Downloaded from http://rsos.royalsocietypublishing.org/ on June 15, 2017
Drastic skin microbiota differences between co-occurring
frogs and newts
Molly C. Bletz, R. G. Bina Perl and Miguel Vences
Article citation details
R. Soc. open sci. 4: 170107.
http://dx.doi.org/10.1098/rsos.170107
Review timeline
Original submission:
Revised submission:
Final acceptance:
6 February 2017
7 March 2017
7 March 2017
Note: Reports are unedited and appear as
submitted by the referee. The review history
appears in chronological order.
Review History
RSOS-170107.R0 (Original submission)
Review form: Reviewer 1
Is the manuscript scientifically sound in its present form?
No
Are the interpretations and conclusions justified by the results?
Yes
Is the language acceptable?
Yes
Is it clear how to access all supporting data?
Yes
Do you have any ethical concerns with this paper?
No
Have you any concerns about statistical analyses in this paper?
No
© 2017 The Authors. Published by the Royal Society under the terms of the Creative Commons
Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use,
provided the original author and source are credited
Downloaded from http://rsos.royalsocietypublishing.org/ on June 15, 2017
2
Recommendation?
Accept with minor revision (please list in comments)
Comments to the Author(s)
This manuscript examines the differences in skin microbiomes between 6 species of frogs and
newts in Germany. I think the results are interesting and it is worth publishing, however, the
authors must address some of these concerns or questions.
1-The title says that species are syntopic. Do these species hybridize? I would like to see more
discussion about which of these species are closely related and how their microbiomes are
similar/different. For example, I would expect the Lissotritons to be more similar to each other
than to Ichthyosaura and Triturus. Although the number of species does not allow for
phylogenetic comparative methods, it would be interesting to see how evolutionary relatedness is
correlated with microbiome composition.
2-I would like to see more information about habitat partitioning in these species.
Minor comments
Line 8-9 “these differences have rarely been analyzed in systems that minimize confounding
factors, such as season, location or host ecology”. Statement is not entirely true see McKenzie et al
ISMEJ, Kueneman et al. MolEco, Belden et al. Frontiers Micro.
Line 14-17 Long sentence and unclear. Rewrite. I did not understand the second set of numbers.
Are those Jaccard distances?
Line 17-19 I do not think that this core analysis is appropriate. The comparisons should be done
with shared OTUs among the 6 species.
Line 19 delete word ‘some’
Line 21 Break sentence after colonizers. Start new sentence about future studies. Also, how
genomic, transcriptomic and metabolomic analyzes are going to help understand chemical and
physical characteristics of the skin environment. What about differences in pH, cell size, structure
etc?
Line 33-34 Not true. See Walke et al Biotropica 2011
Line 43 delete ‘so far’
Line 45 “species from major clades of amphibians consistently differ from species of other such
clades in cutaneous microbial composition or diversity.” This statement needs to be rewritten. Is
the objective to say that there are phylogenetic differences in amphibian microbiomes?
Line 67 Remove capitalized S from spring
Line 135 replace ‘completed’ to ‘performed’.
Line 143-144 I don’t agree with the threshold chosen by the authors for this analysis. If the
comparisons are just between two categories (frogs vs newts), then the OTU should be present in
100% of the individuals of all species of newts/frogs.
Line 151 Here it starts to get confusing, why core microbiome differs from the LDA analyses, then
on line 155 another OTU table is used with a 75% threshold. I would recommend using the same
threshold for all the analyses.
Downloaded from http://rsos.royalsocietypublishing.org/ on June 15, 2017
3
Line 161 I would suggest incorporating pairwise comparisons directly in Fig. 1 instead of having
tables 1 and 2.
Line 208-209 True, but also they might inhabit different areas of the ponds. Any insights on this?
Do you think that ponds are completely homogenous?
Line 222 Please mention which are the 3 OTUs.
Line 226-227 I didn’t see in the methods anything about environmental samples. Please check.
Line 228-231 Maybe move to methods? Seems awkwardly placed.
Line 232 Change “encountered” by “observed”.
Review form: Reviewer 2
Is the manuscript scientifically sound in its present form?
