Discussion
We found clear localization of the microbiome in the S. virgatusgut and reproductive tracts. While localization has been seen in other
reptiles (Colston et
al., 2015; Costello et al., 2010; Keenan et al., 2013; Kohl et al.,
2017), the cloaca in previously sampled reptiles was found to have
relatively high diversity and to host representatives from the upper
regions of the gut. This pattern may be expected as the cloaca is the
terminus of the GI and reproductive systems, and is often assumed to be
both inoculated by feces that pass through and influenced by sexual
transmission during copulation
(Videvall et al.,
2018; Wen et al., 2021; White et al., 2011). Similarly, the
reproductive tract of chickens shows increasing levels of diversity from
oviducts to cloaca, which has the highest diversity (Wen et al., 2021).
In contrast to these previously described patterns, the cloacal
microbiome of S. virgatus females is low diversity, especially at
the Family and Phylum level at which it was dominated byEnterobacteriaceae and Protobacteria, and is distinct from upper
intestine, oviductal, and fecal microbiomes. This pattern may indicate
strong selection for the specific cohort of bacteria found in theS. virgatus cloaca. S. virgatus females deposit beneficial
microbes on their eggs during oviposition that facilitate egg survival
(Bunker et al., in
review). This function could be the driving force behind the winnowing
of the microbiome in the cloaca, especially considering that many
members of the Enterobacteriaceae family are known to have
antifungal properties
(Dhar Purkayastha et
al., 2018; Gutiérrez-Román et al., 2015; Kalbe et al., 1996).
Cloacal swabs taken from a given individual lizard resulted in
communities similar to the lower intestine and the cloacal tissue, and
distinct from higher regions of the gut and reproductive tract. The
cloacal swab community was predominantly Enterobacteriaceae but,
like the lower intestine, had several families that made small but
noticeable contributions to the community and was dispersed slightly
differently than the cloacal tissue microbiome. The upper intestine and
oviduct have different microbial communities than the lower sections of
the intestinal tract but also have differences in major families from
one another. Recent evidence has contradicted the long-accepted idea
that embryonic development is sterile
(Funkhouser &
Bordenstein, 2013). Microbial communities similar to gut microbes have
been found in chicken oviducts
(Shterzer et al.,
2020), and Trevaline et al.
(2018) found
that microbes may colonize bird and lizard eggs from within the oviduct
before the shell develops. The unique community in the oviduct here
could indicate an internal egg microbiome which is seeded during egg
development; the functional significance of this microbiome should be
investigated further.
In contrast to the adequate sampling of the cloacal and lower intestinal
microbial communities by cloacal swabs, fecal pellets contain strikingly
distinct communities from all gut regions, with greatest similarity to
the upper intestine. Pre-defecation cloacal swabs and the fecal samples
taken from the same individuals were found to have different microbial
communities from one another by every metric we examined. As described
above, the swabs were more similar to the cloaca and lower intestine,
and largely dominated by Enterobacteriaceae . The fecal sample
community was most similar to that found in the upper intestine, with a
relatively high abundance of Ruminococcaceae and Bacteroidaceae,but was dominated by Lachnospiraceae which was largely absent
from all regions of the gut, and lacked the high abundance ofEnterobacteriaceae that was found across the gut. Overall, these
patterns caution against the common practice of using fecal samples as a
proxy for the gut microbiome without first validating this approach.
While Study 2 found some changes between pre- and post-defecation, those
changes were largely non-significant, and likely due to increased
variation within the post-defecation swabs, as indicated by differences
in dispersion, more so than composition, between time points. The
variation is due to a distinct bifurcation in the recovered swab
communities from post-defecation swabs, with most of these swabs
mirroring cloacal tissue, with its high abundance of Proteobacteria, and
a smaller portion displaying a more “feces-like” community. This
variation in post-defecation cloacal swabs was also present in the field
swabs of lizards from Study 1; 5 of the 8 cloacal swab communities from
Study 1 were made up of >95% Enterobacteriaceae andHelicobacteraceae , while other samples were dominated byLachnospiraceae and Bacteroidaceae , two feces-associated
taxa. Because recovered communities from feces and pre-defecation
cloacal swabs were distinct, it seems unlikely that the cloaca is being
inoculated with fecal microbes. Rather, a more likely explanation is
that small amounts of fecal material may attach to swabs if they are
taken shortly after defecation, which then masks the much less diverse
cloacal community when it is sequenced. The idea of temporary
contamination is further supported by the fact that the same lizards,
when re-swabbed for a different study, often had comparatively lower
diversity and a greater relative abundance ofEnterobacteriaceae -associated ASVs (Online Resource 3). Some
animals continued to vary between the feces-like and cloacal tissue
phenotype when they were resampled, likely due to repeated defecation
events. Cloacal swabs often have been found to be inconsistent and
unreliable (Videvall
et al., 2018; Williams & Athrey, 2020), and potential contamination by
fecal material could account for some of this variation. Indeed, cloacal
swabs have been used to collect feces in the past
(Stanley et al.,
2015). The fact that these fecal microbes do not colonize and grow in
the S. virgatus cloaca supports the hypothesis that the low
microbial diversity of the cloaca is due to selection, and that there is
a mechanism to maintain stability of the microbiome in that region. This
type of selection has been seen in other species
(Nyholm &
McFall-Ngai, 2004; Zhang et al., 2016), and supports a model in which
function is the driving force of microbiome diversity
(Reese & Dunn,
2018).
At the phylum level, Bacteroidetes and Firmicutes are often considered
to represent the core gut microbiome across vertebrates
(Colston & Jackson,
2016; Ley et al., 2008). However, this dogma is based largely on
studies that rely on fecal sampling in mammals and so may not accurately
represent endogenous microbial communities of diverse vertebrate taxa.
For instance, Bacteroidetes have been found to be relatively rare in
some gut regions of wild reptiles and birds
(Colston et al., 2015;
Hird, 2017; Keenan et al., 2013; Kreisinger et al., 2015), and were
relatively uncommon across all S. virgatus gut regions and even
in fecal pellets, which were dominated by Firmicutes. Firmicutes and
Proteobacteria were more equally represented in the S. virgatusupper intestine, though Proteobacteria were more common, and
Proteobacteria increased in dominance down the GI tract to the cloaca,
which was nearly entirely composed of this phylum. Proteobacteria have
been found to be highly abundant in the GI tract of other non-mammalian
vertebrates (Colston
& Jackson, 2016), particularly in studies that sampled directly from
the GI tract rather than relying on fecal sampling alone, but its
dominance in the S. virgatus cloaca appears to be extreme even in
comparison to those systems. While other studies have found a majority
of the microbiome was composed of Proteobacteria, the lowest relative
abundance of this cloacal community member was 96.9% with greater than
99% on average, and the majority of those reads belonged to a single
family. The cloacal swabs were less consistent, as discussed above,
although still 85% Proteobacteria on average, and seemed to land
between the cloacal tissue community and that of the lower intestine.
Variation in the endogenous microbiome across taxa has been considered
in relation to diet and other aspects of animal life history and ecology
(Colston & Jackson,
2016; Ley et al., 2008). As descriptions of vertebrate microbiomes
continue to accumulate, it will be interesting to examine the potential
influence of reproductive mode (i.e., viviparity vs. oviparity), as this
separates mammals from most non-mammalian vertebrates. We propose that
oviparous species, and especially those without egg-tending, may have
unique selective pressures on the cloacal microbiome to transfer
antifungal or otherwise egg-protective bacteria to eggshells during
oviposition. Future research will compare the cloacal microbiome of
oviparous and viviparous Sceloporus lizards.