Study 1: Is the cloaca a uniquely specialized region of the gastrointestinal and reproductive tracts?
The Shannon diversity value was lowest in the cloacal tissue, on average, but was not statistically different across tissue samples and cloacal swabs (F = 1.32, df = 4,26, p = 0.425, Fig 1a). Similar non-significant patterns were found when comparing richness (F = 2.07, df = 4,26, p = 0.114; Fig 1b) and phylogenetic diversity across tissue types (F = 1.30, df = 4,26, p = 0.297; Fig 1c). When comparing beta diversity, measured by Bray-Curtis distance, there was a significant difference in composition between all groups (F = 1.52, df = 4,33, p = 0.014), although the groups were dispersed similarly (F = 1.04, df = 4,33, p = 0.400, Fig 2a). When clustered using weighted UniFrac distances, community composition tended to differ across all tissue types (F = 1.87, df = 4,33, p = 0.060, Fig 2b), although the groups were dispersed differently (F = 4.12, df = 4,33, p = 0.008), which could account for the difference in distances. When clustered using unweighted UniFrac distances, community membership differed significantly between tissue types (F = 1.66, df = 4,33, p = 0.008, Fig 2c), and the tissue types showed similar dispersion (F = 1.16, df = 4,33, p = 0.346).
The most abundant family in all tissues types wasEnterobacteriaceae (Figure 3; Online Resource 1). In the cloacal tissue, on average, Enterobacteriaceae made up 82.7 ± 10.8% (SE) of the whole community, while the next most abundant family (Helicobacteraceae ) only made up 16.6 ± 10.9% and was > 0.1% in only 2 individuals. No other families made up more than 1% of the composition of the cloacal tissue. The cloacal swabs and lower intestine showed similar patterns:Enterobacteriaceae made up 69.8 ± 14.2% and 64.7 ± 11.5% respectively, and the next most abundant family in both wasHelicobacteraceae. However, both the cloacal swabs and the lower intestine had several families that made up between 1-5% of the community, including Bacteroidaceae , Ruminococcaceae ,Tannerellaceae , Lachnospiraceae , andDesulfovibrionaceae . The upper intestine had the lowest percent composition of Enterobacteriaceae with only 34.9 ± 9.1%, closely followed by Ruminococcaceae which made up 32.9 ± 12.8% on average, and both Bacteroidaceae and Burkholderiaceae made up ~9% (± 7.5, ± 9.0). The oviduct community was 54.9 ± 9.3% Enterobacteriaceae on average and had many families that were between 1-5%, but no others higher than 10%.
A differential abundance analysis showed that the percentage ofEnterobacteriaceae found in the cloacal tissue microbial community was similar to that in the cloacal swabs (t = -1.05 p = 0.304) and lower intestine (t = 0.156, p = 0.372) communities, and was significantly greater than that in the upper intestine (t = -3.09, p = 0.005) and oviduct (t = -2.06, p = 0.049) communities (Online Resource 1). It also showed that the cloacal tissue community had distinctly low percentages of Ruminococcaceae (cloacal swab: t = 4.39, p < 0.001; lower intestine: t = 4.38, p < 0.001; upper intestine: t = 7.94, p << 0.001; oviduct: t = 2.49, p = 0.019) and Bacteroidaceae (cloacal swab: t = 4.88, p < 0.001; lower intestine: t = 4.88, p < 0.001; upper intestine: t = 5.07, p << 0.001; oviduct: t = 3.59, p = 0.001).