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Aggregate sizes regulate the microbial community patterns in sandy soil

profile

Article in Soil Ecology Letters · July 2021

DOI: 10.1007/s42832-021-0095-1

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Soil Ecol. Lett.
https://doi.org/10.1007/s42832-021-0095-1

RESEARCH ARTICLE

Aggregate sizes regulate the microbial community patterns

in sandy soil profile
Yifei Sun1, Meiling Sun1, Guowei Chen2, Xin Chen1, Baoguo Li1, Gang Wang1 ,*

1 Department of Water & Soil Sciences, China Agricultural University, Beijing 100049, China
2 Department of Civil Engineering, Hefei University of Technology, Hefei 230000, China

HIGHLIGHTS GRAPHICAL ABSTRACT

• Relative abundances of microbial commu-

nities were most related to aggregate propor-
tions in clay-layer soils.
• Aggregate content with < 0.053 mm in clay-
layer soil significantly influence the diversity
of soil microbial community.
• Complexity of microbial interactions raised
along increasing precipitation across sam-
pling sites.
• Competition for substrates induced niche
differentiation in deeper soils.

ARTICLE INFO

Article history:
Received October 26, 2020
Revised April 9, 2021
ABSTRACT
Accepted April 17, 2021
Soil microorganisms play a key role in the function of soil ecosystem, yet our knowledge about how
Keywords: microbial communities respond to the typically sandy soil environmental properties along the soil
Aggregate distributions profile is still insufficient. We investigated the soil microbial community patterns from top (0 – 20 cm)
to clay-layer (>80 cm) of the typical sandy soils in three regions in China with different levels of
16S rRNA precipitation, including Lishu County in Jilin Province (LS), Langfang City in Hebei Province (LF) and
Microbial community Zhengzhou City in Henan Province (ZZ). Our findings showed that small-size aggregates ( < 0.5 mm)
rather than large ones (³ 0.5 mm) dominated the soil profile. The relative abundances of
Sandy soil Actinobacteria, Crenarchaeota and Firmicutes were highly related to aggregate proportions of the
Network analysis deep clay-layer soil. The network analysis revealed the distinct community patterns among modules,
Soil profile evidencing niche differentiation along the soil profile. The keystone species OTU_11292 was
observed having migrated clearly into the other module of the clay-layer soil. Different roles of the
OTU_30 (belonging to Gemmatimonadetes) in soil processes might partly explain the different
microbial distribution between top- and clay-layer soils. These findings provided new insights into the
candidate mechanisms of microbial diversity maintenance and community patterning of sandy soils,
which were necessary for better understanding of ecological rules guiding long-term agricultural
practice.
© Higher Education Press 2021

* Corresponding author
E-mail address: [email protected] (G. Wang)
2 Aggregate sizes regulate the microbial community patterns in sandy soil profile

