Spatial heterogeneity of the shorebird gastrointestinal microbiome

The gastrointestinal tract (GIT) consists of connected structures that vary in function and physiology, and different GIT sections potentially provide different habitats for microorganisms. Birds possess unique GIT structures, including the oesophagus, proventriculus, gizzard, small intestine, caeca and large intestine. To understand birds as hosts of microbial ecosystems, we characterized the microbial communities in six sections of the GIT of two shorebird species, the Dunlin and Semipalmated Sandpiper, identified potential host species effects on the GIT microbiome and used microbial source tracking to determine microbial origin throughout the GIT. The upper three GIT sections had higher alpha diversity and genus richness compared to the lower sections, and microbial communities in the upper GIT showed no clustering. The proventriculus and gizzard microbiomes primarily originated from upstream sections, while the majority of the large intestine microbiome originated from the caeca. The heterogeneity of the GIT sections shown in our study urges caution in equating data from faeces or a single GIT component to the entire GIT microbiome but confirms that ecologically similar species may share many attributes in GIT microbiomes.

refer to the microbiota of the large intestine. Meaning that researchers sample feces in order to draw conclusions about the large intestinal microbiome, but the typical word used is often gut microbiome. I don't think anyone believes that the bacterial community will be similar throughout the entire GIT (especially since this term includes everything from the bill to the cloaca). It has also been shown in previous studies that this is indeed the case. I think the authors are correct in stating that the GIT sections are different (which they have evaluated) and that fecal samples do not portray everything in the GIT (which they have not evaluated). But I also want to urge the authors that it might be useful to be slightly careful with this wording, because no one seriously believes you will get an accurate picture of the esophagus microbiome by sampling feces. So the point of feces not representing the entire GIT community becomes a bit meaningless. The point of fecal sampling is not to measure the entire GIT but to evaluate the large intestine non-invasively. • L50: The cited paper has not studied the gizzard microbial community or it's pH. Please revise. • L53: I think "decades" is a bit exaggerated. Probably "years" would fit better. Mammalian microbiome research in its current form is also relatively new. • Methods sampling: It is my understanding that one needs a permit for trapping birds and another permit for collecting birds. Feel free to correct me if I'm wrong. Even if the birds used in this study were accidentally killed during the trapping procedure, don't the authors agree it would be appropriate to state the permit or licenses used for trapping and collecting, since this allowed the authors (or collaborators) to catch the birds in the first place? • L105: As far as I'm aware, there is no MiSeq v4 kit. There are v2 and v3. Probably just a typo.
• L126: Dada2, vegan, phyloseq, DeSeq2, FEAST versions have not been specified. Since these kinds of software often make substantial changes between versions, it would be good to state version used for reproducibility reasons. • L170 Data availability: Royal Society Data Policy states that "Datasets and code should be deposited in an appropriate, recognized, publicly available repository. Where no data-specific repository exists, authors should deposit their datasets in a general repository such as Dryad or Figshare." First of all, I cannot find the sequences or the metatable of this study in the provided Figshare link. Regardless, I believe the 16S sequences in this study should be deposited in appropriate sequence databases such as SRA or ENA or DDBJ to allow for future re-analyses and meta-analyses. Figshare is not an appropriate repository for open sequence data. The metatable can be store on Figshare (in addition to the sequence repository). • Table 1: Misspellings. Please check.
• Table 1: Curious as to why only p-values are provided in Table 1? Where are the effect sizes? pvalues only tell the reader whether the test was significant or not at an arbitrary threshold. As a reader you want to see the results of the analysis. Is the esophagus more diverse than the gizzard? That's not possible to tell from p-values. Please add to the table diversity values, so the reader will at least know which direction the difference is. Consider also stats from the ANOVA test.
