sign? I would recommend to redesign the table for instance by highlighting headlines more and reducing spaces between the content, when possible. l. 179 Please homogenize the manufacturer information and headquarter location l.180 Please rephrase and do not use 16S V3-V4 and similar. ll. 191, 192 s. above. Was each sequencing sample retrieved from a single carapace or have you added some replication at any steps during the workflows? l. 196 Is the V3 procedure also appropriate for the 18S rRNA V7-V8 region? l. 205 s. above l. 232 You already introduced ASV as abbreviation l. 240 How do you identify turtles as "juvenile" or does this mean that the sex-specific phenotypes are not visibly developed? ll. 245-247 I would consider this sentence results and not M&M. l. 261 "Through adonis2" does not sound correct, please rephrase using adonis2() function for instance. lff. 268 14k reads is relatively low considering your average of 80k, have you excluded certain samples? Table 2 As before, I Think you could optimize the table or transfer to supplementary data. l. 276 Your second and first sub header both contain taxonomic assignments, please rephrase or restructure here. l. 279 s above regarding harmonizing italization for taxonomy names. ll. 279-281 Please add some percentages for the most abundant taxa. Figure2 I would recommend to change figures and merge the different samples together and indicate how many communities were included. Since you want to give here a rough estimation about the community composition, I have to admit that the bars are far to small and its really hard to retrieve data, if you want to have a more detailed look on the data. Please explain in more detail what the category "Other" mean. Please distinguish between "low abundant taxa" and "not identifiable". The x axis is missing a legend and I personally like % values more, but that's up to your preferences. If you have cryptic names like KD3-93, please give information about the next higher phylogenetic level if applicable. Example: Chloroflexota_IXD-785. Is the order of family names correlating with the overall abundance of each taxon or based on something else? Llf. 281 It might be an interesting approach to distinguish your 18S rRNA data more between algae and others. And as a comment, I wonder why you did not focus on fungal species when analyzing microbiome data? This needs further explanation. And please add some numbers for the most abundant taxa. Also would like to see some more description of differences between species-based and site-based differences. l. 293 would use "Figure 3X" Figure 3 I personally think that UpSet plots are not very common (or at least in my research area), therefore I would suggest to add some more explanation in the caption to make sure the audience can follow the content more easily. l. 308 reminder for not using "16S community" throughout the manuscript l.322 How were outliers identified? Figure 4. I like the figure design! The 300 m scale should be with another colour to enhance visibility. And there are no A),B)…..signs in the figure. This must be included and think about making signs showing what are 16S and what are 18S data. Figure 5 Please optimize the x-axis regarding the labels. It is a bit unconvenient to retrieve the relevant findings from the data for instance with black bars and below it the descriptor of species or site. Furthermore, I would suggest putting gaps between relevant sections to improve the visibility. The legend regarding the abundance is poorly designed. Please add information of the unit and optimize the aesthetics. Add information of the ASV order on the y-axis. I agree it would be too cloudy to add all ASVs, but it is relevant if you applied clustering for your heatmap or the ASV order is just based on the order in your abundance table. l. 339 If you want to highlight both, species and site-based effects I would use two heatmaps per Community. One with species order (as it is right now) on the y-axis and one with sites. ll.351-354 use Figure 6X Figure 6 If applicable please add percentages on the axis to show the degree of description each axis is contributing. I would delete 0.1, because no -0.1 is written and it confuses. Please add legend of the colours and description of lines in the plot as well as explanation of what are the ellipsoids showing and how they have been generated. ll. 364 "all ANOVA returned significant differences […]" I would suggest rephrasing of this expression. ll. 365, 366 I would recommend to do not use E-5 but X-5 Its not my field of expertise, but please check if F5,78 is a commonly used expression in this research area and explain or rephrase if necessary. And I personally would elaborate or introduce the concept of beta dispersion a bit more to help a broader community that, lack background information, to follow your thoughts. Llf. 373 s. comment above. LLf. 397 Why providing data of significant differences in percentages. Have you considered comparing the centroid position of the total site/species data with each other and then calculating if there is a significant difference? At least I think that would help to provide the information you want, or rephrase the sections if you want to highlight something else. l. 399 I can not retrieve data regarding BP4-S4 from the table. Therefore, I would rephrase of adjust Table 3. Llf. 407 Would recommend to write "the factor species was not recovered as significant…" and respectively for all other factors It might be useful to add here a table or figure to simplify the visualization of findings. l. 423 Please elaborate more on how you got these results and what you aimed for with this? l. 431 Chapter 8. §Figures and table" is empty. Place check if formatting is correct throughout the manuscript. ll. 433-438 Would suggest to add some more distinct facts regarding "host organism phenotype" or other factors which are influenced. l. 443 Would be interesting if you could provide data/ studies of how many kilometers turtle usually travel during their lifetime to access their potential vectoring properties. l. 445 I agree in the point of vectoring, but without any proves or references I would not state that turtle shells act as accumulators as so far no indications are mentioned showing that this is a better habitat as other "random" surfaces. ll. 448 I would recommend to give an estimation and/or more detailed information of the degree of impact that can be derived from turtles within the habitat. l. 455 Are there reference microbiome data showing the "usual" diversity in this habitat or comparable sites? If you want to consider turtle shell as "important microbial accumulators" you should provide data to compare your findings. l. 458 please be more distinct and use numbers instead of "relatively high" l. 461 Interesting findings, I would like to read more information about these genera and if they are ecologically relevant or relevant for certain processes. l.472 I do not know all these eukaryotes by heart, but I think there are also some algae which might cope better with oxygen limitation, but worse with shadows. I think it would be beneficial to try to draw conclusions also based on autotrophic organisms and their abundances. This is out of my scientific knowledge but I would think, that as long as the turtle can survive oxygen limitations, its carapace microbiome should cope with it as well. Llf 472 Please add more references from the literature and try to compare your findings with what is known from other regions which face similar environmental conditions and how the diversity is influenced there. l. 481 Are there eukaryotic data for this statement or would this phenomenon occur for bacteria as well? Llf. 485 Please add some more references regarding turtle shell microbiome, also from other research groups if applicable. Further, it would be beneficial to compare your findings regarding interplay between centroid position and diversity dispersal and beta diversity. ll. 507-508 Would suggest to rephrase and talk about community data cluster together instead of species. l. 531 This statement might be a bit strong, you could consider your lower the harshness l. 534 I do not know if relatedness necessarily correlates with phenotypic properties of turtle shell surfaces. Please check on that and rephrase if necessary. Or add references to support your statement. l. 542 Are there phylogenetic data of your turtle species which you could refer to give estimation of the phylogenetic relatedness of your species? l. 562 Would suggest to further elaborate on ecoSNPs l. 566 Add information on other shell-like structure of other organisms and try to find similarities of strong differences in regard of its microbiome /ecology impacts, if applicable. l. 612 Redundant header
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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