DISCUSSION
Biological understanding of vertebrates is incomplete without a thorough
knowledge of the microbiome. One of the specific goals of evolutionary
biology is to discover and describe biodiversity (see
(Hird, 2017)); herein, 1740
samples from 779 wild birds expand what we know about both birds and
their associated microbes. These samples, from a total of seven body
sites, provide a fundamental description of the microbiomes of diverse
and previously undescribed body sites in over 200 species. The cloacal
and intestinal microbiome results support many previous studies that
show a dominance of Proteobacteria and Firmicutes (Figure 2,
Supplemental Table S2, reviewed in
(Grond, Sandercock,
Jumpponen, & Zeglin, 2018)).
The avian blood microbiome has only been characterized in chickens,
where it was shown to be comprised of 60.58% Proteobacteria, 13.99%
Bacteriodetes, 11.45% Firmicutes, 10.21% Actinobacteria, and 1.96%
Cyanobacteria (Mandal et al.,
2016). Our blood samples exhibited over 20% more Proteobacteria, with
correspondingly smaller percentages of the remaining phyla (Fig. 2,
Supplemental Table S2). The differences between the two findings may be
unsurprising given that wild birds tend to have higher percentages of
Proteobacteria (Grond et al.,
2018). Clostridium was more abundant in the blood than in the
cloaca, intestines, and buccal cavity (Fig. 6a, b, c) and is known to
cause infections in birds
(Crespo, Fisher, Shivaprasad,
Fernández-Miyakawa, & Uzal, 2007). Janthinobacterium was more
abundant in the blood than in the cloaca and buccal cavities (Fig. 6a,
b). This genus has previously been identified in the cloaca of
shorebirds (Santos et al.,
2012). Known to contain pathogenic species, Mycobacterium was
more abundant in the blood than in the cloaca, intestines, gizzard, and
buccal cavity (Fig. 6a, b, c, d) , potentially showing that some birds
were infected in this study or that non-pathogenic species are part of
the healthy microbiota (Dhama
et al., 2011). Phenylobacterium was also more common in the
blood than in the cloaca, intestines, gizzard, and buccal cavity and has
been previously shown to be in the uropygial glands of house sparrows
with malaria (Fig. 6a, b, c, d)
(Videvall et al., 2021).Legionella was more abundant in the blood than in the cloaca and
the buccal cavity (Fig. 6a, b). This genus has been found in the cloaca
of birds and is known to cause opportunistic infections in humans
(Santos et al., 2012;
Fields, Benson, & Besser,
2002). Parvibaculum (more abundant in the blood than in cloaca,
intestines, gizzard, and buccal cavity and more abundant in the buccal
cavity than in the cloaca (Fig. 6a, b, c, d, e)), Planctomyces(more abundant in the blood than in the cloaca, intestines, and buccal
cavity (Fig. 6a, b, c)) and Sediminibacterium (more abundant in
the blood than in the cloaca, intestines, gizzard, and buccal cavity
(Fig. 6a, b, c, d)) were abundant in this study but are not commonly
described members of the avian microbiota.
The buccal microbiome contained over 50% Proteobacteria and smaller
percentages of Firmicutes, Actinobacteria, Tenericutes, and
Bacteriodetes (Fig. 2, Supplemental Table S2). These results are similar
to what was found in the oral microbiome of the Great Tit
(Kropáčková et al., 2017)
but are quite different from those found in the Cooper’s hawk, which
contains higher relative abundance of Firmicutes than the quantities of
Actinobacteria, Bacteriodetes, and Tenericutes
(Taylor et al., 2019).
Further sampling is required to determine how uniform the oral
microbiome is in wild birds. Hylemonella was more abundant in the
buccal cavity than in the blood, gizzard, intestines, and cloaca (Fig.
6a, e, f, g). This genus has been identified in the skin microbiome of
vultures, raising the possibility of preening transferring skin and
buccal microbes (Zepeda
Mendoza et al., 2018). Gallibacterium was more abundant in the
buccal cavity than in the blood, gizzard, intestines, and cloaca (Fig.
6a, e, f, g) and has been identified in the respiratory tract of healthy
and unhealthy birds
(Bisgaard, 1977),
(Mushin, Weisman, & Singer,
1980). Leucobacter was more abundant in the buccal cavity than
in the blood or cloaca in this dataset (Figure 6a, e) and has been
identified in the feces of swiftlets
(Sien, Lihan, Yee, Chuan, &
Koon, 2013). Rothia was more abundant in the buccal cavity than
in the gizzard or cloaca (Figure 6e, g) and is also a common member of
the human saliva microbiome
(Tsuzukibashi et al., 2017).