• Figure 2: I like the colors used. They are easy to tell apart. • L252: The word microbiome is used but I think the authors mean gastro-intestinal tract. • L284: "Decreased alpha diversity and community complexity in the lower GIT could be the result of host filtering of bacteria in the upper GI sections." Can the authors please explain this further how they mean? If the host kills certain bacteria in the upper GI sections with pH, the dead bacteria would still be present in the lower gut community as well due to the downward flow of content. This also does not explain why the diversity of bacteria is higher upper in the gut? Do the authors mean that the higher diversity in the upper gut is most likely derived from the diet and environmentally sourced bacteria? • Discussion: I don't know the word limit of this journal but if possible, I would love to read a little more extended discussion. The authors very briefly touch upon some of the interesting results they found, but there is very little integration on what it means and any comparisons with previous studies. There are a lot of similar studies that have been conducted in grouse, ostriches and sparrows for example. • Overall, I think the manuscript is well-written and easy to read.

22-Oct-2019
Dear Dr Grond, On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-191609 entitled "Spatial Heterogeneity of the Shorebird Gut Microbiome" 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.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Please see below the comments and suggested MINOR revisions made by the individual(s) who reviewed your manuscript. I would like to take your attention to one critical issue raised by the reviewers: 1. The link provided in the manuscript (10.6084/m9.figshare.9792668) is not valid, thus data provided in the link is not accessible. Please provide a valid link or update the link so the data can be easily accessed.
Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) Major comments: Overall this paper is well organized, the methods and analyses are sound, and the results are interesting. I think it will be an important contribution to our field.
My only major concerns are as follows: 1) Some of the statistical analyses would benefit from adjustments to control for repeated measures from a single individual 2) Data does not seem to be available at provided link, and no link is provided for a script that would allow people to replicate your analyses ABSTRACT 23 -alpha and genus diversity? This reads strangely; maybe taxonomic and alpha diversity, or rephrase another way. 26 -The language of "sourcing" and "originating" without the context of your cool analyses is confusing. In the abstract before reading the paper it seems as though it refers to your sampling. Consider add a sentence about the method, or use different language for the abstract.
INTRO 54 -"our knowledge of the diversity and distribution of microbes in the GIT of most bird species" 63 -can you estimate the age of the birds? or just specify juvenile or adult.
METHODS 81 -what kind of string was used? Overall, great job on this section. All of it was very clear and tight.

RESULTS
179 -Did you use a repeated measures ANOVA to compare samples only within individuals? As your samples show considerable variation among individuals, it may be beneficial to account for this statistically. 180 -sentence order is off, I think you meant to have "Shannon's alpha diversity" and "host species" switched 248 -"small intestine" does not need to be capitalized 345 -"have been shown to show" consider rephrasing for less redundancy. Also, consider adding citations to this sentence. DISCUSSION 312 -typo : "have share" FIGURES fig. 1 -consider adding full sample type name, vertically or at an angle on the axis for ease of interpretation table 1 -typo -"proventriculus" is missing a "t" Reviewer: 2 Comments to the Author(s) Grond et al. have evaluated the microbiome of six sections of the GIT in six individuals of two species of shorebirds. This is a good study and I enjoyed reading it. It is useful for the avian microbiome community. It is well written with adequate amount of information in the methods and the results. The figures are nice and well presented. The analyses are appropriate. I don't have any major comments but I hope that my minor comments will be useful to the authors during the revision of the paper.
Minor comments: • Abstract and discussion: In general, I believe the term "gut microbiome" is most often used to refer to the microbiota of the large intestine. Meaning that researchers sample feces in order to draw conclusions about the large intestinal microbiome, but the typical word used is often gut microbiome. I don't think anyone believes that the bacterial community will be similar throughout the entire GIT (especially since this term includes everything from the bill to the cloaca). It has also been shown in previous studies that this is indeed the case. I think the authors are correct in stating that the GIT sections are different (which they have evaluated) and that fecal samples do not portray everything in the GIT (which they have not evaluated). But I also want to urge the authors that it might be useful to be slightly careful with this wording, because no one seriously believes you will get an accurate picture of the esophagus microbiome by sampling feces. So the point of feces not representing the entire GIT community becomes a bit meaningless. The point of fecal sampling is not to measure the entire GIT but to evaluate the large intestine non-invasively. • L50: The cited paper has not studied the gizzard microbial community or it's pH. Please revise. • L53: I think "decades" is a bit exaggerated. Probably "years" would fit better. Mammalian microbiome research in its current form is also relatively new. • Methods sampling: It is my understanding that one needs a permit for trapping birds and another permit for collecting birds. Feel free to correct me if I'm wrong. Even if the birds used in this study were accidentally killed during the trapping procedure, don't the authors agree it would be appropriate to state the permit or licenses used for trapping and collecting, since this allowed the authors (or collaborators) to catch the birds in the first place? • L105: As far as I'm aware, there is no MiSeq v4 kit. There are v2 and v3. Probably just a typo. • L126: Dada2, vegan, phyloseq, DeSeq2, FEAST versions have not been specified. Since these kinds of software often make substantial changes between versions, it would be good to state version used for reproducibility reasons. • L170 Data availability: Royal Society Data Policy states that "Datasets and code should be deposited in an appropriate, recognized, publicly available repository. Where no data-specific repository exists, authors should deposit their datasets in a general repository such as Dryad or Figshare." First of all, I cannot find the sequences or the metatable of this study in the provided Figshare link. Regardless, I believe the 16S sequences in this study should be deposited in appropriate sequence databases such as SRA or ENA or DDBJ to allow for future re-analyses and meta-analyses. Figshare is not an appropriate repository for open sequence data. The metatable can be store on Figshare (in addition to the sequence repository). • Table 1: Misspellings. Please check.
