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Thank you for following through on my suggestions. The current manuscript has a better flow and is ready for publication.
[# PeerJ Staff Note - this decision was reviewed and approved by Konstantinos Kormas, a PeerJ Section Editor covering this Section #]
I agree with the reviewer that the manuscript is almost ready for publication, pending just minor revision and proofreading of the newly added text, and the whole manuscript in general.
Dear Editor,
The authors have made substantial efforts to improve the readability of their manuscript.
The structure has improved, as well as the theoretical embedding of their research.
Methodological issues have been addressed sufficiently, with the main limitations of lack of sequencing controls largely taken away by additional PCRs and electrophoresis gels.
I hope my comments have been helpful to the authors to improve their manuscript on their exciting research project.
I hereby recommend the paper ready for publication. However, another check of grammar on the newly added text may be useful.
with regards,
Pieter van Veelen
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I agree with the concerns and suggestions of the reviewers that albeit the research framework is solid and the manuscript is nicely written, the manuscript requires some major revisions before acceptance. I look forward to reading the revised version of this interesting manuscript.
[# PeerJ Staff Note: Please ensure that all review and editorial comments are addressed in a response letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. #]
This study is an interesting example of microbial variation between body sites in two wild penguin species, with clear hypotheses and results. The authors do a good job of providing a wide range of relevant examples from the literature. Raw sequences are accessible in GenBank, and most ethical permissions are described within the methods (although export permits were not mentioned in the context of sending samples for sequencing).
However, grammar and clarity need substantial improvement. The most significant error is in the name of one of the two study species - the Magellanic penguin is referred to as the "Magellan penguin" throughout the text and in the title. I am listing a number of grammatical errors here, but more remain, and the manuscript would benefit from significant proofreading prior to publication.
Grammatical corrections:
1. The common name for Spheniscus magellanicus is the Magellanic penguin (not Magellan penguin), please correct in the title and throughout the text and figures.
2. Add scientific names for both penguins in the abstract.
3. When using “king penguin” within a sentence, “king” should not be capitalized. You would capitalize Magellanic penguin because it is derived from someone’s name, but king penguin is always lowercase. Please correct throughout the text.
4. Line 28 and title – “Strait of Magallanes” should be either “Strait of Magellan” or “Straits of Magellan” in English. Please choose one and correct throughout title and text.
5. Line 29 - give the full name for “16S sequencing” in the abstract and the first time mentioned in the text - “16S rRNA gene sequencing.”
6. Line 36 – “most relevant taxa” should probably be “most prevalent taxa”, unless you are describing how those taxa are relevant to something else.
7. Line 35, line 42, and elsewhere – refer to these species as either “the king penguin”, or “king penguins” (i.e. when you say “in king penguin” it should actually say either “in the king penguin” or “in king penguins”). Please correct throughout the text.
8. Line 37 – sentence beginning with “Penguin body sites microbiota alpha diversity” needs to be restructured. That phrase could be replaced with something like “Microbial alpha diversity across body sites…” Change present tense to past tense to match the rest of the text.
9. Line 42 – rephrase “…fitted well a neutral community model” to something like “fit well under a neutral community model.”
10. Line 52 – correct “virus” to “viruses”
11. Line 59 – This sentence has some odd spacing that could be a formatting glitch or could be an in-text issue. Also, the sentence is grammatically incorrect and should be rephrased to something like: “…whereas the skin or feathers of birds (especially seabirds) have received less attention.”
12. Line 68 – “continental birds plumage and skin” should be written as possessive; rephrase to “the plumage and skin of continental birds…” Similar problems on lines 77, 78, 310, 429, and in several places in the discussion.
13. Line 105 – “body sites microbiota” should be “body site microbiota.” Correct throughout the text as this occurs in many places.
14. Line 109 – need to rephase the sentence “penguins body sites bacterial communities.” Could replace with “bacterial communities of penguin body sites.”
15. Line 111 – replace “body sites” with “body site”
16. Line 112 – add a dash for “high-throughput”
17. Line 145 – replace “proceed” with “proceeded”, rephrase “this stressed more the animal” to “this increased animal stress”, and replace “change” with “changed”
18. Line 147 – rephrase from “We rubbed five times each penguin body site” to “We rubbed each penguin body site five times”
19. Line 181 – unknown reads should not be discarded; this disguises the degree to which database biases may be influencing the results.
20. Line 200 – there is a partial sentence in here, proofread and correct.
21. Line 211 – correct “alfa” to “alpha”
22. Line 221 – correct “compare” to “compared”
23. Figure 3 – labels should be improved in R for publication (i.e. don’t leave in spreadsheet labels like S_mag_nest; rename instead).