1 Introduction water content is the primary control on how soil structure

changes with depths in the soil profile. Soil C is mainly
Microbial community composition is a key control of a host of correlated with macroaggregates in the surface soil as a
essential processes for soil productivity and sustainability. The substrate supply (Mandiola et al., 2011). Limited oxygen
majority studies of soil microbiology have focused on the diffusion in aggregates generate hotspots coexisting with the
surface soil profiles where microbial numbers and biochemical anoxic and oxic microbes (Long and Or, 2005; Ebrahimi and Or,
activities are higher (Marschner et al., 2003; Drenovsky et al., 2016). According to the hierarchical theory of aggregate
2004; Barber et al., 2017). Soil profiles are often a few to several formation, these elements are distributed unevenly in different
meters deep with large numbers of microorganisms residing in aggregates size fractions and strongly related with aggregates
subsurface horizons (Blume et al., 2002). Studies have shown stabilities (Six et al., 2004). The quality and quantity of nutrients
that microbial community compositions and biological proper- in aggregates with different sizes along the soil profile are likely
ties vary with differing pedogenetic layers as well as the the driving factors regulating the nature and properties of the
microbial biomass and metabolic activities (Ekelund et al., 2001; residing microbial communities (Or et al., 2007; Vos et al., 2013;
Castellazzi et al., 2004; Sanesi and Certini, 2005). Deeper Ebrahimi and Or, 2016).
layers of soil may contain microbial communities that are Soil water content not only varies over depth in soil profile but
specialized for their niches, and could be fundamentally distinct also manipulates soil pore structure associating with drying-
from their shallow neighbors (Fritze et al., 2000; Blume et al., rewetting process, which conspires to limit nutrients (including
2002). Knowledge about the candidate mechanisms for driving oxygen and other gases) diffusion into (and out from)
soil microbial community structure and functions is helpful to aggregates that generate hotspots accommodating microbial
fully understand the soil microbial ecology, yet our under- populations (Wang and Or, 2013; Ebrahimi and Or, 2016).
standing about the nature of the microbial communities across These processes are even highlighted among rapidly water-
the deep soil profile is still insufficient. fluctuating soil, e.g., sandy soil (typically the sandy-clay
Key soil environmental properties, such as soil pH, oxygen, transition layers). Sandy soil is characterized by poor fertility
temperature, nutrient contents and resource availabilities are due to the low water-holding capacity and constriction of root
considered to shape soil microbial communities in deeper layers growth in the subsoil. Most researches on sandy soils have
(Eilers et al., 2012; Kramer et al., 2013; Xu et al., 2021). The ignored the often dense and barely permeable clay layer where
quantity and quality of soil organic matters (SOM) decline water and nutrients could be stored while hardly plant roots
through soil profiles because the input of root exudates and develop as compared with topsoil (Acosta-Martínez et al., 2010;
surface litters are richer in surface than deeper horizons Martirosyan et al., 2013; Sradnick et al., 2013). These harsh
(Trumbore, 2000) and thereby, strongly influencing microbial conditions likely exert environmental stresses on soil microbial
community composition. Microbial community composition also communities, enabling niche differentiations. Therefore, we
shifts along the soil depth, e.g., increasing relative abundance of investigated soil microbial diversity and functional patterns and
bacterial populations along the soil depth that are mainly their associations with the characteristics of sandy soil profiles
attributed to the difference in available phosphorus, exchange- typically crossing sandy-clay layers’ transition profile. Soil
able calcium and soil water availability (Fierer et al., 2003). The aggregate fractions and microbial community characteristics,
total microbial biomass strongly decreases with soil depth due as well as key nutrient indices across soil depth were quantified
to the variation in soil carbon (C) and nitrogen (N) contents to determine the main influencing factor on changes of soil
(Allison et al., 2010). The spatial configurations of soil C and N microorganisms. Microbial functions were evaluated by using
are further intensified by the heterogeneity of soil structural Functional Annotation of Prokaryotic Taxa (FAPROTAX) and
characteristics from pore to aggregates. Soil aggregates are Phylogenetic Investigation of Communities by Reconstruction
considered as the building block of soil structure, whereby of Unobserved States (PICRUSt). We hypothesized that small-
moisture, oxygen (and other gases) and nutrients fluctuate size aggregates fractions instead of environmental properties
among soil profiles, leading to the vertical variations in the regulated the microbial community compositions and that
abundance and composition of soil microbial communities diversities distributed heterogeneously in soil profile. We also
(Eilers et al., 2012). Soil aggregates (often with different sizes) hypothesized that microbial interactions would form different
supply physical protection of substrates utilized by microorgan- ecological niche under different precipitation, and that niche
isms at different degrees, thereby inducing different degree of differentiation would occur in deeper layers as well as that the
carbon accumulation and turnover in these size fractions associated microbial functions were due to the resource
(Kandeler et al., 2002). The aggregates distributions along soil availability and environmental stress.
vertical profile vary with ambient conditions and resources, thus
influence microbial population and community composition
2 Materials and methods
(Eilers et al., 2012; Fierer et al., 2010). For example, studies
have shown that enzyme activities related to C decomposition
are higher in macroaggregates, and organic carbon concentra- 2.1 Site description and soil sample collection
tion in microaggregates is generally higher than that of
macroaggregates (Qin et al., 2010; Nie et al., 2014). Soil Soil samples were taken separately from three croplands in
Yifei Sun et al. 3