Yes
Are the interpretations and conclusions justified by the results?
Yes
Is the language acceptable?
Yes
Is it clear how to access all supporting data?
Yes
Do you have any ethical concerns with this paper?
No
Have you any concerns about statistical analyses in this paper?
Yes
Recommendation?
Accept with minor revision (please list in comments)
Comments to the Author(s)
The paper analyses microbiota composition of frogs and salamanders collected from two
different localities for patterns of differences between phylogenetically distinct and related taxa.
Differences in species richness and diversity between frogs and salamanders were found and
these were consistent across the two localities.
The cutaneous microbiome of amphibians is affected by many factors and teasing apart the
possible roles of host and environmental factors on microbiota variation by examining syntopic
species, as this study shows, can provide useful results. However, specific comparisons between
the life histories and corresponding habitat usage of the salamanders and frogs should be
considered to interpret the observed patterns of micobiota variation.
The study might be of more general interest if were to include results of screening for the
presence of amphibian pathogens known to affect amphibian population viability and
microbiota. Screening for Batrachochytrium dendrobatidis (Bd) and/or Batrachochytrium
Downloaded from http://rsos.royalsocietypublishing.org/ on June 15, 2017
4
salamandrivorans (Bsal) from swabs now is relatively routine. Availability of such data would be
very useful in interpreting the results.
Specific comments
Given that the study emphasises comparisons among syntopic species, low samples sizes (or
complete absence) of some species from some localities in the dataset reduces the generality of
the conclusions. Why were R. dalmatina and L. helveticus not represented from one locality, and
why were sample sizes of I. alpestris so low?
Sample collection (lines 77-79): Please mention the distance between the localities. Also mention
details on geographical variation and possible anthropogenic factors that may affect the collection
sites with potential consequences on skin microbiota.
Statistical analyses (lines 135-136) need to be explained in more detail. There are many different
types of ANOVA. What model was used, and using what software?
ANOVA results are not properly presented (line 164). How many degrees of freedom in the
comparison, and what error term was used? Also P is indicated with unjustifiable precision.
Have authors screened Bd/Bsal from swab samples? This would provide a possible basis for
understanding patterns of cutaneous microbiota variation within and between species/families.
Authors have quoted the evidence that Bd can alter skin microbiome (lines 36 and 310), so might
expand their paper if they have such data available from their swabs.
Decision letter (RSOS-170107)
01-Mar-2017
Dear Miss Bletz
On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-170107 entitled
"Drastic skin microbiota differences between syntopic frogs and newts" has been accepted for
publication in Royal Society Open Science subject to minor revision in accordance with the
referee suggestions. Please find the referees' comments at the end of this email.
The reviewers and handling editors have recommended publication, but also suggest some minor
revisions to your manuscript. Therefore, I invite you to respond to the comments and revise your
manuscript.
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5
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on behalf of Kevin Padian
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Reviewer comments to Author:
Reviewer: 1
Comments to the Author(s)
This manuscript examines the differences in skin microbiomes between 6 species of frogs and
newts in Germany. I think the results are interesting and it is worth publishing, however, the
authors must address some of these concerns or questions.
1-The title says that species are syntopic. Do these species hybridize? I would like to see more
discussion about which of these species are closely related and how their microbiomes are
similar/different. For example, I would expect the Lissotritons to be more similar to each other
than to Ichthyosaura and Triturus. Although the number of species does not allow for
phylogenetic comparative methods, it would be interesting to see how evolutionary relatedness is
correlated with microbiome composition.
2-I would like to see more information about habitat partitioning in these species.
Downloaded from http://rsos.royalsocietypublishing.org/ on June 15, 2017
7
Minor comments
Line 8-9 “these differences have rarely been analyzed in systems that minimize confounding
factors, such as season, location or host ecology”. Statement is not entirely true see McKenzie et al
ISMEJ, Kueneman et al. MolEco, Belden et al. Frontiers Micro.
Line 14-17 Long sentence and unclear. Rewrite. I did not understand the second set of numbers.
Are those Jaccard distances?
Line 17-19 I do not think that this core analysis is appropriate. The comparisons should be done
with shared OTUs among the 6 species.
