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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: No

Reviewer #4: Partly

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: N/A

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

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Reviewer #1: This study provides valuable evidence on how nesting strategies—wet-nesting and dry-nesting—affect both the quantity and community structure of bacteria adhering to the eggshells of wetland-dwelling bird species. By presenting empirical data that align with intuitive expectations, the study effectively demonstrates the impact of nesting strategies on eggshell bacterial communities, thereby contributing new insights to the field of avian microbial ecology. The progression from objectives and experimental methods to results and conclusions is exceptionally orderly and logically structured. This clarity enhances the significance of the research findings, emphasizing that the results are not merely speculative but hold meaningful evidence. However, there are several significant shortcomings.

Comments

1.It is regrettable that this study had a small sample size, ultimately failing to present statistically significant data. Due to the limited number of samples, a detailed evaluation of other environmental factors influencing bacterial communities (such as temperature, humidity, types of nesting materials, seasonal fluctuations, and annual variations) was insufficient. In future studies, it is recommended to increase the sample size and expand the number of samples for dry-nesting species to more accurately assess the impact of nesting strategies. Enhancing data reliability by increasing the number of dry-nesting species samples is crucial.

2.The authors independently reported results from both culture-independent (direct DNA extraction and ARISA analysis) and culture-dependent (bacterial culturing) methods. However, by detailing the relationship and differences between the bacterial communities detected via ARISA analysis and the culturing results, the complementary roles of both methods can be clarified. In addition, it would be beneficial to discuss the extent to which the culture-independent method captures bacterial diversity compared to the culture-dependent method, and whether there are any biases towards specific bacterial communities.

3.The introduction explains vertical transmission of eggshell bacteria in birds (transmission from the mother within the oviduct before eggshell formation). However, this study does not clearly distinguish whether the eggshell bacteria are derived from maternal contact or from other environmental sources. A discussion including comparisons with the bacterial communities present on the exterior and interior of the mother bird is necessary. Please discuss comparative analyses between the mother's bacterial communities (such as those in the cloaca or skin microbiota) and those on the eggshell to identify maternally transmitted bacteria.

4.The terms "wet-nesting" and "dry-nesting" are inconsistently hyphenated throughout the manuscript. It is recommended to standardize the hyphenation for consistency.

Reviewer #2: Summary:

In this manuscript, van Dongen and Brandl et al. seek to characterize the differences in microbial communities between dry- and wet-nesting bird species in wetland environs. The authors collected sample swabs from nests of both dry- and wet-nesting species to analyze the bacterial populations associated. They demonstrate an increased bacterial abundance on wet-nesting species eggs than dry-nesting eggs. They also use ARISA to show distinct bacterial populations among wet and dry-nesting eggs. Overall, this small study is meticulously controlled and reported, however lacks the depth which the field has come to expect from studies on microbial communities, and the exact techniques chosen are perhaps not ideal for the questions being asked. The authors should attempt to use more modern and accurate techniques to better dissect their hypothesis.

Major Comments:

• The authors determine bacterial abundance by selective and non-selective plating on bacterial growth plates. While this is fine for certain bacterial taxa, there is a risk of the isolated colonies not being representative of the actual microbial community, only the microbes capable of being cultured on agar. Performing 16S qPCR would be more accurately indicative of true bacterial abundance.

• The authors use automated ribosomal intergenic space analysis (ARISA) to detect and report OTUs from their sampled nests. While an established assay, ARISA sports few benefits over newer, more powerful techniques such as 16S sequencing or shotgun metagenomics. These techniques would allow the authors to better characterize the exact differences between the microbial communities under research.

Minor Comments:

• Line 144 – the word “media” is missing.

• Figure 1 – it would be helpful to include a legend in the figure itself indicating that white bars are dry-nesting birds and black bars are wet-nesting birds.

• Figure 1 – Statistical significance would be helpful to report in the figure itself using the * system for noting p-values.

• Figure 2 – Authors should note in the text the differences between figures 2a, 2b, and 2c.

