Results
A total of 535430 bacterial 16S rRNA V4 amplicon sequence were obtained
from 9 samples covering 3 biotopes: soil, phyllosphere and faeces in the
grazed temperate steppe in Inner Mongolia and further clustered into
2136 OTUs. Simultaneously, 840851 ITS gene sequence and 614820 16S gene
sequence were also generated and clustered into 1340 fungal and 621
archaeal OTUs respectively.
The α-diversity revealed distinct
differences in richness, diversity and evenness of communities among
biotopes of soil, phyllosphere and faeces (Table S1). The Shannon and
Simpson index indicated that the soil fungal and bacterial diversity
were induced in phyllosphere and faeces contrasted to the soil while
fungi and bacteria showed diversity at the lowest level in faeces. The
ace and chao index demonstrated the highest fungal richness in the soil
than in the other two biotopes. The fungal and bacterial richness also
diminished in the faeces.
All the microbiotas showed significant different distributions among the
biotopes of soil, phyllosphere and faeces. The microbial communities
were visualized via polar coordinate analysis
(PCoA)
(Fig. 1a, 1b and 1c). The PCoA graphs indicated that distinct clusters
were shaped among the biotopes of soil, phyllosphere and faeces for
fungal, bacterial and archaeal phyla (Fig. 1a, 1b and 1c). These
clusters were confirmed by the sample within-between analysis which
consisted of permutational multivariate analysis of variance (ADONIS)
and similarity analysis (ANOSIM) (Table 1). These examinations also
revealed that bacterial community varied among the three biotopes more
significantly than fungi and archaea. The Hierarchical clustering tree
at order level, based on bray- curtis distance, further supported these
microbial phylogenetic differentiations among the three biotopes (Figure
S1).
The network like venn analysis revealed that soil was the potential
microbial source reservoir of phyllosphere while faeces was a much less
relavant biotope to them. Figure. 2a indicated that phyllosphere shared
large amount of OTUs with soil. However, faeces shared much fewer OTUs
with the other two biotopes, especially rarely with the soil. The OTUs
shared by all the three biotopes also contributed a fraction of low
percentage (Fig. 2a). To be precise, the venn analysis of fungi,
bacteria and archaea (Fig. 2b, 2c and 2d) showed that the main
proportion of shared OTUs are contributed by fungi and bacteria. The 916
and 419 OTUs were shared by soil and phyllosphere for fungi and bacteria
respectively while only 26 OTUs for archaea. However, with respect to
the OTUs between soil and faeces, only 20 occurred in fungi, and both
the OTUs numbers in bacteria and archaea are 0. The OTUs shared among
all the three biotopes were also mainly in fungi. The three biotopes
communal OTUs numbers are 104, 33 and 6 for fungi, bacteria and archaea
successively.
The microbial composition also showed remarkable differentiation among
the biotopes of soil, phyllosphere and archaea. For the fungi, samples
from the soil biotope were more diverse than those of the phyllophere
and faeces biotopes. Ascomycota (62.94%) was the dominant phylum
followed with Zygomycota (14.97%) and Basidiomycota (10.01%) in the
soil. Ascomycota was also the most predominant phylum in phyllosphere
while its relative abundance increased to 89.69% and it was 2.63% and
5.99% for Zygomycota and Basidiomycota respectively. Moreover,
Ascomycota (97.36%) almost completely dominated in the faeces fungal
microbiome (Fig. 3a). For the bacterial community, the fecal microbiota
was not as diverse as the soil and phyllosphere microbiota. The dominant
bacterial phyla in the soil and phyllosphere were both Actionobacteria,
Fimicutes, Proteobacteria,
Bacteroidetes,
Acidobacteria, Chloroflexi and Cyanobacteria. Actinobacteria (soil:
44.02%, phyllosphere:34.69%) contributed most in both biotopes. The
relative abundance of Bacteroidetes
(soil: 0.92%, phyllosphere:10.99%)
and Acidobacteria (soil: 20.29%, phyllosphere:1.83%) increased or
decreased most acutely from the soil to phyllosphere biotopes.
Nevertheless, Firmicutes (61.71%) and Bacteroidetes (24.63%) dominated
in the fecal microbiota (Fig. 3b). In the archaeal community, the
dominant phyla in the soil and phyllosphere biotopes were Thaumarchaeota
while Euryarchaeota dominated in the faeces biotope
(Fig. 3c).
To further understand the association of the microbial structure among
the soil, phyllosphere and faeces, the fungal, bacterial and archaeal
composition of three biotopes were visualized by ternary analysis
(Fig.
1d, 1e and 1f). A plenty of fungal classes were shared between the soil
and phyllosphere with low abundance. Few fungal classes coexisted
between faeces and the other two biotopes. Several fungal classes with a
certain amount of relative abundance were communal among three biotopes.