Line 19 delete word ‘some’
Line 21 Break sentence after colonizers. Start new sentence about future studies. Also, how
genomic, transcriptomic and metabolomic analyzes are going to help understand chemical and
physical characteristics of the skin environment. What about differences in pH, cell size, structure
etc?
Line 33-34 Not true. See Walke et al Biotropica 2011
Line 43 delete ‘so far’
Line 45 “species from major clades of amphibians consistently differ from species of other such
clades in cutaneous microbial composition or diversity.” This statement needs to be rewritten. Is
the objective to say that there are phylogenetic differences in amphibian microbiomes?
Line 67 Remove capitalized S from spring
Line 135 replace ‘completed’ to ‘performed’.
Line 143-144 I don’t agree with the threshold chosen by the authors for this analysis. If the
comparisons are just between two categories (frogs vs newts), then the OTU should be present in
100% of the individuals of all species of newts/frogs.
Line 151 Here it starts to get confusing, why core microbiome differs from the LDA analyses, then
on line 155 another OTU table is used with a 75% threshold. I would recommend using the same
threshold for all the analyses.
Line 161 I would suggest incorporating pairwise comparisons directly in Fig. 1 instead of having
tables 1 and 2.
Line 208-209 True, but also they might inhabit different areas of the ponds. Any insights on this?
Do you think that ponds are completely homogenous?
Line 222 Please mention which are the 3 OTUs.
Line 226-227 I didn’t see in the methods anything about environmental samples. Please check.
Line 228-231 Maybe move to methods? Seems awkwardly placed.
Line 232 Change “encountered” by “observed”.
Downloaded from http://rsos.royalsocietypublishing.org/ on June 15, 2017
8
Reviewer: 2
Comments to the Author(s)
The paper analyses microbiota composition of frogs and salamanders collected from two
different localities for patterns of differences between phylogenetically distinct and related taxa.
Differences in species richness and diversity between frogs and salamanders were found and
these were consistent across the two localities.
The cutaneous microbiome of amphibians is affected by many factors and teasing apart the
possible roles of host and environmental factors on microbiota variation by examining syntopic
species, as this study shows, can provide useful results. However, specific comparisons between
the life histories and corresponding habitat usage of the salamanders and frogs should be
considered to interpret the observed patterns of micobiota variation.
The study might be of more general interest if were to include results of screening for the
presence of amphibian pathogens known to affect amphibian population viability and
microbiota. Screening for Batrachochytrium dendrobatidis (Bd) and/or Batrachochytrium
salamandrivorans (Bsal) from swabs now is relatively routine. Availability of such data would be
very useful in interpreting the results.
Specific comments
Given that the study emphasises comparisons among syntopic species, low samples sizes (or
complete absence) of some species from some localities in the dataset reduces the generality of
the conclusions. Why were R. dalmatina and L. helveticus not represented from one locality, and
why were sample sizes of I. alpestris so low?
Sample collection (lines 77-79): Please mention the distance between the localities. Also mention
details on geographical variation and possible anthropogenic factors that may affect the collection
sites with potential consequences on skin microbiota.
Statistical analyses (lines 135-136) need to be explained in more detail. There are many different
types of ANOVA. What model was used, and using what software?
ANOVA results are not properly presented (line 164). How many degrees of freedom in the
comparison, and what error term was used? Also P is indicated with unjustifiable precision.
Have authors screened Bd/Bsal from swab samples? This would provide a possible basis for
understanding patterns of cutaneous microbiota variation within and between species/families.
Authors have quoted the evidence that Bd can alter skin microbiome (lines 36 and 310), so might
expand their paper if they have such data available from their swabs.
Author's Response to Decision Letter for (RSOS-170107)
See Appendix A.
Downloaded from http://rsos.royalsocietypublishing.org/ on June 15, 2017
9
Decision letter (RSOS-170107.R1)
07-Mar-2017
Dear Miss Bletz,
I am pleased to inform you that your manuscript entitled "Drastic skin microbiota differences
between co-occurring frogs and newts" is now accepted for publication in Royal Society Open
Science.