Reviewer #3: Review: PONE-D-24-57432 - Bacterial assemblages on eggs reflect nesting strategies in wetland-associated birds

Dear Authors. When I read the abstract of your manuscript, I was excited about it because, as you mention, it is the first comparative study looking at the eggshell bacterial assemblage across wetland species with different nesting strategies – wet and dry. However, once I started reading the sampling methodology and statistical testing, my enthusiasm waned slightly. While I find the results regarding the differences in the bacterial community between water samples and on eggs of wet-nesting species mainly interesting supporting the strong nesting microhabitat effect of these wet-nesting strategy species, I have methodological doubts about the study that do not allow for the robust conclusions you state, e.g. that species identity does not play a large role in dry-nesting species. I think this result is more of an artifact of not having a study controlled for the age of the clutch from which you sampled eggshell microbiota from only one egg. Why not from the whole clutch, i.e. each egg, since you didn't control for clutch age and individual eggs at all? I consider this a major shortcoming and either you should add how you checked this, but since you do not mention the nesting stage at which you sampled the eggs at all in the "Material and Methods", rather you did not take this into account at all, you should discuss these limitations and poor sampling within the species sufficiently. I also don't consider it a correct approach to include species in the dry-nesting category that differ significantly in their nesting strategies - e.g. most species nest on the ground, while only a few have elevated nests. Also, ducks deposit their own down-feathers in the nest and also partially incubate the brood, whereas this is no longer typical of other species that represent this dry-nesting group. I suggest a couple of ways to reduce this variability in your data, at least for bacterial load/abundance testing, since you take this into account for ARISA-based OTUs community structure, but not for eggshell bacterial load testing.

Your discussion also needs thorough re-writing– mostly toning down your strong conclusions that are unfortunately based on inappropriate sampling and discussion of your sampling and methodological limitations.

I list my concerns and a possible partial solution below. I hopefully they will help to make your data and outcomes more robust.

Abstract:

Last sentence of abstract sounds a little vague. I understand what you want to express, but better will be use some sentence which stimulate the future research on antimicrobial protection of eggs across habitat humidity gradient or try to be more generalist in your expressing and make some future suggestions.

Material and Methods:

Line 128 – why you did not swab all egg in the nest to have some “common” bacterial assemblage?? Did you candle eggs to assign development stage of each nest-eggs? Difference between first and last egg in the nest may be 7 or more days – especially in the case of ducks. So if you take lastly laid egg (randomly) it can have lower bacterial load compared to first laid egg in the clutch. So, sampling eggshell of only one egg from clutch is really not good sampling approach. Please note, eggshell bacterial assemblage may significantly differ with respect to nesting/incubation period - bacterial assemblages on eggs reflect and vary in fresh or continuously incubated eggs. Moreover, considering that your sampling design is strongly imbalanced (e.g. 48 Great-crested grebe nests to compared to one Moorhen nest), I see the fatal flaw in your study as you did not control nest/egg age or incubation phase during the sampling period which was quite extensive (May-July). All these factors may strongly affect both, microbial load and assemblage. How you were able to control for this confounding effect in your study. Please justify. Otherwise, your results cannot be considered as reliable with omitting this variability into account and indices of bacterial load and communities may be very biased preventing comparison between individual nests (wet-, dry- breeders).

Line 144 – cultivation of bacteria – you did not run in replicates? How did you count CFU? Manually? Using SW from photography? Be more detailed in description of your technique?

- ARISA- please as this method is not such precise as sequencing, use term ARISA based-OTUs throughout the whole manuscript as term OTUs per se is emerged from more precise sequencing technique than ARISA.

Statistical analyses

To have you sampling design, I first inspect within species variability in bacterial load/abundnace. I am lacking basic exploratory data analysis (min, max, range, mean SD, SE – i.e. expressing variability of data within species. I guess it will be quite extensive. You can do at least for Fulica atra and Podiceps cristatus where you have nice sampling.

- another factor is covering the egg with the feathers – ducks (Mallards and Aythya spp.) use their own body feathers as nest lining material and cover eggs with these feathers. This might be other factor that can strongly affect eggshell microbial assemblage. To see your species and dataset. I am for to extend your statistics from glmer approach to also pairwise comparison of grebes or only Podiceps cirstatus with only Fulica atra as representative species from wet-, dry -nesting strategy continuum. I see grouping of ducks, Savi warblers and herons as not intuitive with respect to their nest building and nest material specifities. Also, most species are ground nesters but what about heron and Ixobrychus and Savi warbler??? I think you mixed too much together without appropriate controlling of confounding effects.

I am strongly for make more species groups – e.g. put ducks using feathers in the nest together as they really have specific nest building habits via incorporating own body down feathers into the nest and cover the eggs with them. You can do this statistic as additional testing that can uncover another specifities in eggshell bacterial load. Now I see you did for bacterial communities’ statistics, but why you did not do also for bacterial load (abundance) data??? E.g in Figure 1 I like to see boxplots where wet-nesters will be depictes separately, e.g. ducks also separately, and other species as well – except of these having n=1). I think it will be more reliable than to mix all dry-nesters together into one group consisting of many species with distinct nesting strategy.

