Discussion
Previous studies focused on the biotopes of soil, phyllosphere and
faeces in a grazed steppe ecosystem respectively while the connections
of microbiota among these biotopes, which played a role to carry out the
energy flow in the ecosystem, were neglected. This study investigated
the diversity, structure and the interaction of the microbial
communities in these biotopes at the molecular level. It provided direct
evidence to reveal the microbial distribution, migration and ecosystem
function of the microbiota among these biotopes.
Soil, phyllosphere and faeces all maintained distinct microbiota and the
type of biotopes explaining the majority of this microbial variation. As
for the α-diversity, the soil fungal richness was higher contrasted to
the fungal microbiota from the other biotopes (Table S1). This community
diversity variation was likely due to the complicate and distinctive
soil environment. Soil encompasses numerous microcosms to harbour
distinct microbial communities rather than one single environment.
Although these soil environments were limited in the only micrometer or
millimeter scale, they provided niches of unique abiotic habitat and
biotic communities to filter the microbial activities, diversities and
compositions (Fierer, 2017). In addition, compared to soil bacteria,
soil fungi were considered more adaptive to the vast different
environments. Fungi were regarded as the mediators of slower carbon
cycling because of their low growth rate and tolerance of low
temperature and aridity (Rinnan & Baath, 2009; Rousk & Bååth, 2007;
Sun, Li, Avera, Strahm, & Badgley, 2017). Furthermore, the great
priority effect influenced on fungi and made it easier to survive in the
extensive environments (Peay, Belisle, & Fukami, 2012). Meanwhile,
fecal richness was of the lowest level in the fungal and bacterial
community among all the biotopes (Table S1). The fecal microbial
communities were considered similar in those residing to the livestock
intestinal tract (Romero-Perez, Ominski, McAllister, & Krause, 2011).
Obviously, the extreme environment of intestinal tract limited the gut
and fecal fungal and bacterial richness via filtering the microbiota
with pH, moisture and temperature stress and anaerobic environment. With
respect to the β-diversity, all the microbiome also showed distinct
microbial clusters among the three biotopes. This was due to the
environmental filter driving the microbial community assembling in a
dynamic ecosystem. Rothschild et al revealed that the environment
dominated in shaping microbiome although it was only considered as a
minor factor in the previous studies (Rothschild et al., 2018). The
biotope preferences of the microbiota were associated with the
environmental and ecological characteristics such as physicochemical
properties and physiological capabilities (Bates et al., 2011; Jiao, Xu,
Zhang, & Lu, 2019; Stams & Plugge, 2009).
Although all the three biotopes harboured distinct microbial communities
from each other, the overlaps among these microbiotas revealed the
microbial migration in the grazed steppe ecosystem (Fig. 2). The largest
proportion of OTUs overlap occurred between soil and phyllosphere. These
overlaps mainly contributed by bacteria and fungi suggested that the
majority of phyllosphere bacteria and fungi originated from the soil. In
another words, soil showed its potential as a microbial source reservoir
of phyllophere (Zarraonaindia et al., 2015). This was consistent with
the common view that phyllosphere microbiota was influenced by the
microbiota of surrounding environmental source initially and then they
were selected by the leaf taxa (Copeland, Yuan, Layeghifard, Wang, &
Guttman, 2015; Lajoie, Maglione, & Kembel, 2020; Maignien, DeForce,
Chafee, Eren, & Simmons, 2014). However, the specific environmental
source influencing phyllosphere microbiota most strongly depends on the
growing condition of the leaf species. Each leaf shaped a unique
environment to thrive for specific microorganisms (Vacher et al., 2016;
Vorholt, 2012). For instance, Copeland found the phyllosphere microbiota
of several annual crops mainly originated from soil microbiota in the
field condition (Copeland et al., 2015). Nevertheless, on the A.
thaliana leaves, the phyllosphere microbiota mirrored airborne
microbiota initially in the sterile soil (Maignien et al., 2014). A
small proportion of fecal OTUs also occurred in phyllosphere while most
of them were at low level of abundance. Phyllosphere microbiota was
colonized on the leaf surface exposed to rapidly fluctuating
temperature, moisture and relatively alternative humidity and nutrient
resource
(S.