You can expect to receive a proof of your article in the near future. Please contact the editorial
office ([email protected] and [email protected]) to let us know if
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As the issue version of your paper will be the only version to be published I would advise you to
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In order to raise the profile of your paper once it is published, we can send through a PDF of your
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On behalf of the Editors of Royal Society Open Science, we look forward to your continued
contributions to the Journal.
Best wishes,
Alice Power
Editorial Coordinator
Royal Society Open Science
[email protected]
AppendixDownloaded
A
from http://rsos.royalsocietypublishing.org/ on June 15, 2017
Response: Thank you both for your constructive feedback on our manuscript.
Reviewer comments to Author:
Reviewer: 1
Comments to the Author(s)
This manuscript examines the differences in skin microbiomes between 6 species of frogs and newts in
Germany. I think the results are interesting and it is worth publishing, however, the authors must address
some of these concerns or questions.
1-The title says that species are syntopic. Do these species hybridize? I would like to see more
discussion about which of these species are closely related and how their microbiomes are
similar/different. For example, I would expect the Lissotritons to be more similar to each other than to
Ichthyosaura and Triturus. Although the number of species does not allow for phylogenetic comparative
methods, it would be interesting to see how evolutionary relatedness is correlated with microbiome
composition.
2-I would like to see more information about habitat partitioning in these species.
Response: We used “syntopic” here with the meaning of “joint occurrence of two species in the same
habitat at the same time”. While we believe the reviewer is not uncomfortable with the term itself, we have
replaced it in the manuscript with “co-occurring”. We have furthermore emphasized that these species, in
the two ponds, co-occur strictly within the very same small pond area and thus are certainly exposed to
the same environmental reservoir of bacteria in their terrestrial habitat. We have furthermore emphasized
that these species in general do not hybridize, with exceptional hybridization recorded from the two
Lissotriton. We have also added a table of the pair-wise species distances to show the microbial
distinctness patterns with respect of more and less closely related host species. Briefly, for the sampled
newts there was no clear pattern of congeners being more similar than species from different genera. We
have also briefly mentioned this in the discussion.
Minor comments
Line 8-9 “these differences have rarely been analyzed in systems that minimize confounding factors, such
as season, location or host ecology”. Statement is not entirely true see McKenzie et al ISMEJ, Kueneman
et al. MolEco, Belden et al. Frontiers Micro.
Response: We have adjusted the wording of this sentence to make it less strong. It now reads: “however,
few studies have analysed these differences in systems that minimize confounding factors, such as
season, location or host ecology.” It’s important to note, that the three references mentioned here are
included within the introduction when introducing the concept of host-specific communities.
We acknowledge that other studies have also addressed this; however, we see only the Mckenzie et al
paper as fully removing the stated factors, and this study focused on larval amphibians. Belden et al
compares amphibian species with very different habitat usages (terrestrial and arboreal frogs), and
Kueneman et al again studies species that will differ in host microhabitat usage.
Line 14-17 Long sentence and unclear. Rewrite. I did not understand the second set of numbers. Are
those Jaccard distances?
Response: We have rewritten this as follows to improve its clarity: “While frogs and newts had similar
cutaneous bacterial richness, their bacterial composition strongly differed. The average Jaccard distances
between frogs and newts >0.5, while between species within these groups the Jaccard distances were
only 0.387 and 0.407 for frogs and newts, respectively.”
Line 17-19 I do not think that this core analysis is appropriate. The comparisons should be done with
shared OTUs among the 6 species.
Downloaded from http://rsos.royalsocietypublishing.org/ on June 15, 2017
Response: We believe there has been a slight misunderstanding here as a result of ambiguity
surrounding our use of the word “core”. In effort to clarify this we have removed the word “core” in the
abstract, and worked to clarify this within the methods on LEfSe and core computation.
To explain further, in the context of the LEfSe, we used the “compute_core_microbiome.py” script to
generate lists of common OTUs for each species as a way to focus the LEfSe analysis on true
autochthonous member and minimize the number of comparisons being performed. The lists from each
species were compiled and then the OTU table composed of all samples from all species was filtered to
maintain the OTUs from the compiled list. These calculations were not meant to define the core
communities.
