https://doi.org/10.5061/dryad.m0cfxppbt

These files are used for the analyses and data filtering of the microbiota sequence data analyzed in the paper. The raw sequence data associated with these files have been deposited in the Sequence Read Archive (BioProject ID: PRJNA578383, Accession numbers in the file: "Barn_owl_nestling_paper_SRA_Accession_numbers.xlsx"). The script "Owl microbiome data filtering.R" deals with filtering the sequence data. The other scripts are for analyzing the resulting filtered data and constructing figures.

Description of the data and file structure

File name: Ta_Israel_microbiome_run3_6-30-20_and_Run2_owls_not_in_run3_updated_1-26-23_combined_R1_and_R2_seqtab.rds

This is a table of amplicon sequence variants saved as an R object file. The pipeline of Callahan et al (2016a) was used to process the raw 16S rRNA gene sequence data. 
The initial 10 bases of each read were removed, and DADA2 (Callahan et al. 2016b) was used to infer ASVs from a combined pool of all of the sequence data. Then, paired reads were merged, and chimeric sequences were filtered out. This file is the resulting sequences. This is one of the input files for the script: " Owl microbiome data filtering.R."

File name: Ta_Israel_microbiome_run3_6-30-20_and_Run2_owls_not_in_run3_updated_1-26-23_combined_R1_and_R2_taxtab.rds

This is a taxonomy table associated with the amplicon sequence variants saved as an R object file. Sequences were classified with the SILVA 138.1 taxonomy database (Quast et al. 2012; Glöckner et al. 2017). This is one of the input files for the script: " Owl microbiome data filtering.R."

File name: Ta_Israel_microbiome_run3_6-30-20_and_Run2_owls_not_in_run3_updated_1-26-23_combined_R1_and_R2_treeML_GTR_fitGTR.rds

This is phylogeny for the amplicon sequence variants saved as an R object file. The sequences were aligned with DECIPHER (Wright 2015) in order to infer a maximum likelihood phylogeny in phangorn (Schliep 2010). This is the output from: 1) the phangorn function pml, which "computes the likelihood of a phylogenetic tree given a sequence alignment and a model" according to the phangorn manual; and 2) the phangorn function optim.pml which "optimizes the different model parameters" according to the phangorn manual. We specifically used the following R code when running phangorn to give this object: optim.pml(fitGTR, model="GTR", optInv=TRUE, optGamma=TRUErearrangement = "stochastic", control = pml.control(trace = 0)). This is one of the input files for the script: " Owl microbiome data filtering.R"

Filename: Barn_owl_run3_metadata_8-28-23.txt

This is the metadata associated with the barn owls in this study. This is one of the input files for the script: "Owl microbiome data filtering.R." The 52 columns in the table are as follows.

1. File_order = a field to order the data file.

2. Sample_name_genetic_data = the sample name associated with the DNA sequence data

3. SampleID = the sample name associated with the owl

4. Sequencing_run = the numerical code for the set of samples that were run together on the same lane of sequencing. Most samples in this dataset were on sequencing run 3, but a few of them were sequenced at an earlier date on sequencing run 2.

5. Negative_control = a sample that was included to detect contaminants during the DNA extraction or library prep process.

6. Redo_pair_of_samples = this field identifies samples that were sequenced twice, with the first one being the “original” and the second one being the “redo.”

7. Remove = samples that a priori needed to be removed from the analyses of the owls (i.e. negative and positive control samples).

8. Oral_swab = whether the sample was an oral swab. Yes = an oral swab, no = a cloacal swab.

9. PCR_concentration = The V4 region of the 16S rRNA gene was amplified by PCR, and this is the concentration of the PCR product.

10. Library_run_number = the numerical code for the set of samples that underwent PCR and library prep together.

11. Duplicate = whether the sample was a duplicate with another sample in the dataset.

12. Date_extracted = the date of the DNA extraction for the sample.

13. Adult_Nestling = whether the bird was an adult owl or a nestling owl.

14. Sex = the sex of the bird with “M” = male and “F” = female

15. Band_number = the unique band number assigned to the bird

16. Recap_filter_female_to_match_offspring_sampling_time_updated = This field is to identify and remove duplicate adult female samples. The goal was to keep adult female samples that were collected at a similar time as the nestling samples. However, the offspring were sampled twice, so sometimes the best sample for the adult female differed. “Remove" = a sample to remove and “keep” = a sample to keep. The adult female sample that best matched the sampling of the young nestlings is demarcated as “young” and the sample that best matched the sampling of the old nestlings is demarcated as “old.”