• Table 1: Curious as to why only p-values are provided in Table 1? Where are the effect sizes? pvalues only tell the reader whether the test was significant or not at an arbitrary threshold. As a reader you want to see the results of the analysis. Is the esophagus more diverse than the gizzard? That's not possible to tell from p-values. Please add to the table diversity values, so the reader will at least know which direction the difference is. Consider also stats from the ANOVA test.
• Figure 2: I like the colors used. They are easy to tell apart. • L252: The word microbiome is used but I think the authors mean gastro-intestinal tract. • L284: "Decreased alpha diversity and community complexity in the lower GIT could be the result of host filtering of bacteria in the upper GI sections." Can the authors please explain this further how they mean? If the host kills certain bacteria in the upper GI sections with pH, the dead bacteria would still be present in the lower gut community as well due to the downward flow of content. This also does not explain why the diversity of bacteria is higher upper in the gut? Do the authors mean that the higher diversity in the upper gut is most likely derived from the diet and environmentally sourced bacteria? • Discussion: I don't know the word limit of this journal but if possible, I would love to read a little more extended discussion. The authors very briefly touch upon some of the interesting results they found, but there is very little integration on what it means and any comparisons with previous studies. There are a lot of similar studies that have been conducted in grouse, ostriches and sparrows for example. • Overall, I think the manuscript is well-written and easy to read.

11-Nov-2019
Dear Dr Grond, It is a pleasure to accept your manuscript entitled "Spatial Heterogeneity of the Shorebird Gastrointestinal Microbiome" in its current form for publication in Royal Society Open Science.
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Dear editors,
Below we addressed the comments of Dr. Tezel and the two reviewers. We hope we have adequately addressed their concerns, and have modified our manuscript to be satisfactory for publication. We thank Dr. Tezel and the reviewers for their constructive comments and compliments. Sincerely,

Kirsten Grond
• Ethics statement If your study uses humans or animals please include details of the ethical approval received, including the name of the committee that granted approval. For human studies please also detail whether informed consent was obtained. For field studies on animals please include details of all permissions, licences and/or approvals granted to carry out the fieldwork. >> We added an ethics section at the end of the manuscript with all permits and approvals.
• Data accessibility It is a condition of publication that all supporting data are made available either as supplementary information or preferably in a suitable permanent repository. The data accessibility section should state where the article's supporting data can be accessed. This section should also include details, where possible of where to access other relevant research materials such as statistical tools, protocols, software etc can be accessed. If the data has been deposited in an external repository this section should list the database, accession number and link to the DOI for all data from the article that has been made publicly available. Data sets that have been deposited in an external repository and have a DOI should also be appropriately cited in the manuscript and included in the reference list. >> the data accessibility section is updated Associate Editor Comments to Author (Dr Ulas Tezel): Dear Dr. Kirsten Grond: Please see below the comments and suggested MINOR revisions made by the individual(s) who reviewed your manuscript. I would like to take your attention to one critical issue raised by the reviewers: 1. The link provided in the manuscript (10.6084/m9.figshare.9792668) is not valid, thus data provided in the link is not accessible. Please provide a valid link or update the link so the data can be easily accessed. >> Our apologies for the broken link. We updated the link for figshare, and added the SRA BioProject number for NCBI.