E. Lindow & Brandl, 2003). Thus, although the fecal microbiota
originated from a specific environment with extreme stresses quite
different from most of other biotopes, there were still some OTUs shared
by these two biotopes since phyllosphere could offer a temporary
microenvironment similar to the fecal microbiota. However, the drastic
fluctuation of environment condition made phyllosphere an extreme and
hostile habitat for the microbial community (S. E. Lindow & Brandl,
2003). For instance, a large majority of bacterial colonist had to face
threat of being washed or killed by
water,
peroxide
and UV light (Beattie & Lindow, 1995; Wilson & Lindow, 1994b). The
availability of nutrient source such as glucose, fructose, and sucrose
were also limited for the phyllosphere epiphytic colonization (Wilson &
Lindow, 1994a). Hence, phyllosphere microbiota maintained at a low level
of abundance because it had to tolerate such environment. Nevertheless,
OTUs rarely occurred in both soil and faeces biotopes. Soil lacked of
the drastic fluctuation of environmental condition like phyllosphere to
fit the extreme environment condition of faeces. To put it another way,
the rapid fluctuation of environment condition limited the growth and
activity of phyllosphere microbiota, but it made phyllosphere a most
extensively adaptive biotope for microbiota. Therefore, phyllosphere
played the role of bridge to link the microbial communities among
biotopes of soil, phyllosphere and faeces in such a grazed steppe
ecosystem.
Referring to the community structure, the microbial migration among
biotopes was predominated by specific taxa. ADONIS and ANOSIM described
the increasing community similarity among three biotopes with the
successive order of bacteria, fungi and archaea (Table 1). The
similarity for bacteria was mainly contributed by the shared taxa
between soil and phyllosphere. With high relative abundance,
Actinobacteria and Proteobacteria occurred in both biotopes of soil and
phyllosphere as the dominant phyla (Fig. 3). Whipps and
Laforest‐Lapointe also found α‐ and γ‐Proteobacteria the most common
classes within phyllosphere. However, the proximity of the relative
abundance of these two phyla in phyllosphere varied from the low
abundance of Actinobacteria within tree phyllosphere while the patterns
of some Conifer species were consistent with our study
(Laforest-Lapointe, Messier, & Kembel, 2016; Whipps et al., 2008).
Actinobacteria were the most dominant bacteria in the soil biotope to
decompose the plant litters and debris (Ventura et al., 2007). In
addition, the genus of Streptomyces , which belonged to
Actinobacteria, played role in inhibiting the fungal pathogens within
rhizosphere. It was supposed to perform the similar function in the
biotope of phyllosphere (Kunova et al., 2016). Nevertheless, the
bacteria in the faeces biotopes showed significant discrepancy from the
other two biotopes. The dominant phyla Firmicutes and Bacteroidetes
occurred rarely in the soil and phyllosphere. The observed dominance of
these phyla was due to the extreme environment in the intestinal tract
of livestocks. Firmicutes were resistant to harsh environmental
conditions, especially the desiccation stress, through their
Gram-positive cell walls and spore-forming ability (Schimel, Balser, &
Wallenstein, 2007; Van Horn et al., 2014). Bacteroidetes were the
primary gateway to take advantage of glycans and carbonhydrate for the
metabolism of the microbiota in the anaerobic and extreme environment in
intestinal tracts (Faith, McNulty, Rey, & Gordon, 2011; Fischbach &
Sonnenburg, 2011; Sonnenburg et al., 2010; Wu et al., 2011). In contrast
to the diversity in bacteria domain, the archaea showed homogeneous
within the three biotopes.
Thaumarchaeota
predominated in both soil and phyllosphere. This distribution was
similar to the previous study in the two biotopes respectively (Fierer
et al., 2012; Rinta-Kanto et al., 2016; Taffner, Cernava, Erlacher, &
Berg, 2019).
Thaumarchaeota
were known as a novel phylum serving for the aerobic ammonia oxidization
and the nitrification in archaea independent to Crenarchaeota and
Euryarchaeota (Brochier-Armanet, Gribaldo, & Forterre, 2012; Jiao et
al., 2019; Konneke et al., 2005). In the fecal archaea, Euryarchaeota
replaced Thaumarchaeota as the dominant phylum. Methanobacteria and
Methanomicrobia (belonged to Euryarchaeota) composed the primary
assemblages. Both these classes were methanogenic archaea colonized by
gut to reduce carbon dioxide into methane through present hydrogen in
the intestinal tracts (Roccarina et al., 2010; van de Pol et al., 2017).