17. Date = the calendar date at which the nestling was swabbed to collect a sample of its microbiota.

18. Julian_Swab_Date = the number of days after the first day of the year at which the nestling was swabbed to collect a sample of its microbiota.

19. box = The code for the nest box associated with the individual, which is the nest box used by the adult owls for reproduction and the natal nest box for the nestlings.

20. X_UTM = The UTM X coordinate for the nest box.

21. Y_UTM = The UTM Y coordinate for the nest box.

22. Mass = the mass of the bird when it was sampled in grams.

23. laying_date = the date on which the first egg was laid in the nest box.

24. clutch_size = the number of eggs laid in the nest box.

25. N_eggs_not_hatching = the number of eggs that did not hatch

26. Brood_size_at_hatching = the number of nestlings at the time of hatching.

27. brood_size_53days_old = the number of nestlings that survived to 53 days of age.

28. Nestling_rank = the order at which nestlings hatched within a clutch.

29. Nestling_age = the nestling age at which it was sampled.

30. Nestling_age_group = the nestling age group, with “young” being for nestlings before they began to develop adult feathers and “old” being for nestlings before they fledged and began to leave the nest. The young nestlings were 26–34 days of age (mean = 30.27, standard deviation = 2.56) and the old nestlings were 49–56 days of age (mean = 53.11, standard deviation = 1.81).

31. All_age_group = the same as the Nestling_age_group field except that a category for adults is included.

32. Crossfoster_nestling = a “yes” in this column designates a nestling involved in the crossfostering experiment, whether as a control or crossfoster nestling.

33. Experiment = the experimental treatment assigned to the nestlings, which was to be in an

unmanipulated nest, a crossfostered nestling moved to a new nest, or a control nestling that stayed in its nest while some of its siblings were moved to a new nest.

34. First_box = The code of the nestbox that the nestling initially came from.

35. Second_box = The code of the nestbox after the cross-fostering experiment. It may or may not be the same as the first_box, depending on whether the individual was switched to a new nest.

36. Experimental_group = The codes of the two nestboxes that were paired for the crossfostering experiment. Codes are just given for the nestlings in this field.

37. Experimental_group_with_adults group = The codes of the two nestboxes that were paired for the crossfostering experiment. Codes are just given for both the nestlings and adults in this field.

38. Nestbox_numerical = A simplified numerical code of the nestbox from that sampling time point.

39. Female_at_Date = the band identification number of the female at the nest when sampling occurred.

40. Natal_F = the band identification number of the biological mother of the nestling

41. Male_at_date = the band identification number of the male at the nest when sampling occurred.

42. Natal_M = the band identification number of the biological father of the nestling

43. Crossfoster_F = the band identification number of the female at the nest after the crossfostering treatment. Therefore, this female may or may not be biologically related to the offspring at the nest because it was a partial cross-fostering experiment.

44. Crossfoster_M = the band identification number of the male at the nest after the crossfostering treatment. Therefore, this male may or may not be biologically related to the offspring at the nest because it was a partial cross-fostering experiment.

45. median_dist_14days = For each night, median displacement in meters was calculated for each individual from the straight-line distances between each ATLAS localization and the owl's nest location. Then these nightly values were average over the two week period.

46. Initial_Date_14days = the initial calendar date at which movement data was collected.

47. Last_Date_14days = the final calendar date at which movement data was collected.

48. PercAtBox_14days = The percentage of time at the nest box was calculated by dividing the number of observations in which the owl was within a 100 m radius of the nest by the total number of observations. We summarized the movement data during a 13-day window that began the day after the capture of the adult owl to prevent any effect of capture on subsequent movement behavior from impacting the results. 

49. Effective_days_recap1_14days = This column gives the effective number of days of movement data for a 13 day window after the day the owl was captured. When summarizing the movement data, we set a minimum threshold level of data per day by excluding any days with less than 20% of the expected number of localizations.

50. median_dist_7days = For each night, median displacement in meters was calculated for each individual from the straight-line distances between each ATLAS localization and the owl's nest location. Then these nightly values were average over the one week period.

51. PercAtBox_7days = The percentage of time at the nest box was calculated by dividing the number of observations in which the owl was within a 100 m radius of the nest by the total number of observations. We summarized the movement data during a 6-day window that began the day after the capture of the adult owl to prevent any effect of capture on subsequent movement behavior from impacting the results. 