24. Figure 3 caption – replace “pointed” with “indicated.” Add “the” for “the orange bar”, “the yellow bar”, etc.
25. Italicize bacterial genera within the figures
26. Line 355 – what do you mean by “Alike king penguin”? Should either be rephrased to “Like the king penguins,” or “Unlike the king penguins,” depending on the intended meaning.
27. Line 438 - remove “in”
28. Line 441 – remove “On the other hand,”
29. Line 442 – Replace “Yet,” with “However,” and replace “find” with “found”. Check that you stay in past tense throughout the discussion.
30. Line 447 – rephrase: “The main roles of preen oil in continental birds are feather…” etc.
31. Significant proofreading is needed throughout the discussion section to improve sentence transitions, remove unnecessary commas, and correct grammatical errors similar to those indicated earlier in the text.
The research questions are clear, hypotheses are stated in the beginning and again in the discussion, and the low number of skin microbiome studies in seabirds makes this study an interesting contribution. The methods section is descriptive, but several details still need to be added (see below). The sequencing read analysis is generally strong. However, unclassified reads should not be removed from the analysis, and it would be useful to see how large the proportion of unclassified reads are compared to classified reads. I recommend including unclassified reads in the analysis for this manuscript prior to publication.
A significant problem is the lack of experimental controls in the study. All microbiome studies should make use of both positive and negative controls, but this is particularly essential in low biomass studies such as this one. However, I recognize that adding controls at this point would be a significant burden, and will leave it up to the editor how much weight to place on this issue. Authors, make sure to include positive and negative controls for all microbiome studies in the future.
Additional points:
1. Line 47 – explain a bit more in the abstract how this might be relevant to ocean health.
2. Line 88 – the association between microbes and microbes? This is unclear, please clarify the gap this research is addressing.
3. Line 146 – so you are comparing chest feathers to back swabs? This seems like it would artificially alter the results between body sites. At the very least, this should be expanded on within the discussion.
4. Methods section – a description of the nest sampling method is needed.
5. Lines 155-160 – more detail is needed on the DNA extraction and sequencing. How much time elapsed between sample collection and DNA extraction? Did you use all of the swabs collected for each individual, or only one each? What was the DNA yield for the extractions? Did yield significantly vary between feathers vs. swabs? Or penguin swabs vs. nest swabs? Did you have to concentrate samples? How much DNA was sent to the sequencing company?
6. Line 161 – so you exported the samples to Korea? What permits were in place for exportation and importation?
7. Did you control for any confounding variables within your PERMANOVAs such as age or sex? Clarify this in the text and justify your decision either way.
The sample size for this study is very small, but within an adequate range for the statistical analyses performed. The small sample size and lack of covariates limits inferences from this study, but it is still a useful addition to the avian microbiome literature. Results and conclusions clearly address the research questions present throughout the text.
I enjoyed reading this descriptive paper on penguin body site microbiomes. Prior to publication, I think the authors should include unclassified reads in the analysis, carefully correct all grammatical errors throughout the text (particularly the naming issues), and include additional details in the methods section (listed above). The lack of sampling/extraction/sequencing controls is concerning, but this problem would be difficult to resolve within a reasonable timeframe. In my opinion, making these changes would improve the manuscript sufficiently for publication.
I have reviewed the manuscript by Ochoa-Sánchez et al. titled “Body site microbiota of Magellan and King penguins inhabiting the Strait of Magallanes follows species-specific patterns”. The authors have made substantial efforts in fieldwork to collect samples from two species of penguin to analyze the microbiota of different body parts and nests of a single species. The authors have presented a comprehensive analysis with statistically suitable methodology, and visualized the results in an appropriate way. This effort in itself is worthy of congratulation for the massive amount of effort.
Yet, to be considered for publication in PeerJ, in particular, the ecological context from the avian ecology perspective is largely lacking from the introduction. Where the discussion covers many aspects of avian biology, and presents interesting ideas, these topics deserve a better embedding in the introduction to make the presented results more effectively contributing to the field. While Penguins are unique among birds, the expectations of microbiota differences between body sites, between penguin species, and their relationship to Penguin’s unique traits and behavioural ecology could be improved. In a Major Revision, the authors are invited to embed their hypotheses better in that context. While the study appears to be carried out well, the sequencing effort lacks proper controls (both negative and mock positive), which makes judging data quality difficult. A Major Revision should therefore include additional statistical evidence to argue that the resulting inferences from 16S rRNA gene amplicon data are not resulting from contamination, which is a serious concern with low biomass samples taken with swabs.
Basic reporting
Language and writing style
To benefit the readership most, the scientific writing in professional English language needs an additional round of fine-tuning. In various cases throughout manuscript, the Spanish mother tongue of the authors conflicts somewhat with the grammar and sentence structure, or choice of words, such as in Line 145-146. Specifically, I suggest in general to rewrite ‘assemble’ to ‘assembly’ and to replace ‘derive’ to ‘result in’. In addition, some sentences are not finished properly, such as in Line 200. The results section in particular may need to be revised to report results in past tense. Prior to submission for another round of review, the manuscript quality would benefit language editing before it can be considered acceptable for publication. A suggestion would be to ask a fluent English-speaking scientist to assist with this task.