METHODS 81 -what kind of string was used? >> we used regular cotton string, and did not use the tissue and content directly next to the string. We added this to the methods.
Overall, great job on this section. All of it was very clear and tight.

RESULTS
179 -Did you use a repeated measures ANOVA to compare samples only within individuals? As your samples show considerable variation among individuals, it may be beneficial to account for this statistically. >> see above 180 -sentence order is off, I think you meant to have "Shannon's alpha diversity" and "host species" switched >> We thank the reviewer for noticing this mistake, and have rewritten the sentence.
248 -"small intestine" does not need to be capitalized >> We removed the capitalization of small intestine. 345 -"have been shown to show" consider rephrasing for less redundancy. Also, consider adding citations to this sentence. >> We replaced 'have been shown to show' with 'show', and added a citation by Videlvall and al 2018.
DISCUSSION 312 -typo : "have share" >> We removed 'have' in this sentence. FIGURES fig. 1 -consider adding full sample type name, vertically or at an angle on the axis for ease of interpretation >> We added the full sample type name to figure 1. table 1 -typo -"proventriculus" is missing a "t" >> We corrected this mistake.
Reviewer: 2 Comments to the Author(s) Grond et al. have evaluated the microbiome of six sections of the GIT in six individuals of two species of shorebirds. This is a good study and I enjoyed reading it. It is useful for the avian microbiome community. It is well written with adequate amount of information in the methods and the results. The figures are nice and well presented. The analyses are appropriate. I don't have any major comments but I hope that my minor comments will be useful to the authors during the revision of the paper.
Minor comments: • Abstract and discussion: In general, I believe the term "gut microbiome" is most often used to refer to the microbiota of the large intestine. Meaning that researchers sample feces in order to draw conclusions about the large intestinal microbiome, but the typical word used is often gut microbiome. I don't think anyone believes that the bacterial community will be similar throughout the entire GIT (especially since this term includes everything from the bill to the cloaca). It has also been shown in previous studies that this is indeed the case. I think the authors are correct in stating that the GIT sections are different (which they have evaluated) and that fecal samples do not portray everything in the GIT (which they have not evaluated). But I also want to urge the authors that it might be useful to be slightly careful with this wording, because no one seriously believes you will get an accurate picture of the esophagus microbiome by sampling feces. So the point of feces not representing the entire GIT community becomes a bit meaningless. The point of fecal sampling is not to measure the entire GIT but to evaluate the large intestine non-invasively. >> We have changed gut microbiome to GIT microbiome throughout the paper. Technically, the gut microbiome represents the entire GIT, but with its use in current literature we agree with the author that it could be a confusing term.
Although we agree with the author that the comparison of esophagus and fecal microbiomes are meaningless, we do want to emphasize in our paper that data from feces or single GIT sections do not represent the GIT microbiome. Although gut microbiome is often used interchangeably with the large intestinal microbiome, this is rarely mentioned in publications. I have seen a number of publications equating fecal or even cloacal samples to represent the gut, with no specification of what the gut represents in their paper.
However, I have used fecal sampling in a number of my own studies and I agree it is the only non-invasive method available for evaluating the large intestine microbiome. I added a sentence to the conclusion section to clarify this: "Although fecal samples are unlikely to capture the entire GIT microbiome community and variety, it is often the only non-invasive method available for investigating the microbiome of wild animals. Therefore, we advise authors that use fecal samples to clearly define which microbiome their samples represent." • L50: The cited paper has not studied the gizzard microbial community or it's pH. Please revise. >> Our apologies for the mistake. We changed the sentence to reflect the hypothesis of the authors that the acidic stomach causes the microbial filtering from face to intestine.
• L53: I think "decades" is a bit exaggerated. Probably "years" would fit better. Mammalian microbiome research in its current form is also relatively new. >> We removed 'several decades', which changed the sentence to: "After lagging behind mammalian microbiome research" • Methods sampling: It is my understanding that one needs a permit for trapping birds and another permit for collecting birds. Feel free to correct me if I'm wrong. Even if the birds used in this study were accidentally killed during the trapping procedure, don't the authors agree it would be appropriate to state the permit or licenses used for trapping and collecting, since this allowed the authors (or collaborators) to catch the birds in the first place? >> We agree with Reviewer 2 and we added our permit numbers for trapping and collecting to the new required Ethics section.