Lishu County of Jilin Province (123°45′E, 43°02′N, LS), Kit for Soil (MPBIO Laboratories Inc., CA, USA). The
Langfang City of Hebei Province (116°38′E, 39°28′ N, LF), concentration and quality of extracted DNA were examined
and Zhengzhou City of Henan Province (112°42′E, 34°16′N, and determined through the absorption of DNA at 260 nm
ZZ), three kinds of typical sandy soil (according to Chinese using a NanoDrop® ND-2000c UV-Vis spectrophotometer
Soil Texture Classification) of unirrigated agricultural lands (NanoDrop Technologies, Wilmington, DE, USA). Then all the
with the average annual precipitations of 490 mm, 555 mm extracted DNA samples were stored under –80°C for further
and 662 mm in LS, LF and ZZ, respectively. The average molecular biology analysis. The V3 + V4 region of bacterial
annual temperatures are 6.4°C, 11.9°C and 14.3°C in LS, LF 16S rRNA gene was amplified with the primers barcoded–
and ZZ, respectively. The cropping systems are continuous 515F (GTGCCAGCMGCCGCGGTAA) and 806R (CCCCGY-
maize in LS and ZZ and wheat/maize rotation in LF. These CAATTCMTTTRAGT), traditionally used by the Earth Micro-
three sampling sites formed an increasing gradient in average biome Project (EMP). PCR reactions performed in a 50 μL
annual precipitation and temperature, but had similar profile mixture containing 25 μL Premix Ex Taq (Takara Biotechnol-
pattern and cropping system. Field area in each sampling site ogy), 5 μL of each primer (2 μM), 2 μLof diluted template DNA
was about 300 m2 (30 m  10 m). Soil samples were collected (1–10 ng) and 13 μL sterilized water. Thermal-cycling
from the top down to a clay layer (>80 cm) and separated as conditions were listed as follows: an initial denaturation of 3
follows: topsoil (0–20 cm), rooted zone beneath the plough min at 94°C, six touchdown cycles of 45 s at 94°C, 60 s from
layer (40–50 cm) and clay-layer soils (80–100 cm). Five 65°C to 58°C, 70 s at 72°C, followed by 22 cycles of 45 s at
composite soil samples from each depth were collected at 94°C, 60 s at 58°C, 60 s at 72°C with a final elongation of 10
each sampling site in May 2016, and each soil sample was min 72°C. PCR products were purified using a Wizard SV Gel
taken by mixing ten adjacent soil cores on each depth of each and PCR Clean-up system (Promega, San Luis Obispo, CA,
plot. The combined soil samples were immediately placed in USA). Concentrations of PCR products were fluorometrically
ice bag and sent to laboratory for further analysis of their quantified by Qubit dsDNA HS Assay Kit (Invitrogen,
environmental properties characterization and molecular Carlsbad, CA, USA) before being sequenced on the Miseq
biology. To this end, they were divided into two aliquots and platform (Illumina, San Diego, CA, USA) at Novogene, Beijing,
stored at 4°C and –80°C respectively. China.

2.2 Soil properties and aggregate fractions 2.4 Bioinformatics analysis

To determine soil water content of each sample, five-gram Raw sequences were processed in QIIME 1.7.0 (Caporaso et
soils were weighed and placed in an oven (101–00BS, China) al., 2010). The reads that could not be assembled were
at 105°C for 24 h. Soil organic matter (OM) was measured by removed, so did the chimeric reads. Sequences were quality
potassium dichromate oxidation methods (Nelson and Som- trimmed and clustered into operational taxonomic units
mers, 1982). Total nitrogen (TN) and total phosphorus (TP) (OTUs) at a 97% identity threshold using UCLUST (Edgar,
were quantified through combustion in a Thermo Scientific 2010). Taxonomic assignment was carried out through OTU
FLASH 2000 NC Analyzer (Vario EL III, Elementar, Germany). with the RDP Classifier (Wang et al., 2007). Alpha diversity
Soil available potassium (AK) was extracted with NH4OAc was characterized by richness (Shannon index) and even-
and determined using a flame photometer (M425, Sherwood, ness (Pielou index). Beta diversity was evaluated by
England) (Bao, 2000). calculating the Bray-Curtis distances. The Miseq sequencing
50-gram soils were wet-sieved using the method modified data are available online (http://www.ncbi.nlm.nih.gov/geo/).
from Six et al. (2000) to determine the water-stable aggregate The accession number for the MiSeq data is SRR9098510-
proportions. Briefly, soil sample was first placed on a 1 mm SRR9098555.
sieve and slaked by submersion in deionized water for 5 min.
The sieve was then gently moved up and down for 10 min.
2.5 Construction and characterization of phylogenetic
Remaining materials on sieve were rinsed into containers,
molecular ecological networks
while materials passing through 1.00 mm sieve mesh were
transferred to 0.50 mm, 0.25 mm and 0.053 mm sieves in turn Network analysis can evaluate the ecological interaction
for further fractionation, ultimately generating five aggregate patterns among microbial species in different environments
fractions (>1.00 mm, 0.50–1.00 mm, 0.25–0.50 mm, 0.053– (Ma et al., 2016; Jiang et al., 2017; Sung et al., 2017). The
0.25 mm, < 0.053 mm). Each fraction was dried (105°C) dominant species were often regarded as functionally
and weighed, then used to determine proportions of macro- important species in a community (Smith and Knapp, 2003).
(³ 0.5 mm) and microaggregates ( < 0.5 mm) in soils. In ecosystem, dominant taxa were thought to be more
functionally important than other taxa (Walker et al., 1999).
2.3 DNA extraction and sequence processing Keystone species in a network are important microbes that
may lead to a dramatic change of an ecosystem once
According to the manufacturer’s instruction, 0.5 g freezing- removed (Ze et al., 2013), which were often defined by the
dried soil was used to isolate total DNA with a FastDNA Spin degree of node-specific interactions for taxa within microbial
4 Aggregate sizes regulate the microbial community patterns in sandy soil profile

