Bacterial community composition and diversity
Across all samples, bacteria from 37 phyla were detected. Firmicutes,
Proteobacteria, Bacteroidetes, and Actinobacteria composed 91.13% of
the total reads; 5.8% of the reads belonged to the 33 remaining phyla
and 3.07% of reads did not align to any known bacterial phyla (Figure
1A). Clostridia (Phylum Firmicutes), Gammaproteobacteria(Phylum Proteobacteria), and Bacteroidia (Phylum Bacteroidetes)
were the most abundant classes, representing 70.16% of all reads. The
mean abundance of most phyla and classes differed between initial
sampling in The Bahamas and subsequent samplings in Michigan (Figure 1B,
Table S2). The birds shifted from a Firmicutes dominated microbiome in
The Bahamas (mean abundance per individual 39.82% [SD, ±13.97%])
and Michigan following arrival (38.12% [SD, ±16.41%]) to
Proteobacteria as the most abundant phylum in the second Michigan
recapture period (47.07% [SD, ±27.90%]). Bacteroidetes and
Actinobacteria were also proportionally more abundant in The Bahamas
than in the second Michigan recapture period. Notably, Cyanobacteria
represented 1.91% (SD, ±5.93%) of the total microbiota in The Bahamas,
but decreased to 0.05% (SD, ±0.23%) by the second recapture period in
Michigan.
Alpha diversity was not significantly affected by year, host age or host
sex (Table S3). However, the three sampling periods significantly
differed (Type III ANOVA with Satterthwaite’s method; Observed richness:
F2,116.34 = 14.76, P <0.0001; Shannon
Diversity: F2,126.91 = 29.22, P<0.0001). All Bonferroni corrected pairwise comparisons on the
fitted values from the linear model were significantly different from
each other (Observed: Bahamas vs. each recapture period both P<0.0001, first vs. second recapture period, P = 0.002;
Shannon Diversity: all comparisons: P <0.0001). Birds
in The Bahamas showed increased bacterial diversity compared to either
recapture period in Michigan, demonstrated through a comparison of all
samples (Figure 2A) as well as with paired sampling of the same
individuals (Figure 2B). In the birds sampled in triplicate, alpha
diversity varied between first and second Michigan recaptures (Figure
2C). A comparison of tagged and randomly captured birds in the first
Michigan sampling period of 2018 revealed no significant differences in
alpha diversity (pairwise t-test; Observed: p = 0.13, Shannon Diversity:
p = 0.22). We observed decreased alpha diversity through the first three
days after arrival in Michigan followed by a slight increase over the
following six days (Figure 3).
Our results indicate that beta diversity was not significantly affected
by age or sex of the birds within the full dataset or individual
sampling periods (Table 2), with the exception of age in the second
Michigan resampling period (unweighted UniFrac: PERMANOVA p = 0.0128,
PERMDISP p = 0.2213). Community composition of the microbiota
significantly differed by year in the full dataset and at each sampling
period (Table 2, Figure 4A). Additionally, our PERMANOVA results suggest
that sampling period exerted a significant effect on the microbiota
composition (Bray-Curtis: p = 0.0002, unweighted UniFrac: p = 0.0001),
though the significant unweighted UniFrac result can be explained
through variation in spread of the sample composition, rather than with
significantly different centroids such as with the Bray-Curtis
dissimilarity matrix (PERMDISP; Bray-Curtis p = 0.7104, unweighted
UniFrac p = 3.71e-6). This indicates that although the abundances of
microbiota are significantly different during sampling periods, the
taxonomic variation of bacterial lineages present are not. The effect of
sampling period on the gut microbiota explained 2.5% and 2.4% of the
variation in microbiota composition for Bray-Curtis and unweighted
UniFrac respectively. Taken together, all variables tested (Sampling
period, Year, Sex, Age) explained less than 5% of the total variation
in the microbiome (Bray-Curtis: 4.91%, unweighted UniFrac: 4.6%). No
consistent changes were observed in the beta diversity of the birds
sampled in triplicate (Figure 4B, Figure S3).
Across the three sampling periods, six ASVs and six genera were
identified by ANCOM as differentially abundant. GeneraSolirubrobacter , Nocardioides , and Rubrobacter were
significantly associated with The Bahamas, Endobacter andCandidatus Hamiltonella associated with first recapture period in
Michigan, and Serratia with second recapture period. ASVs within
families Beijernickiaceae and Pseudomonadaceae were
significantly more abundant during first recapture in Michigan and
families Enterobacteriaceae , Beijernickiaceae , andSynergistaceae contained ASVs associated with the second
recapture in Michigan. No ASVs were statistically associated with The
Bahamas.