Theoretical embedding
The descriptive aspect of bird microbiota and the fundamental knowledge gap of drivers of animal associated microbiota variation is valid and given the unique ecological conditions penguins experience, the data are relevant and contain novelty. Yet, to make the reported data more valuable and to place the results in the context of the field, I invite the authors to strengthen the theoretical background of the study with ecological hypotheses. In its current presentation, the data presentation is highly descriptive. For example, the authors claim to present data on the microbiota of first subaquatic foraging seabirds, but do not provide any mechanistic interpretation or hypotheses on how this feeding behavior in birds affects microbiota assembly. In its current form, the background lacks both a mechanistic context on penguin microbiota assembly processes, as well as microbial ecological theory background. For example, the authors do not compare the microbiota of these birds to seabird microbiota that are foraging at ocean surface.
To provide context for the chosen sample types, and species as experimental design, I recommend the authors to strengthen the introduction by explaining how the authors think that:
1. Penguin microbiota assemble, and to what extent and which role the nest environment plays
2. The stochastic processes contrast deterministic processes on and by the bird body/physiology
3. how these stochastic and deterministic processes are driven by the Penguin’s biology and behavior, in the context of how the two penguin species differ behaviorally.
4. anthropogenic influences affect all the penguin-associated microbial ecology in general, and also practically for the two penguin species, to accommodate the Special Issue context that the study is intended to fit
Experimental design
Line 103-105: The sentence implies that nest microbiota data is analysed for both species. This does not fit the dataset, and I suggest the authors make that clear here. Accordingly, Penguin microbiota versus nest microbiota is not explored for King Penguins, so the dataset is not very useful make inferences on how different nest microclimates affect penguin microbiota. This needs to be made clear from the start, in the last paragraph of the introduction section.
Sequencing controls
The authors are invited to supply more information on the molecular analysis quality control. Current state of the art practical instructions for microbiome research stress the importance of robust sample processing, particularly by including positive and negative controls for the sequencing process. Since inclusion of these controls are not reported, it is difficult to judge the technical quality of the 16S rRNA gene amplicon sequence data. The risk of contamination is particularly pertinent with the use of swabs, for example see Salter et al. 2014 [http://www.biomedcentral.com/1741-7007/12/87 ] and Dyrhovden et al. 2021 [https://doi.org/10.1128/mbio.00598-21].
If blank DNA extractions are not performed, I would like to ask the authors for including (supplementary) information on DNA concentrations of the swabs, which are presumably low-biomass samples, an aspect that is impossible to conclude on without the DNA concentration data. With that data, I would like to invite the authors to perform a post hoc statistical analysis to identify likely contaminant ASVs, such as by using DECONTAM (https://doi.org/10.1186/s40168-018-0605-2 ) to show convincingly that the inferences on the microbial players are not biased by contaminant taxa, or by showing the insignificant degree of contaminant effects.
Outside of the lack of sequencing controls, the findings may well be valid. To support the claim that Penguins are unique in their subaquatic marine life, for discussion purposes, the authors are invited to compare to auks, cormorants, grebes, Aythya, Somateria and Melanitta ducks, to name a few, that also spend considerable time under (sea) water. A notable difference is the unique feather structure. It would make the paper stronger if the authors expand on this difference, the potential different roles microbiota could play, perhaps in association of preen waxes. A link to anything on evolutionary, behavioural, physiological differences between penguins and other birds and the potential microbial ecological differences would benefit this manuscript. Various of the mentioned topics have been discussed in the Discussion section, but as mentioned earlier in this review, these topics could be touched upon in the Introduction.
General comments
Specific comments:
Line 59: Remove front tab
Line 86-88: I would like to invite the authors to make a more nuanced statement on the importance of the study. I specifically regard an overstatement that this ‘must be addressed to understand.. [avian microbiota]”. It would be nice if the authors could hypothesize instead how the feather and skin microbes of penguins may contribute to the subaquatic lifestyle, which is relatively unique in birds.
Line 96-97: I cannot understand what this means
Line 100: Please explain why nest site of open nests in King penguin would be less affecting than the burrows. Incubation, and chick brooding at the open nest also incurs prolonged nest contact.
Line 211: change alfa into alpha
Confidential notes
In Table 2:
- the F statistic should be renamed as pseudo-F statistic in PERMANOVA.
In Table 3:
- the columns stating % average abundances are often >>100%. Please recalculate these values or change the column name to the appropriate unit.
- Also, please unify the amount of decimal numbers in these columns.
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