Therefore, with the environmental filtering and functional selection,
bacteria and archaea migrated between the biotopes of soil and
phyllosphere, rather than faeces.
The only communal phyla migrating among all the three biotopes were
Ascomycota
and a small amount of Basidiomycota belonging to fungi. It indicated
that these fungal phyla possessed robust environmental resistance and
extensive environmental adaptability. In most of the previous study
focusing on the fungal communities of soil, phyllosphere and faeces,
Ascomycota all occupied the significant place of the dominant phyla
order (Jia, Wang, Fan, & Chai, 2018; Perazzolli et al., 2014; Yelle,
Ralph, Lu, & Hammel, 2008). In all the three biotopes of soil,
phyllosphere and faeces, the Ascomycota behaviours were saprophytic and
played a role in decomposing the various substrates such as plant and
animal residues to transform them into nutrition for the direct usage of
relative biota. Meanwhile, Nectriaceae, a parasitic and saprophytic
family belonging to Ascomycota, generally occurred in the livestocks and
monocotyledons. Nectriaceae were pivotal for maintaining the balance of
the grazed grassland ecosystem because of their function of regulating
the host genotypes and environments (Jia et al., 2018). Only a few
sequences classified into Basidomycota were shared by all the three
biotopes. Most of them belonged to Cryptococcus .Cryptococcusoccurred commonly in the animal to cause the systemic infection and its
primary risk factor was HIV infection (Cafarchia et al., 2006; Idnurm et
al., 2005). TheCryptococcusfrom animal faeces could be carried into other biotopes such as soil and
phyllosphere with the niches of sufficiently utilizable carbon and
nitrogen source (Nielsen, De Obaldia, & Heitman, 2007). However,
whether the overlap of Cryptococcus could be applied to other
terrestrial area was not clear since this overlap occurred rarely in
taxa classification and this result may depend on the timing of the
sampling. Thus, Ascomycota was the definitely phylum migrating in the
ecosystem to link all the biotopes. Yet more types of livestock should
be brought into the survey to confirm this result since only sheep were
investigated in this study.
Physical environmental factors explained more variation of the microbial
abundance among the biotopes while nitrogen was the most remarkable
nutrient source for the microbial distribution.
SEM
(Fig. 6) showed that temperature explained the most of the impacts on
fungi and bacteria abundance while pH was the only factor influenced
fungi, bacteria and archaea. Mantel test (Table S4) also demonstrated
that temperature influenced microbial diversity significantly. This
discrepancy might be owing to the environmental sensitivity of the
dominant species such as Actinobacteria, Zygomycota and Thaumarchaeota,
so they were replaced by more extensively adaptive and stress-tolerant
phyla such as Firmicutes, Ascomycota and Euryarchaeota in the intestinal
tracts with harsh environmental conditions (Egidi et al., 2019; Jiao et
al., 2019; Roccarina et al., 2010; Schimel et al., 2007; van de Pol et
al., 2017; Van Horn et al., 2014). Nitrogen was also found to be the
vital nutrition factor revealed by SEM (Fig. 6). This was mainly caused
by Ascomycota, the communal and dominant phylum migrating among all the
biotopes since it was highly associated with the nitrogen immobilization
(Egidi et al., 2019). In addition, Fusarium oxysporum, Fusarium
solani, Cylindrocarpon tonkinense and Gibberella fujiuroii, belonging
to Ascomycota, all performed function of denitrification (Levy-Booth,
Prescott, & Grayston, 2014).
Although the soil showed its potential of microbial reservoir for
phyllosphere, only fungi migrated among all the biotopes of soil,
phyllophere and faeces. Thus, the biotopes in an ecosystem were linked
by the energy flow through the fungal community. De Vries et alreported the fungal-based process and the carbon and nitrogen cycle
governed led weak resilience rather than strong resistance of grassland
ecosystem (de Vries et al., 2012). Our network analysis (Fig. 4)
confirmed it by exhibiting the significant discrepancy between the
microbial community of grazed and ungrazed grassland after long-term
moderate grazing. It was also noteworthy that fungi showed the most
marginal variation in the co-occurrence networks. This was consistent
with the slower response of fungi due to their lower growth rate and
extensively adaptability in contrast to bacteria (Bardgett & van der
Putten, 2014; Rinnan & Baath, 2009; Rousk & Bååth, 2007; Sun et al.,
2017). Therefore, the fungal migration among biotopes made the ecosystem
robust for the short-term but instable for the long-term.