networks (Fisher and Mehta, 2014). Constructing and dissimilarity matrices between top and clay-layer soils. R
analyzing the microbial network provide a deeper under- (version 3.5.2) was used in data processing and statistical
standing of microbial communities at various circumstances, analysis. Network analysis was performed to visualize the
particularly the interrelationships between different species. changes of microbial interactions, and mantel test was to
Microbial interaction networks constructed based on 16S evaluate the effect of environmental elements on microbial
rDNA sequencing data were defined as phylogenetic mole- communities and interactions. The networks were visualized
cular ecological networks (Deng et al., 2012a). OTUs (at 97% with Cytoscape 3.6.1.
sequence identity) occurring in 80% of the total samples were
applied for network computation. The correlation between two
detected microorganisms was measured by Spearman 3 Results
correlation coefficient (r value). Only the similarity values
higher than a specific threshold were kept for calculating 3.1 Soil aggregates and environmental properties distribution
matrix eigenvalues. To allow comparison, an identical cut-off along soil profile
of 0.90 was used to construct the interaction networks.
Positive associations in networks indicated common preferred The distribution of soil aggregate size fractions was depicted
environmental conditions, interspecies cross-feeding, co- in Fig. 1. In general, microaggregates ( < 0.5 mm) dominated
aggregation or niche overlapping, whereas the negatives in all three samples, with macroaggregates (³ 0.5 mm)
suggested competition, niche partitioning or resistance to be accounting for 11.2% – 14.93% and 13.17% – 19.9% for the
grazed by predator in a food web (Deng et al., 2016). The top- and clay-layer samples, respectively. Specifically, 0.25 –
network construction and statistical analysis were performed 0.5 mm aggregates dominated in LS samples, comprising
using the existing pipeline available at http://ieg4.rccc.ou.edu/ 47.16% and 31.96% of the total aggregates in the top- and
mena (Deng et al., 2012b). clay-layer soils, respectively. As for the ZZ samples, aggre-
OTUs within a module are highly connected among gates of 0.053 – 0.25 mm were the most abundant group,
themselves but have less connections with OTUs outside occupying 47.95% and 42.85% in total aggregates in the top-
the group. Species within the same module are considered to and clay-layer soils, respectively. Microaggregates (0.053 –
have the same niche. Modularity (M) demonstrated a network 0.25 mm and < 0.053 mm) were of the majority for the LF
which could be naturally divided into communities or modules. samples, accounting for 35.27% and 43.82% in the top-layer
Random networks were used to evaluate whether the soils, and 39.31% and 36.49% in the clay-layer soils,
constructed networks were random, with the number of respectively.
nodes and links being constant between random and In the top-layer soils, water content (W) was 4.25%, 5.84%
experimental networks (Deng et al., 2012b). According to and 6.17% for the LS, LF and ZZ samples, respectively
the within-module connectivity (Zi) and among-module con- (seeing in Supplementary Fig. S1). Water content in the
nectivity (Pi), four topological roles of nodes were classified: deeper-layer soils at LS and LF remained nearly unchanged
(a) peripheral nodes (Zi£ 2.5, Pi£ 0.62), always linking to the as compared with those top-layer ones, while decreased from
nodes within their modules; (b) connectors (Z i £ 2.5, 6.17% to 3.56% for the ZZ samples. The organic matter (OM)
Pi > 0.62), highly linking to several modules; (c) module hubs reached at the highest value of 11.15 g kg–1 in the deeper-
(Zi > 2.5, Pi£ 0.62), which have a few links to many nodes in layer soils from ZZ, followed by the LS samples of 5.45 g kg–1
their own modules; and (d) network hubs (Zi > 2.5, Pi > 0.62), and LF ones of 5.33 g kg–1. Total nitrogen (TN) content
which act as both module hubs and connectors. seemed stable in LS and ZZ profiles, while significantly
decreased from top- to clay-layer soils. The mean TN content
in LF soil was 0.50 g kg–1, about 64% and 4% higher
2.6 Ecological and statistical methods
than those in LS and ZZ soils. Soil total phosphorus (TP)
The results in our study are shown as mean value, and declined with soil depth in all three sites with the lowest value
statistical significance is accepted at *P < 0.05, **P < 0.01 and of 1.1 g kg–1 in the clay-layer soil from LF. The average values
***P < 0.001. Pearson correlation analysis was used to assess of soil available potassium (K) were 133.27, 113.33 and
the correlations between soil properties and soil microbial 93.62 mg/ kg–1 in LF, LS and ZZ, respectively. The TN value
communities using SPSS 25. Based on the normalized significantly correlated with aggregates at size of 0.25 –
resample OTU table of 16S rRNA gene, we obtained alpha 0.5 mm (r = –0.499, P < 0.05), and the TP value with
diversity (Shannon index), canonical correlation analysis aggregates of 0.25 – 0.5 mm (r = 0.589, P < 0.01)
(CCA), function annotation for 16S rRNA gene by using and < 0053 mm (r = – 0.441, P < 0.05).
FAPROTAX (Louca et al., 2016) and PICRUSt (Langille et al.,
2013). A one-way ANOVA was used to identify significant 3.2 Variation of bacterial community compositions and
changes in soil properties and microbial community diversi- diversity patterns
ties. Permutation multivariate analysis of variance (PerMA-
NOVA) was used to evaluate the significant change of The RDP classifier with a threshold of 0.5 was applied to
microbial community compositions based on Bray-Curtis identify the dominant bacterial sequences to differentiate
Yifei Sun et al. 5