52. ndays_7days == This column gives the effective number of days of movement data for a 6 day window after the day the owl was captured. When summarizing the movement data, we set a minimum threshold level of data per day by excluding any days with less than 20% of the expected number of localizations.

File name: Ta_run3_6-30-20_and_Run2_owls_not_in_run3_updated_1-26-23_combined_R1_and_R2_9900reads.rarefied_decontam_prevelance_threshold0.5_updated_8-28-23

This file serves as the input for all the analyses and figures in the paper. It is an R object file in phyloseq format, so the phyloseq R-package (McMurdie and Holmes 2013) should be activated in R before reading in the file. This file contains the ASV table, the taxonomic information, the phylogeny, and metadata about the samples for subsequent analyses of the data. It is the output of the "Owl microbiome data filtering.R" script that is also included here. Thus, it is the microbiota data that has gone through filtering steps such as restricting the taxa to only Bacteria, removing sequences likely to be contaminants, and standardizing the number of reads per sample to 9900 reads. It is the input file for the data analysis scripts.

Filename: distance.to.parents.tests.combined.old.nestling.data.3-1-24.txt

This is a data table of metrics of the distances between the microbial communities of nestlings and their parents. It is the output of data processing performed in the script: "Fig 6, S6 Owl microbiota distance between nestlings and parents tests.R." Two of the distance metrics (unweighted UniFrac and weighted UniFrac) change slightly with different runs of the script, so this file is included to be able to exactly reproduce the results in the paper. It is the input file into two scripts:  "Fig 6, S6 Owl microbiota distance between nestlings and parents tests.R." and " Owl microbiota distance to parents with date filter.R" The table is used to assess how different the microbiomes of nestlings (at the old sampling time point) are to their mothers versus their fathers (e.g. Fig. 6B in the paper). The 12 columns in the table are as follows.

1. band.id = the unique band number assigned to the nestlings

2. sex = the sex of the nestlings with “M” = male and “F” = female

3. experiment = the experimental treatment assigned to the nestlings, which was to be in an

unmanipulated nest, a crossfostered nestling moved to a new nest, or a control nestling that stayed in its nest while some of its siblings were moved to a new nest.

4. mass.old = the mass of the nestling when it was sampled at the old time point

5. Natal_F = the band identification number of the biological mother of the nestling

6. Natal_M = the band identification number of the biological father of the nestling

7. jaccard.distance = the distance between the nestling and parents microbiota as measured by the Jaccard metric  

8. bray.distance = the distance between the nestling and parents microbiota as measured by the Bray-Curtis metric  

9. unifrac.distance = the distance between the nestling and parents microbiota as measured by the unweighted UniFrac metric 

10. wunifrac.distance = the distance between the nestling and parents microbiota as measured by the weighted UniFrac metric

11. parent = which parent the nestlings microbiome was being compared to, either its mother or father.

12. nestling.parent.swab.date.difference = the number of days in which the nestling was swabbed (at the old time point) after its parent had been swabbed to collect a sample of its microbiota.

Filename: distance.to.parents.tests.old.new.data.3-1-24.txt

This is a data table of metrics of the distances between the microbial communities of nestlings and their parents. It is the output of data processing performed in the script: "Fig 6, S6 Owl microbiota distance between nestlings and parents tests.R." Two of the distance metrics (unweighted UniFrac and weighted UniFrac) change slightly with different runs of the script, so this file is included to be able to exactly reproduce the results in the paper. It is the imput file into two scripts: "Fig 6, S6 Owl microbiota distance between nestlings and parents tests.R." and " Owl microbiota distance to parents with date filter.R" The table is used to assess how different the microbiomes of nestlings (at the old sampling time point) are to their mothers and their fathers (e.g. Fig. 6C and Fig S6C-D in the paper). The 30 columns in the table are as follows.

1. band.id = the unique band number assigned to the nestlings

2. sex = the sex of the nestlings with “M” = male and “F” = female

3. experiment = the experimental treatment assigned to the nestlings, which was to be in an

unmanipulated nest, a crossfostered nestling moved to a new nest, or a control nestling that stayed in its nest while some of its siblings were moved to a new nest.

4. mass.old = the mass of the nestling when it was sampled at the old time point

5. Julian_Swab_Date = the number of days after the first day of the year at which the nestling was swabbed to collect a sample of its microbiota.

