1. Symbiotic microbes may mediate songbird chemical signals Danielle Whittaker Kevin Theis Photo by Marine Drouilly

2. Symbiotic hypothesis for chemical communication

3. Symbiotic microbes in spotted hyena scent pouches are responsible for odors in scent marks Structure (Bray-Curtis) ANOSIM: R = 0.36, P = 0.002 Fig Tree Southern Comfort Mara River Emarti Hill Bacterial community structure varies with clan membership (Theis et al. 2012)

4. Preen oil is an odor source in birds Oil secreted from the uropygial gland contains volatile compounds that give birds an odor

5. Photo by Danielle Whittaker Volatile compounds in preen oil vary with: Species Sex Population Relatedness Breeding condition Quality …and could play a role in mate choice.

6. Photo by Marine Drouilly Do avian preen glands harbor odor-producing bacteria? Do adults transmit these bacteria to their offspring during the nestling phase?

7. Dark-eyed junco (Junco hyemalis) Mountain Lake Biological Station, VA

8. Dark-eyed Juncos Seasonal breeders Socially monogamous ~30% extra-pair fertilization (EPF) rate Females incubate eggs, both males and females feed nestlings Nestlings fledge at day 12 Photo by Marine Drouilly

9. Photo by Dawn O’Neal Field Methods 13 nests, 64 juncos Swab samples from preen glands: all nestlings (2-4, mean = 2.9) at age 11-12 days both parents at all but 2 nests

10. Photo by Marine Drouilly Extracted DNA from swabs using MO BIO PowerSoil Kits Illumina MiSeq platform, targeting the V4 region of bacterial 16S rRNA gene Processed using mothur Each sample subsampled to 5000 sequences Bacterial sequences clustered based on 97% nucleotide similarity to define OTUs Sequencing Methods

11. Photo by Flickr user Super Bay Bacterial communities in junco preen glands have very high levels of diversity Top 20 OTUs account for ~45% of sequences, with no OTU accounting for more than 5% In hyena scent glands, the top 20 OTUs account for ~90% of sequences, with the top OTU accounting for ~45%

12. Top 20 OTUs in junco preen glands: Burkholderiaceae, Burkholderia Burkholderiaceae, Ralstonia Clostridiaceae, Clostridium Clostridiales Family XI, Incertae Sedis XI, Anaerococcus Comamonadaceae, unknown Enterobacteriaceae, unknown Enterococcaceae, Enterococcus Halomonadaceae, Kushneria Halomonadaceae, Salinicola Kineosporiaceae, Kineococcus Micrococcaceae, unknown Moraxellaceae, Alkanindiges Moraxellaceae, unknown Unknown, unknown Pseudomonadaceae, Pseudomonas Rhodobacteraceae, unknown Sphingomonadaceae, Sphingomonas

13. Other prominent OTUs associated with odor: Corynebacterium: human axillary odor Porphyromonas: malodorous breath Bacteroides, Finegoldia & Fusobacterium: common volatile fatty acid producers associated with many vertebrates

14. Bacterial community structure varied by nest Nests significantly different (70/78 pairwise comparisons) NPMANOVA, all: F = 3.751, P = 0.0001 (Bray-Curtis) Nestlings only: F = 6.946, P = 0.0001 Photo by Nicole Gerlach

15. all individuals, NPMANOVA, F = 3.751, P = 0.0001

16. nestlings only, NPMANOVA, F = 6.946, P = 0.0001

17. Nestlings were more similar to mother than father Wilcoxon’s test, N = 34, W = 564, P < 0.0001

18. Junco preen gland bacterial communities Highly diverse Cluster by nest Nestlings closely resemble each other and mother Reliable transmission across generations via physical contact

19. Acknowledgments Photo by Nicole Gerlach Tracy Teal Arvind Venkataraman Ellen Ketterson Samuel Slowinski