Fig. 1 Distribution of soil aggregate size fractions between surface (A) and clay-layer (B) in three sampling sites. LF: Langfang City (yellow);
LS: Lishu County (blue); ZZ: Zhengzhou City (green).

phylogenetic bacterial taxa levels. The relative abundances of PERMANOVA analysis revealed that the bacterial commu-
bacteria at the phylum and class levels between top and clay- nities for the ZZ soils differed significantly between the top-
layer soils from sampling sites were listed in Fig. 2. The results and clay-layer soils (P < 0.05). In addition, mantel tests and
indicated Proteobacteria, Actinobacteria, Thaumarchaeota, canonical correlation analysis (CCA) were performed to
Acidobacteria, Bacteroidetes, Chloroflexi, Planctomycetes investigate the relationship between environmental attributes
and Firmicutes were the most superior phyla, accounting for and the structure of microbial communities. Mantel tests
³ 85% of the total sequences (Fig. 2A). Generally, Proteo- based on Bray-Curtis distances demonstrated that OM was
bacteria dominated bacterial communities with the abundance significantly correlated with bacterial communities for the ZZ
of 23.3% and 21.41%, 19.54% and 23.67%, 22.34% and soils (Fig. 3C). The first two canonical axes explained 11.37%,
24.5% between top- and clay-layer from LS, LF and ZZ soils, 27.51% and 29.2% of the microbial communities from the LS,
respectively. Firmicutes was more abundant in LS than that in LF and ZZ soils, respectively. No significant change in
LF and ZZ soils, while contrary tendency was observed in diversity and evenness were observed within soil profiles in
Thaumarchaeota. Actinobacteria and Acidobacteria showed three sampling sites, in terms of Shannon index and Pielou
no significant changes between top- and clay-layer soils from evenness (Supplementary Fig. S2). Shannon index value of
three sampling sites. The relative abundance of Proteobac- bacterial communities for the LF soils ranged at 5.83– 6.30,
teria (r = – 0.832, P < 0.05) and Crenerchaeota (r = 0.814, which was higher than those of the LS and ZZ soils. The
P < 0.05) significantly correlated with the TN value, and highest Shannon index values of top-layer (6.86) and clay-
Actinobacteria (r = – 0.884, P < 0.05) was strongly affected layer (7.03) soils were observed in the ZZ soils, while the LF
by the OM. The relative abundance of Acidobacteria strongly soils hosting the lowest Pielou index values in both the top-
correlated with aggregates at size of 0.5 – 1 mm (Table 1) in layer (0.77) and clay-layer (0.84) soils. A generally negative
the top-layer soils. Crenerchaeota and Firmicutes had relationship was found between the Shannon index value and
significant relationship with aggregates at size of 0.053 – the percentage of aggregates under 0.053 mm for the clay-
0.25 mm and >1 mm in the clay-layer soils, respectively. layer soils yet the top-layer soils (Table 2).
Actinobacteria was obviously influenced by aggregates at
size of 0.5 – 1 mm in the top-layer and 0.25 – 0.5 mm in the 3.3 Soil microbial communities and interaction patterns
clay-layer soils. The most abundant class was Actinobacteria among sampling sites
(Fig. 2B) with the range of 11.93%–14.85% and 12.84%–
14.38% in top- and clay-layer soils, respectively. The relative The co-occurrence ecological network constructed by OTUs
abundances of Chloroplast and Bacilli were higher in LS than in different modules was depicted in Fig. 4. One module
those in LF and ZZ. LS had the highest abundance of suggested that the microbial communities within it should
Betaproteobacteria (6.53%) in topsoil and the lowest abun- have similar ecological niches. Hence, the soil microbial taxa
dance of Alphaproteobacteria (6.39%) in clay-layer soil. in LS were grouped into eight major ecological clusters
6 Aggregate sizes regulate the microbial community patterns in sandy soil profile