The gizzard samples were composed mostly of Firmicutes, Proteobacteria,
and Cyanobacteria (Fig. 2, Supplemental Table S2). This is not
consistent with the only published study on gizzard microbiomes that
were mainly composed of Bacteroidetes, Cyanobacteria, Planctomycetes,
Verrucomicrobia, and Alpha and Gammaproteobacteria
(García-Amado et al., 2018).
The liver and spleen microbiomes were both dominated by Firmicutes and
Proteobacteria (Fig. 2, Supplemental Table S2); this is similar to the
microbiome of wild mouse spleens which are also composed primarily of
Firmicutes and Proteobacteria
(Ge, Guo, Ge, Yin, & Yin,
2018). The relative abundances of these two phyla were very different
in the liver and spleen, as compared to the blood. Spleens filter blood,
so similarities between these two sample types may be expected. However,
the vast differences between them show that these environments may be
hospitable to different communities of bacteria, although this requires
further confirmation, as our spleen and liver sample sizes were quite
low.
The majority of our samples were of the blood, buccal, gizzard,
intestines, and cloaca. These sample types were significantly different
from each other in all three beta diversity metrics measured
(p <0.001) and the variation explained by each of these
metrics was relatively high (6.7%- 17%). This shows that the types of
taxa in each sample type are different from each other and that taxa
those that are more phylogenetically distinct are more abundant. These
measurements are lower than some other bird body site studies
(Grond, Guilani, & Hird,
2020), but perhaps to be expected as this study incorporates dozens to
hundreds of bird species that may have distinct microbiomes.
Many ornithologists are interested in studying the microbiome without
harming the bird and therefore many have asked whether non-destructive
sampling (e.g., oral swabs, cloacal swabs, feces) is adequate to
describe the gut microbiome
(Videvall, Strandh,
Engelbrecht, Cloete, & Cornwallis, 2018). At the ASV level, our
cloacal and intestine samples had substantial, and roughly equivalent,
unique components; however, at the level of the sequencing reads, almost
all the diversity was shared (Fig. 5b). Therefore, the unique ASVs
contribute far less to the total microbiota than the shared ASVs.
Compositionally, ten genera were more abundant in the cloaca than in the
intestines: Acinetobacter , Aerococcus ,Cloacibacterium , Cupriavidus , Limnohabitans ,Micrococcus , Propionibacterium , Rheinheimera ,Staphylococcus , and Stenotrophomas (Figure 5a). Notably,Cloacibacterium has been found in avian blood
(Mandal et al., 2016),Micrococcus in healthy conjunctiva and nasal passages
(Silvanose, Bailey, Naldo, &
Howlett, 2001), and Staphylococcus can cause infections in birds
(Hermans, Devriese, De Herdt,
Godard, & Haesebrouck, 2000). Six genera were more abundant in the
intestines than in the cloaca: Balneimonas , Enterococcus ,Lactobacillus , Lactococcus , Psychrobacter , andRickettsiella (Figure 5a). Enterococcus has been found in
the cloaca of birds
(Jørgensen et al., 2017) andPsychrobacter has been found in the throats and guts of birds
(Kämpfer et al., 2015),
(Kämpfer et al., 2020).Lactobacillus and Lactococcus have been previously
identified in the cloaca of birds
(Allegretti et al., 2014;
Gunasekaran, Trabelcy,
Izhaki, & Halpern, 2021), but we found them at higher abundances in
the intestines, which is similar to previous comparative studies (Hird
et al., 2015; Capunitan et al., 2020). Enterococcus was more
abundant in the intestines than the cloaca, gizzard, blood, and buccal
cavity and more common in the blood than the gizzard (Figs. 5a, 6c, d,
f, i). This genus has been found in the cloaca of chickens
(Jørgensen et al., 2017).Lactobacillus similarly was more common in the intestines than
the cloacal, gizzard, blood, and buccal cavity (Figs. 5a, 6c, f, i).
This matched a finding in parrots that found Lactobacillus in
their cloaca (Allegretti et
al., 2014). In a comparison of body site microbiota in ostriches,
several families that include our differentially abundant intestinal
microbes were also significantly higher at internal gastrointestinal
sites, as compared to the cloaca (Lactobacillaceae ,Streptococcaceae , Enterococcaceae ; Videvall et al., 2018).