• L105: As far as I'm aware, there is no MiSeq v4 kit. There are v2 and v3. Probably just a typo. >> This is indeed a typo, our apologies. We used the v2 kit and changed this in the manuscript.
• L126: Dada2, vegan, phyloseq, DeSeq2, FEAST versions have not been specified. Since these kinds of software often make substantial changes between versions, it would be good to state version used for reproducibility reasons. >> We agree with reviewer 2, and added the version numbers of the programs and packages used, with the exception of FEAST. We mistakenly identified FEAST as a package, when it is custom code described in the citation provided.
• L170 Data availability: Royal Society Data Policy states that "Datasets and code should be deposited in an appropriate, recognized, publicly available repository. Where no data-specific repository exists, authors should deposit their datasets in a general repository such as Dryad or Figshare." First of all, I cannot find the sequences or the metatable of this study in the provided Figshare link. Regardless, I believe the 16S sequences in this study should be deposited in appropriate sequence databases such as SRA or ENA or DDBJ to allow for future re-analyses and meta-analyses. Figshare is not an appropriate repository for open sequence data. The metatable can be store on Figshare (in addition to the sequence repository). >> we updated the figshare link and provided the SRA bioproject number that contains the raw sequences. Our apologies for the broken link.
• Table 1: Curious as to why only p-values are provided in Table 1? Where are the effect sizes? p-values only tell the reader whether the test was significant or not at an arbitrary threshold. As a reader you want to see the results of the analysis. Is the esophagus more diverse than the gizzard? That's not possible to tell from p-values. Please add to the table diversity values, so the reader will at least know which direction the difference is. Consider also stats from the ANOVA test. >> We added the diversity values to the table for clarification. Since we performed a TukeyHSD test we did not have test statistics to add for this.
• Figure 2: I like the colors used. They are easy to tell apart. >> Thank you! • L252: The word microbiome is used but I think the authors mean gastro-intestinal tract.
>> We meant the microbiome in general as a collection of microbial communities, but since our paper focuses on the GIT we realize this causes confusion. We changed microbiome to gastrointestinal tract for clarity.
• L284: "Decreased alpha diversity and community complexity in the lower GIT could be the result of host filtering of bacteria in the upper GI sections." Can the authors please explain this further how they mean? If the host kills certain bacteria in the upper GI sections with pH, the dead bacteria would still be present in the lower gut community as well due to the downward flow of content. >> In humans, nucleic acids were shown to be digested in the stomach with a pH of 1.3-3.5 (Liu et al. 2016. Scientific Reports). Birds have a pH from 1-3 (Beasley et al. 2015. PloS One). We therefore believe that the avian proventriculus and gizzard play a role in host filtering by not only killing bacteria, but also (partially) degrading their DNA. We added this information to our paper.
This also does not explain why the diversity of bacteria is higher upper in the gut? Do the authors mean that the higher diversity in the upper gut is most likely derived from the diet and environmentally sourced bacteria? >> We indeed mean that the upper GIT has a wider microbial exposure to environment and diet, which likely results in the higher alpha diversity. We added a sentence to clarify this.
"Higher alpha diversity in the upper GIT is likely due to the influx of a larger diversity of microorganisms that are associated with environment and diet." • Discussion: I don't know the word limit of this journal but if possible, I would love to read a little more extended discussion. The authors very briefly touch upon some of the interesting results they found, but there is very little integration on what it means and any comparisons with previous studies. There are a lot of similar studies that have been conducted in grouse, ostriches and sparrows for example. >> We have added a couple sections to the discussion further explaining our results(see highlighted sections. We also added data from Videvall et al. concerning ostrich microbiomes, but our comparative ability was limited as they did not investigate microbiomes of the upper GIT. We searched for the grouse and sparrow GIT section publications mentioned by the reviewer but were unable to find any relevant papers unfortunately.

•
Overall, I think the manuscript is well-written and easy to read. >> Thank you!