Discussion

Line 316-320 – Sorry but this conclusion is too strong considering your sampling effort one to three nest per most of the dry-nesting species. Based on your dataset and fatal flaw with ignoring incubation phase in sampling egg, you cannot rule out the role of species identity on eggshell bacterial assemblage. Tone down this your conclusions and rather discuss thoroughly possible limitations of your study that more robust sampling controlled for nesting phase of sampling eggs in necessary to test these assumptions – i.e. rule out or test the effect of species identity on eggshell bacterial load. and discuss all these shortcomings of your study.

Lines 327-329 – also this conclusion needs to additional testing as I suggested. Use only dry-nesting species where appropriate number of individuals you have or cluster together ducks that have very specific nesting – incorporating of own body down feathers into the nest lining, egg covering and partial incubation..I think it will help to dilute all confounding effects that were omitted in your comparison and clustering all dry-nesting species in one group…

Lines 336 –337 - of course if you sample one to three nest per species and sample only one egg per clutch. It is not representative eggshell microbial assemblage per species, sorry, but you need more robust sampling to see some nesting ecology-based clustering.

Line 338 – yes, and also cover their eggs with nesting material full of down feathers that may have thermoregulatory and also antimicrobial properties. Also intermitently incubate their eggs before starting full incubating which affect both, eggshell, but also egg white bacterial load – see e.g. Effect of intermittent incubation and clutch covering on the probability of bacterial trans-shell infection | Semantic Scholar, or Laying date, incubation and egg breakage as determinants of bacterial load on bird eggshells: experimental evidence | Oecologia

You should take into account also this very specific nesting ecology before choosing species for your study…or at least take this into account during testing your hypotheses and discuss these specifications thoroughly.

Lines 343-344 and further – tone down thoroughly.. You really don´t have data on these strong conclusions.

Lines 346-348 – and also more thorough sampling of dry-nesting wetland species…

Lines 365-376 – but there is also studies that did not find and effect and in general there is no study testing direct effect of trans-shell penetration and embryo mortality. Discuss more precisely. It is not such straightforward as you described. Most of eggshell bacterial are not able to live inside the eggs etc. etc…

Line 357 – yes, incubation, and how did you control this in your study??? Try to put this infor into the context of your study. It is not discussion, just plain informative manner which you used here..

Line 366 – your findings may highlight something but are not such robust, unfortunately..So try to tone down that your findings may serve as some initial stimuli for further more thorough testing…

Reviewer #4: Specific comments:

-English proofreading is extremely recommended.

-You should double check the references style according the submission guidelines.

-Where are the references you followed for these experiments?

-What is the point of cultivating the samples on agar media that isn't selective?

-For clarity, please provide a concluding paragraph at the end of the study.

-In order to increase clarity and conciseness, I would like to combine the discussion and results sections into a single section. This is because the sections are extremely disorganized and lack sufficient data.

-Since you brought up the eggshells' antibacterial qualities (J L 44, 62, 72, 80, 360, and 362), your study never touched this point.

-In order to make the results so compelling and connected to the clinical findings, it would also be helpful to discuss the most common disease in these species or the chicks after hatching.

-Additionally, I would advise outlining how susceptible these bacterial species are to antibiotics in order to determine whether they are commensal or dangerous infections (MDR pathogens), against which certain bird species are immune.

-Why did you limit your attention to bacteria? How about fungi?

- Where is the raw date for the genetic findings?

-Fig 1: the error bars are so weired, please explain it.

-Please divide Table 1 into dry and wet categories with distinct data for each since it is worthless. Please explain what t/p stands for as well.

L48: Be specific and use birds instead of animals.

L82: Bacterial first, then genetic.

L180: Please provide an illustrative figure to demonstrate the steps taken in a sequential manner, as the sampling numbers are extremely unclear.

L195: Throughout the manuscript, use the same first line space.

L200: Findings? For clarification, please specify which figure.

L244: For wet or dry nesting samples, is 35.2%?

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

**********

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Attachments

Attachment

Submitted filename: Review PONE-D-24-57432.pdf

Dear Dr. van Dongen,

plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Petr Heneberg

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments :

Academic Editor:

- While the introduction and discussion mention vertical transmission, the study does not provide data to distinguish it from environmental acquisition. This limits conclusions about maternal contribution to eggshell microbiota.

- Nesting strategy (wet vs. dry) is used as a broad grouping variable, but species within each group vary significantly in nesting materials, water contact, and incubation behavior. This undermines the strength of clustering conclusions based on nest type alone.

- One Egg per Nest Sampled**: Sampling only one egg per clutch does not account for intra-clutch variation, which can be substantial and could bias results - discuss this in the Limitations section.