6. Natal_F = the band identification number of the biological mother of the nestling

7. Natal_F_mom_info = the band identification number of the biological mother associated with the nestling data. This was recorded to ensure that it matched the Natal_F column.

8. Natal_M = the band identification number of the biological father of the nestling

9. Natal_M_dad_info = the band identification number of the biological father associated with the nestling data. This was recorded to ensure that it matched the Natal_M column.

10. jaccard.old.father.distance = the distance between the nestling and its father’s microbiota as measured by the Jaccard metric

11. bray.old.father.distance = the distance between the nestling and its father’s microbiota as measured by the Bray-Curtis metric

12. unifrac.old.father.distance = the distance between the nestling and its father’s microbiota as measured by the unweighted UniFrac metric 

13. wunifrac.old.father.distance = the distance between the nestling and its father’s microbiota as measured by the weighted UniFrac metric

14. jaccard.old.mother.distance = the distance between the nestling and its mother’s microbiota as measured by the Jaccard metric

15. bray.old.mother.distance = the distance between the nestling and its mother’s microbiota as measured by the Bray-Curtis metric

16. unifrac.old.mother.distance = the distance between the nestling and its mother’s microbiota as measured by the unweighted UniFrac metric  

17. wunifrac.old.mother.distance = the distance between the nestling and its mother’s microbiota as measured by the weighted UniFrac metric

18. father.median_dist_14days = For each night, median displacement in meters was calculated for each individual from the straight-line distances between each ATLAS localization and the owl's nest location. Then these nightly values were average over the two week period. This is the data for the father at the nest.

19. father.PercAtBox_14days = The percentage of time at the nest box was calculated by dividing the number of observations in which the owl was within a 100 m radius of the nest by the total number of observations. We summarized the movement data during a 13-day window that began the day after the capture of the adult owl to prevent any effect of capture on subsequent movement behavior from impacting the results. This is the percentage of time the father was at the nest box.

20. father.Effective_days_recap1_14days = This column gives the effective number of days of movement data for the father in the 13 day window after the day the father was captured. When summarizing the movement data, we set a minimum threshold level of data per day by excluding any days with less than 20% of the expected number of localizations.

21. father.median_dist_7days = For each night, median displacement in meters was calculated for each individual from the straight-line distances between each ATLAS localization and the owl's nest location. Then these nightly values were average over the one week period. This is the data for the father at the nest.

22. father.PercAtBox_7days = The percentage of time the father was at the nest box was calculated by dividing the number of observations in which the owl was within a 100 m radius of the nest by the total number of observations. We summarized the movement data during a 6-day window that began the day after the capture of the adult owl to prevent any effect of capture on subsequent movement behavior from impacting the results. 

23. father.Effective_days_recap1_7days = This column gives the effective number of days of movement data for a 6 day window after the day the owl was captured. When summarizing the movement data, we set a minimum threshold level of data per day by excluding any days with less than 20% of the expected number of localizations. This is the data for the father at the nest.

24. father.swab.date = the number of days after the first day of the year at which the father was swabbed to collect a sample of its microbiota.

25. mother.median_dist_14days = For each night, median displacement in meters was calculated for each individual from the straight-line distances between each ATLAS localization and the owl's nest location. Then these nightly values were average over the two week period. This is the data for the mother at the nest.

26. mother.PercAtBox_14days = The percentage of time at the nest box was calculated by dividing the number of observations in which the owl was within a 100 m radius of the nest by the total number of observations. We summarized the movement data during a 13-day window that began the day after the capture of the adult owl to prevent any effect of capture on subsequent movement behavior from impacting the results. This is the percentage of time the mother was at the nest box.

27. mother.Effective_days_recap1_14days = This column gives the effective number of days of movement data for the father in the 13 day window after the day the mother was captured. When summarizing the movement data, we set a minimum threshold level of data per day by excluding any days with less than 20% of the expected number of localizations.

28. mother.swab.date = the number of days after the first day of the year at which the mother was swabbed to collect a sample of its microbiota.

29. nestling.mother.swab.date.difference = the number of days in which the nestling was swabbed (at the old time point) after its mother had been swabbed to collect a sample of its microbiota.

30. nestling.father.swab.date.difference = the number of days in which the nestling was swabbed (at the old time point) after its father had been swabbed to collect a sample of its microbiota.

Literature Cited

Callahan, B. J., K. Sankaran, J. A. Fukuyama, P. J. McMurdie, and S. P. Holmes. 2016a. Bioconductor workflow for microbiome data analysis: from raw reads to community analyses. F1000Res 5:1492.

