Results

Cowbird versus redwing blackwing redbird cloacal beta diversity

The multivariate dispersion of cowbird cloaca microbial communities was greater than that for the blackwing redbird based on pairwise weighted UniFrac distances between samples (p-value 0.007, "betadisper" function Vegan (Oksanen 2015) (Anderson 2006) ).

Our comparison of cloacal microbiome diversity between cowbirds and blackbirds revealed that while the alpha diversity of the microbial community from individual samples was similar in cowbirds and blackbirds, the beta diversity was significantly higher for the cowbird samples (Fig. 1). This pattern did not appear to be driven by either gender of the individual (male versus female Fig. 2a) or by region of the country where the individual’s range was (MD versus MI, Fig. 2b). 

Comparing paired cowbird gut and cloaca microbial community membership and structure

Cowbird microbial samples generally clustered by environment and not by individual (Figure XX). However, the cloaca community of bird "CB15" clustered with gut communities and the gut community of bird "CB2" clustered within other cloaca communities. Invariably, all cowbird gut communities were dominated by the same OTU ("OTU.2"). OTU.2 belongs to the Enterococcus genus. The closest match for the OTU.2 centroid sequence (XX% shared sequence identity) in the Living Tree Project database is XXX. XXX is a BLAH BLAH BLAH (TODO, classic bird genus?). OTU.2 also matches many sequences in the NCBI nucleotide database with XX% identity from numerous environmental microbiology studies of BLAH BLAH BLAH environments. OTU.2 is not found above XX% relative abundance in any cloacal samples.

Cowbird gut samples were less even than cloaca samples from the same individual (Figure XX). Additionally, cowbird gut samples were less diverse as measured with the Shannon diversity index than cloaca samples (P-value X.XX, paired t-test of Shannon indices). 

Our comparison of gut and cloacal diversity revealed that with the exception of two individuals (CB15 and CB2) the gut and cloacal microbiome communities sorted independently of each other (Fig. 3). For the two individuals that did sort by individual not by sample location, one individual (CB2) did sort together (i.e. cloacal and gut microbiomes had similar membership) while one did not (CB15). Thus, in general the cloacal microbiome was a poor proxy for gut microbiome for the seven cowbirds where we sampled both cloacal and gut microbiomes from the same individual.
We also compared diversity of the cloacal versus the gut microbiome within cowbirds and then among all birds sampled using the Shannon diversity index. The cloacal microbiome had higher variance in alpha diversity and was significantly more diverse than the gut microbiome. This result held when the cowbird was considered alone and when the blackbird cloacal samples were include in the analysis (Fig. 4). 

Cowbird gut microbial communities are less diverse than cloaca microbial diversities