If we restrict the LEfSe comparison to only shared OTUs we will lose important information about OTUs
that are perhaps abundant on one group and nearly absent in the other.
Line 19 delete word ‘some’
Response. This change has been made.
Line 21 Break sentence after colonizers. Start new sentence about future studies. Also, how genomic,
transcriptomic and metabolomic analyzes are going to help understand chemical and physical
characteristics of the skin environment. What about differences in pH, cell size, structure etc?
Response: We have broken the sentence where suggested and expanded the future studies piece to
include specific investigations into the chemical composition of the skin. We have chosen to keep the
statement of the omics methods as it pertains to both host and microbiota sequencing. For example, with
host transcriptomics we can begin to understand what is expressed in skin.
Directed efforts to understand how the chemical composition of the mucosal environment differs among
host and the specificity of amphibian antimicrobial peptides against potential colonizers as well as
genomic, transcriptomic and metabolomic analyses of both hosts and their microbiota will help to better
understand this intriguing pattern.
Line 33-34 Not true. See Walke et al Biotropica 2011
Response: We have adjusted this sentence to include this reference. It now reads: “While vertical
transmission may provide initial colonizers for some amphibian species [9]; maintenance of amphibian
skin microbial communities likely results from continual exchange from the environment[5]”
Line 43 delete ‘so far’
Response: This change has been made.
Line 45 “species from major clades of amphibians consistently differ from species of other such clades in
cutaneous microbial composition or diversity.” This statement needs to be rewritten. Is the objective to
say that there are phylogenetic differences in amphibian microbiomes?
Response: We have rewritten this sentence in effort to make it clearer.
It now reads, “However, field studies have often compared species from different genera; it remains
unexplored whether cutaneous microbial composition or diversity consistently differ to the same extent
when comparing species from within major amphibian clades or genera as when comparing species from
between these major clades (i.e. does host phylogeny play a role in amphibian microbial differences?).”
Line 67 Remove capitalized S from spring
Response: This change has been made.
Line 135 replace ‘completed’ to ‘performed’.
Response: This change has been made.
Line 143-144 I don’t agree with the threshold chosen by the authors for this analysis. If the comparisons
are just between two categories (frogs vs newts), then the OTU should be present in 100% of the
individuals of all species of newts/frogs.
Response: We believe, as explained above, there was misunderstanding associated with this analysis. If
we restrict the LEfSe comparison to only shared OTUs across all groups we will lose important
Downloaded from http://rsos.royalsocietypublishing.org/ on June 15, 2017
information about OTUs that are perhaps abundant on one group and nearly absent in the other. We
believe this confusion is associated with our use of the word “core”, and we have now, throughout the
manuscript, used core only with respect to our description of the core community (OTUs found on 90% of
individuals).
Line 151 Here it starts to get confusing, why core microbiome differs from the LDA analyses, then on line
155 another OTU table is used with a 75% threshold. I would recommend using the same threshold for all
the analyses.
Response: We have recalculated these distances using the same threshold as the core analyses (90%).
Line 161 I would suggest incorporating pairwise comparisons directly in Fig. 1 instead of having tables 1
and 2.
Response: Thanks for this suggestion. We have incorporated the main effect statistical details (Table 1)
into the figure. We have chosen to keep table 2 (now Table 1) as it is difficult to display these pair-wise
comparisons when we have the two locations presented separately.
Line 208-209 True, but also they might inhabit different areas of the ponds. Any insights on this? Do you
think that ponds are completely homogenous?
Response: We have added a sentence here to acknowledge that we cannot fully separate differences
associated with microhabitat use; however, it’s important to note that individuals of frogs and newts are
often found in the same exact habitat in the shallow edges of the ponds. The sentence we added says
“While we cannot fully exclude differences in microhabitat preferences between frogs and newts as factor
influencing cutaneous bacterial composition, individuals of both groups are often found in the shallow
edges of the ponds.”
Line 222 Please mention which are the 3 OTUs.
Response: The names of these taxa have been added to the discussion.
Line 226-227 I didn’t see in the methods anything about environmental samples. Please check.