Callahan, B. J., P. J. McMurdie, M. J. Rosen, A. W. Han, A. J. A. Johnson, and S. P. Holmes. 2016b. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods 13:581–583.

Glöckner, F. O., P. Yilmaz, C. Quast, J. Gerken, A. Beccati, A. Ciuprina, G. Bruns, P. Yarza, J. Peplies, R. Westram, and W. Ludwig. 2017. 25 years of serving the community with ribosomal RNA gene reference databases and tools. Journal of Biotechnology 261:169–176. Elsevier.

McMurdie, P. J., and S. Holmes. 2013. phyloseq: An R Package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE 8:1–11.

Quast, C., E. Pruesse, P. Yilmaz, J. Gerken, T. Schweer, P. Yarza, J. Peplies, and F. O. Glöckner. 2012. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41:D590–D596.

Schliep, K. P. 2010. phangorn: phylogenetic analysis in R. Bioinformatics 27:592–593.

Wright, E. S. 2015. DECIPHER: Harnessing local sequence context to improve protein multiple sequence alignment. BMC Bioinformatics 16:2–14. BioMed Central.

Sharing/Access information

Links to other publicly accessible locations of the data:

https://www.ncbi.nlm.nih.gov/sra/PRJNA578383

Code/Software

The script "Owl_microbiome_data_filtering.R" deals with filtering the sequence data. It filters out contaminant sequence variant, excludes mitochondria, chloroplast and non-bacterial sequences, and rarefies the data to 9912 reads. The input files are: "Barn_owl_run3_metadata_8-28-23.txt", "Ta_Israel_microbiome_run3_6-30-20_and_Run2_owls_not_in_run3_updated_1-26-23_combined_R1_and_R2_taxtab.rds", "Ta_Israel_microbiome_run3_6-30-20_and_Run2_owls_not_in_run3_updated_1-26-23_combined_R1_and_R2_seqtab.rds"Ta_Israel_microbiome_run3_6-30-20_and_Run2_owls_not_in_run3_updated_1-26-23_combined_R1_and_R2_treeML_GTR_fitGTR.rds". The output file of the script is: "Ta_run3_6-30-20_and_Run2_owls_not_in_run3_updated_1-26-23_combined_R1_and_R2_9900reads.rarefied_decontam_prevelance_threshold0.5_updated_8-28-23" 

The following files are all R-scripts for analyzing the data in the paper and for generating the figures and tables. The file names reflect the figures or tables that they generated, with a prefix of "S" for supplementary figures and tables. A general description of the analyses conducted within the scripts is also provided in the file name. In general, the input into the scripts is the file: " Ta_run3_6-30-20_and_Run2_owls_not_in_run3_updated_1-26-23_combined_R1_and_R2_9900reads.rarefied_decontam_prevelance_threshold0.5_updated_8-28-23.rds". However, two of the scripts ("Fig 6, S6 Owl microbiota distance between nestlings and parents tests.R." and " Owl microbiota distance to parents with date filter.R") read in two additional data files in order to exactly reproduce the results reported in the paper (they read in "distance.to.parents.tests.old.new.data.3-1-24.txt" and " distance.to.parents.tests.combined.old.nestling.data.3-1-24.txt"). These extra files are necessary because two of the distance metrics (unweighted UniFrac and weighted UniFrac) change slightly with different runs of the script.

Fig_2_A-C__Fig_S3A_Owl_microbiota_alpha_diversity_sex_and_developmental_effects.R

Fig_2_D_E_F__Fig_S3C_Owl_microbiota_sex_and_developmental_differences_PERMANOVA.R

Fig_3__6A_Owl_microbiota_Alpha_diversity_analyses_8-29-23.R

Fig_4_and_S4_Owl_microbiota_ordination_7-3-23.R

Fig_5_Owl_microbiota_ordination_plots_by_nest_and_old_vs_young_no_labels.R

Fig_6__S6_Owl_microbiota_distance_between_nestlings_and_parents_tests.R

Fig_S1_S2_Owl_microbiota_oral_and_cloacal_ordination_and_barplots.R

Fig_S3A-C_Owl_microbiota_alpha_diversity_analyses_oral_swabs.R

Fig_S5_Owl_microbiota_unmanipulated_owl_nestlings_ordination.R

Owl_microbiota_distance_to_parents_with_date_filter.R

Table_1_and_S1_Owl_microbiota_Permanova_tests_across_groups_of_nests.R