The comparison of body sites also identified many additional taxa common
to bird microbiomes and/or which are known pathogens:Campylobacter(Kapperud & Rosef, 1983;
Hird et al., 2018),Cloacibacterium(Mandal et al., 2016).Comamonas (Kropáčková
et al., 2017), Enhydrobacter(Kreisinger, Čížková,
Kropáčková, & Albrecht, 2015), Methylotenera(Boukerb et al., 2021),Pseudomonas (Oprea,
Crivineanu, Tudor, łOGOE, & Popa, 2010), Psychrobacter(Kämpfer et al., 2015),
(Kämpfer et al., 2020).Sphingobacterium(Gunasekaran et al., 2021),Streptococcus(Devriese et al., 1994),Curtobacterium(Giorgio, De Bonis,
Balestrieri, Rossi, & Guida, 2018), Kocuria(Braun, Wang, Zimmermann,
Boutin, & Wink, 2018), Brevundimonas(Giorgio et al., 2018),
Kingella (Foster et al.,
2005), Micrococcus(Silvanose et al., 2001),Staphylococcus(Hermans et al., 2000),Lactococcus(Gunasekaran et al., 2021).Methylobacterium was found to be more abundant in the blood and
buccal cavity more than the gizzard and more abundant in the buccal
cavity than in the intestines and cloaca (Figure 6d, e, f, g). This is
noteworthy as methylobacterium is known to be a contaminant in
kits (Salter et al., 2014).
Although alpha diversity did not vary significantly across anatomical
sites, we did identify specific microbial taxa that were differentially
abundant between sites. (Fig. 3). Together with the beta diversity
results, this shows that while the sample types contain similar levels
of diversity, the composition of those communities is different across
the body sites.
The microbiome is a trait of the host that may not be independent of the
underlying phylogeny. Strictly speaking, phylogeny describes the
evolutionary history of organisms. For a variety of reasons, phylogeny
captures more than just evolutionary history and more closely related
organisms frequently have more similar traits (“phylogenetic signal”).
Therefore, comparisons of microbial “traits” across species need to
control for phylogeny of the hosts.
Bird weight and one third of the alpha diversity measurements contained
significant phylogenetic signal, as assessed by Pagel’s Lambda (Table 1:
two blood, one buccal, two intestine). When subsetting to include only
Passeriformes, only three of the 15 tests contained significant
phylogenetic signal: two buccal and one cloacal (although the three
gizzard tests were likely affected by extremely low sample size). This
shows an inconsistent or low level of association between phylogeny and
microbiota richness and diversity, meaning that factors beyond phylogeny
impact the microbial communities. This is similar to previous
comparative work that found a White Noise model (of no phylogenetic
signal) may fit the avian microbiome better than a neutral model or a
model that includes selection (Capunitan et al., 2020).
How does size of a host influence the richness and diversity of the
microbiota? Birds can be conceptualized as “islands” containing
communities of microorganisms and their composition could potentially be
driven by the Theory of Island Biogeography. We found eight significant
correlations between bird size and microbiota, when using phylogenetic
comparative methods (Table 1). In the full dataset, the blood samples
show a significant negative correlation between their microbial
diversity and the average host weight for all three of the diversity
metrics (p <0.001), indicating that larger birds
actually have significantly lower diversity in their blood than smaller
birds.
Because bird orders diversified quickly, there can be different
associations of host traits within and across orders
(Harmon et al., 2010).
Therefore, we restricted analyses to a single order (Passeriformes) and
reran the PGLS analyses. In the subset data, all three of the microbiome
metrics for the cloacal samples exhibited significant negative
correlations (p <0.05) (Table 1, Supplemental Fig. S3).
The Theory of Island Biogeography predicts that larger islands will
house more diversity; our results appear to show the opposite: either a
significant negative correlation or no correlation at all.
In broader terms, our results show that as host “islands” increase in
size, the number of microbial taxa immigrating decreases and/or the
number of extinction events grows larger, indicating that regulatory
mechanisms associated with host body size may be influencing the
Species-Area Relationship. For example, avian microbiomes may be more
prone to colonization by bacteria that inhibit conspecific
proliferation, the immune systems of the host may be more sensitive to
new “immigrant” members of the microbiome, different anatomical sites
may be limited by nutrients, or other possibilities may be working to
prevent a significant and large difference in alpha diversity due to
host average weight. However, and importantly, even when thep -values showed there was a negative or positive significant
correlation between average host weight and alpha diversity, the value
was small in every case. This suggests that there are only minor
significant changes in the alpha diversity corresponding to average host
weight. Taken together, our findings do not support an unexamined
application of traditional Species-Area Relationships to the avian
microbiome.