Response: Indeed, thank you for noticing this. We failed to mention this in the methods. We have now
added this to the methods. We have also added a brief summary in the results to document the major
taxa present in the pond water.
Line 228-231 Maybe move to methods? Seems awkwardly placed.
Response: Agreed. The details of this sentence have been moved to the methods.
Line 232 Change “encountered” by “observed”.
Response: This change has been made.
Reviewer: 2
Comments to the Author(s)
The paper analyses microbiota composition of frogs and salamanders collected from two different
localities for patterns of differences between phylogenetically distinct and related taxa. Differences in
species richness and diversity between frogs and salamanders were found and these were consistent
across the two localities.
The cutaneous microbiome of amphibians is affected by many factors and teasing apart the possible
roles of host and environmental factors on microbiota variation by examining syntopic species, as this
study shows, can provide useful results. However, specific comparisons between the life histories and
corresponding habitat usage of the salamanders and frogs should be considered to interpret the observed
patterns of micobiota variation.
Downloaded from http://rsos.royalsocietypublishing.org/ on June 15, 2017
The study might be of more general interest if were to include results of screening for the presence of
amphibian pathogens known to affect amphibian population viability and microbiota. Screening for
Batrachochytrium dendrobatidis (Bd) and/or Batrachochytrium salamandrivorans (Bsal) from swabs now
is relatively routine. Availability of such data would be very useful in interpreting the results.
Response: We agree Bd/Bsal survey data are of interest. We do have such data, however, they are
currently within another manuscript that has recently been accepted for publication in ISMEJ. We have
added a sentence in the discussion noting the presence of Bd at the sampled ponds.
Specific comments
Given that the study emphasises comparisons among syntopic species, low samples sizes (or complete
absence) of some species from some localities in the dataset reduces the generality of the conclusions.
Why were R. dalmatina and L. helveticus not represented from one locality, and why were sample sizes
of I. alpestris so low?
Response: Unfortunately the ability to have all species at the same site is quite difficult. Rana dalmatina
and Lissotriton helveticus are not known to occur in Kleiwiesen, and we have now added a sentence to
Materials and Methods specifying this. It also is worth mentioning that finding exact biological replicates
of these species co-occurring in precisely the same pond is difficult. The pond in the Elm is in fact the
only site known to us, and rather exceptional, in that all four Central European newt species occur here at
exactly the same spot.
In this study, our focus was on minimizing environmental effects by strictly focusing on amphibian
individuals collected in the same period of time from the same sites, and this led to somewhat uneven
sample numbers, as is now mentioned in Materials and Methods. For instance, the sample sizes for I.
alpestris at Kleiwiesen is low because during these sampling dates very few individuals were captured.
Additional individuals were sampled on later dates (March 24 and April 1), however, to minimize the time
window of sampling they were not included in this study.
Sample collection (lines 77-79): Please mention the distance between the localities. Also mention details
on geographical variation and possible anthropogenic factors that may affect the collection sites with
potential consequences on skin microbiota.
Response: We have added a description of the sampling locations to the methods.
Statistical analyses (lines 135-136) need to be explained in more detail. There are many different types of
ANOVA. What model was used, and using what software?
Response: Additional details on the performed ANOVAs have been added to the methods.
ANOVA results are not properly presented (line 164). How many degrees of freedom in the comparison,
and what error term was used? Also P is indicated with unjustifiable precision.
Response: We have added in the degrees of freedom, and rounded the p-value to 3 decimal places.
Have authors screened Bd/Bsal from swab samples? This would provide a possible basis for
understanding patterns of cutaneous microbiota variation within and between species/families. Authors
have quoted the evidence that Bd can alter skin microbiome (lines 36 and 310), so might expand their
paper if they have such data available from their swabs.
Response: As mentioned above, we agree Bd/Bsal survey data are of interest. We do have these data
however, they are currently within another manuscript that has recently been accepted for publication in
ISMEJ. In any case, although we have evidence for Bd in the system, its prevalence is low (about 6%
overall among the newts, across species and seasons) and therefore it is unlikely that our results in the
present study would have been strongly influenced by the presence of the pathogen in some samples.
This is now also briefly mentioned in the Discussion.