- Bacterial communities change with incubation, and the study did not assess incubation stage or egg laying order. Without controlling for this, variability may mask true differences due to nesting strategy - discuss this in the Limitations section.

- Many dry-nesting species have sample sizes of 1–3 nests, making any comparison across species or nesting strategies tenuous - discuss this in the Limitations section.

- Use of Outdated Genetic Method (ARISA). While ARISA is a valid fingerprinting technique, it has been largely supplanted by 16S rRNA sequencing and shotgun metagenomics, which offer finer resolution. The authors acknowledge this but should clearly frame ARISA's limitations.

- Avoid Overstating Conclusions. Many conclusions regarding the effect of nesting strategy are too strong given the limitations in design. These should be reworded to reflect the exploratory nature of the study.

- Clarify Figures. Figures should include clearer legends and visual indicators (e.g., nesting strategy symbols or species groupings). Consider separating duck species from other dry-nesters in visualizations.

- Specify that future research should explore egg antimicrobial traits, control for incubation stage, and incorporate 16S sequencing to identify microbial taxa involved in pathogenic vs. commensal roles.

- Line 23: Change “Animals host diverse bacterial assemblages” to “Birds host diverse bacterial assemblages” to reflect study focus.

- Line 144: Add “the” before “media”: “…onto the media.”

- Hyphenation: Standardize the use of “wet-nesting” and “dry-nesting” throughout the manuscript.

- Tone Down Phrasing: Replace strong language such as “our findings are significant” with “our findings suggest” or “indicate.”

- Line 196: Improve clarity of sampling description—consider a flowchart or table to summarize sample numbers.

- Line 430-432: Phrase such as “highlight important differences” should be reworded to “suggest potential differences.”

- Ensure consistent italicization of all Latin species names throughout the manuscript, including the References.

- The manuscript references older papers for methods but does not cite newer alternatives (e.g., 16S sequencing), which should at least be discussed.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

Reviewer #4: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: (No Response)

Reviewer #3: Dear authors. I am fully satisfied with the revised version of your manuscript. I think you improved the clarity and comprehensively toned down your conclusions and now the study is really useful as a stimuli for futures studies focused on eggshell bacterial assemblages and/or bacterial trans-shell penetration in the context of breeding ecology of birds.

I have only a few minor comments and suggestions which you can find below:

Technical comment:

Why you changed corresponding author in the revised version of the manuscript and why one of the co-authors (L. Rubáčová) has been removed from the author team to Acknowledgements only? Did she agree with this and it was discussed with her thoroughly? It is not usual such dramatically change author membership and status of authors?

To your comments regarding clutch age (my (Reviewer 3) Comment 1) and effect on eggshell bacterial assemblage. Yes, you considered sampling date and describe that effect might be linked with changed environmental factors. But, I am considering incubation of the clutch and resulting eggshell assemblage. As you described on lines 150-152, you sampled one egg from nest when you found it, but although you record the precise date of nest finding, you were not aware of the age of the clutch (i.e. how long the clutch has been incubated already). Moreover, it is quite common that 1) nesting parasitism occurred in Mallards and also coot and grebes and also that 2) intermittent incubation of partial not-completed clutch occurred before the full incubation and clutch completion (at least in ducks it is very common, and I think also swans or grebes are known for this behaviour). In practice, simple fieldwork “candler” (inspection of egg inner structure under the light) is used for determination of embryo age which correspond to intensity of incubation. And as you mentioned in you text of the revised ms that incubation may affect eggshell bacterial assemblage (and quite a lot), this discrepancy in eggs age and developmental stage within the clutch may cause bias if your chosen only one egg from the complete clutch for sampling. I think you should somehow touch of this possible bias in your data caused by the different clutch age, role of intermittent incubation and/or brood parasitism in your focal species.

But in general, it is nice you added additional GLMMs testing for the effect of clutch size and sampling date, as it is really highly probably that enlarged clutches may be incubated before clutch completion or being parasitized by brood parasites. This additional GLMMs made your conclusions more robust. Yet, still, as your study is pioneering in study of bacteria on eggshell with respect to breeding ecology, I am for to include possible factors that might be behind the differences between dry- and wet-nesters. Although I understand your point of view and you want to keep the line of the study as simple as possible, I thin a few sentences in the discussion with other potential factors cannot be distracting from the major message of your study.

Line 141 – “more holistic overview”., I suggest to replace holistic by “comprehensive”. Holistic is really to strong meaning in your case study.

Line 142 - …in isolation. I suggest to replace by “separately” or some other equivalent.