Most of these classes belonged to phylum Ascomycota and some classes
with low abundance belonged to Basidiomycota. As for bacteria, classes
shared between the soil and phyllosphere were also common while much
fewer classes occurred between faeces and phyllosphere. Furthermore,
there was no bacterial class between the soil and faeces. In the
archaeal community, all the classes shared by the soil and phyllosphere
belonged to Thaumarchaeota. A few classes belong to
Euryarchaeota
were shared by faeces and phyllosphere but it was at a low level of
abundance in phyllosphere. The archaeal classes did not coexist between
soil and faeces either (Fig. 1d, 1e and 1f). The radar analysis and
coxcomb analysis of the microbial composition also supported these
statements. It was found that Ascomycota and
Basidiomycota
were communal among three fungal microbiotas but the relative abundance
of Basidiomycota was lower than Ascomycota (Fig. 3d and S2). The random
forest model further classfied the fungal distribution among the three
biotopes. The communal genuses with the top relative abundance such as
unclassified Dothideales, Neostagonospora, unclassified
Sporopmiaceae, unclassified Xylariales and Pyrenophora all
belonged to the fungal lineages within Ascomycota (Fig. 3f).Actinobacteria, Proteobacteria and Acidobacteria were the shared
bacterial phyla between the soil and phyllosphere while Bacteroidetes
were shared between phyllosphere and faeces. As for archaea,
Thaumarchaeota abounded in both soil and phyllosphere biotopes.
Euryarchaeota abounded in faeces but it occurred rarely in phyllosphere
(Fig. S2).
To identify the influence of faeces input to the soil and phyllosphere
microbiota, the microbial community of soil and phyllosphere biotopes
from grazed and ungrazed grassland were presented via network analysis.
Multiple network topological metrics were employed to reveal the
remarkable difference between the microbial assemblages of grazed and
ungrazed grassland. Although the co-occurrence networks constructed by
significant correlation indicated a larger proportion of bacterial and
fungal OTUs were included in the networks of grazed grassland, there
were similar edges in these assemblages with grazing (Fig. 4a and 4b).
The average weighed degree, which indicated the normalized number of
connections to a node, diminished under the grazing conditions. The
decreasing cluster coefficient and increasing average path length also
exhibited the attenuated connectivity of fungal and bacterial networks
in the grazed grassland. Likewise, the margin attenuation of fungal and
bacterial networks density under grazing conditions reflected that
enormous potential connection between nodes of fungal network lost
efficacy in the grazed grassland. Nevertheless, the modularity index
indicated that the bacterial and fungal assemblages were more modular in
the grazed grassland than ungrazed grassland while it was reverse in the
archaeal assemblages (Table S2).
To clarify the influence factor of the microbiota among biotopes of the
soil, phyllosphere and faeces, the environmental characteristics were
determined (Table S3). The Mantel Test revealed that total nitrogen
(TN), pH and temperature influenced all the fungal, bacterial and
archaeal microbiota highly significantly and total organic carbon (TOC)
also influenced fungi very remarkably (Table S4). Although all the
environmental factors such as TOC, TN, total phosphorus (TP), nitrate
nitrogen
(NO3--N),
ammonia nitrogen
(NH4+-N),
carbon nitrogen ratio (C/N), pH, temperature and moisture could
influence the fungal and bacterial communities, there were only TOC, TN,
pH and temperature exerting impact on archaeal community. The canonical
correspondence analysis (CCA) indicated that there was no predominant
influence factor in these environmental variables (Fig. 5). Carbon,
nitrogen and phosphorus impacts were strongly and positively correlated
while they were negatively correlated to pH impact. Moisture and ammonia
nitrogen were in another strongly positive correlated impact pair while
they were significantly and negatively correlated to nitrate nitrogen.
Notably, soil bacteria were most strongly influenced by nitrate nitrogen
and the phyllosphere bacterial community were most correlated to
temperature.
The structure equation model (SEM) was also constructed to assess the
direct and indirect environmental impacts on the microbiota of the
grazed grassland ecosystem which consisted of biotopes of soil,
phyllosphere and faeces (Fig. 6). The multigroup modelling approaches
were applied to assess the hypothesized relationships among significant
environmental physicochemical factors and microbial communities.
Temperature directly and strongly influenced fungal and bacterial
compositions. Nevertheless, moisture and carbon were weakly correlated
to fungal compositions. Nitrogen directly influenced fungal and
bacterial compositions, meanwhile, it was impacted by carbon,
temperature, moisture, fungal and bacterial compositions. The only
factor directly linked to fungal, bacterial and archaeal compositions
was pH. The fungal impact on bacterial community was weak while there
was strong correlation between bacterial and archaeal community.