Line 290 – “….of bacteria on eggshells on wet-nesting..“ rephrase it please.

Line 295 – “coliforms” without capital please

Lines 359-366 – I am still wondering if it cannot be partially caused by pre-incubation behaviour of dry-nesters – covering of clutch by down feathers in mallard and other duck species, intermittent incubation I have mentioned above..I think you should take this into account in discussion of your results (mostly differences between dry and wet-nesters). You already did for covering the clutch by feathers, and even if you disagree to do for incubation (see response to my Comments 18). I think it can help to untangle the all possible confounding factors that have to be taken into account in future studies of eggshell or trans-shell bacterial assemblages.

Line 380 The aim of this study…..I suggest to rephrase… “…bacterial abundance and community structure vary based on……”.

Your Response to Comment 17 of Reviewer 3 – for example this study has show no effect of bacterial trans-shell penetration on hatching success: Effect of intermittent incubation and clutch covering on the probability of bacterial trans‐shell infection - Javŭrková - 2014 - Ibis - Wiley Online Library

Reviewer #4: After reviewing the revisions and responses, I find that all my comments have been adequately addressed, and I have no further comments.

**********

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Reviewer #1: Yes:  Akihiko UDA

Reviewer #3: No

Reviewer #4: No

**********

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Attachments

Attachment

Submitted filename: Review PONE-D-24-57432_R1.pdf

Dear Dr. Brandl,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Sep 13 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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Petr Heneberg

Academic Editor

PLOS ONE

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Additional Editor Comments:

The manuscript has been substantially improved after the previous round of revisions and now covers most of the points that were previously raised. However, there are still a number of issues that should be addressed before it is ready for publication. These remaining points concern interpretation, precision of wording and clarity of figures and data presentation rather than additional data collection.

The conclusions still extend beyond what the data can support. Several sections of the abstract and discussion state or imply that wet-nesting results in higher bacterial loads and that this has consequences for embryonic mortality. This study is observational and does not test cause and effect or hatching outcomes. These implications should be presented as hypotheses for future research rather than results supported by the current data. In particular, the discussion around lines 443 to 444 and the associated summary sentences in the abstract need to be reworded to reduce the strength of the claims.

The categorization of wet-nesting versus dry-nesting species is too broad. This point was acknowledged but the language in the results and discussion still reads as if general conclusions across these categories can be made. The sample of wet-nesting species is restricted to two closely related species and the dry-nesting group is heterogeneous in water contact and nest materials. Each time the results are generalized across these categories, an explicit caveat about these limitations should be included.

The possibility that phylogeny influences bacterial clustering has not been addressed with sufficient emphasis. Both wet-nesting species belong to the same family. Clustering patterns could therefore reflect shared evolutionary history rather than nest characteristics. The discussion should explicitly highlight that these factors cannot be separated in the present dataset and that the effect of nesting strategy should be interpreted with caution.

The clarity of the figures remains an issue. Figure 2 in particular is difficult to interpret due to overlapping symbols. Although the legend provides color codes, the visual separation of species is poor. Symbols could be made partially transparent, larger, or shown in separate panels for wet and dry nesters. The figure caption should also report the stress values for the multidimensional scaling. These were previously requested but have not been implemented.

The description of patterns in bacterial similarity between eggs and water could be misinterpreted. The text attributes differences mainly to ecological filtering, yet differences in the sampling methods and the inherent biases of ARISA are also plausible explanations. These limitations should be clearly distinguished from ecological interpretations.

The study relies on ARISA and culture-based methods, and while this is acceptable for exploratory work, these approaches provide low resolution. The methods section and the discussion mention these limitations, but they should be restated more clearly in the abstract and conclusion. This would help to place the findings in the context of recent research that uses higher resolution molecular approaches.

Finally, one of the earlier reviewer requests to visually separate ducks from other dry-nesting species in the figures was declined by the authors. If this decision is retained, the text should guide the reader through the interpretation of symbol codes more explicitly so that the influence of ducks on clustering patterns can be evaluated by readers.

In summary, the manuscript contributes useful new data on eggshell-associated bacteria in wetland birds. It now acknowledges many of the limitations of the study but still needs further softening of causal language, an explicit discussion of phylogenetic influences, clearer presentation of figures and more careful integration of limitations into the conclusions. Addressing these points will strengthen the work and ensure that the results are interpreted within the constraints of the study design.

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Bacterial assemblages on eggs reflect nesting strategies in wetland-associated birds

PONE-D-24-57432R3

Dear Dr. Brandl,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Petr Heneberg

Academic Editor

PLOS ONE

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