Fig. 2 Relative abundances (%) of microbial communities (phylum and class levels) across three sampling sites. The numbers shown in the
bar plot (left) indicated the relative abundance of the corresponding phyla; Different pattern represented microbial community based on
phylum level. LF: Langfang City; LS: Lishu County; ZZ: Zhengzhou City.

Table 1 P value of Pearson correlation between relative abundance of top 10 classified microbial phyla and aggregate size proportions of the top-
and clay-layer soils.
Gemmati-
Soil Aggregate Acidob- Actinob- Bacteroi- Chlor- Crenarch- Firmi- Nitro- Plancto- Proteobac-
mona-
depth sizes acteria acteria detes oflexi aeota cutes spirae mycetes teria
detes
Topsoil >1mm 0.462 0.412 0.691 0.797 0.920 0.707 0.627 0.414 0.649 0.591
a
0.5–1mm 0.022 0.029 0.868 0.762 0.479 0.267 0.932 0.855 0.910 0.968

0.25–0.5mm 0.105 0.155 0.742 0.636 0.353 0.140 0.806 0.981 0.784 0.842

0.053–0.25mm 0.615 0.665 0.232 0.126 0.157 0.370 0.296 0.509 0.274 0.332

< 0.053mm 0.346 0.296 0.807 0.913 0.804 0.592 0.743 0.530 0.765 0.707
Clay- >1mm 0.233 0.283 0.614 0.508 0.225 0.013 0.678 0.891 0.656 0.714
layer
0.5–1mm 0.669 0.719 0.178 0.072 0.211 0.424 0.242 0.455 0.220 0.278
soil
0.25–0.5mm 0.079 0.028 0.925 0.819 0.536 0.324 0.989 0.798 0.967 0.975

0.053–0.25mm 0.482 0.533 0.365 0.259 0.024 0.237 0.429 0.642 0.407 0.465

< 0.053mm 0.184 0.134 0.969 0.925 0.642 0.430 0.905 0.692 0.927 0.869
a
The bold ones indicate the significance of the relationship between microbial abundances and aggregate sizes distribution.
Yifei Sun et al. 7

Fig. 3 Canonical correspondence analysis (CCA) plots of microbial community composition at the OTU level in three sampling sites (A: LS;
B: LF; C: ZZ). LF: Langfang City; LS: Lishu County; ZZ: Zhengzhou City. Red triangles and green circles represented samples in surface and
clay-layer soils, respectively. “*” indicated the environmental properties significantly affected the microbial community compositions analyzed
by PERMANOVA (*P < 0.05).

Table 2 P value of Pearson correlation between microbial alpha-diversity and aggregate size proportions of the top- and clay-layer soils
Topsoil Clay-layer soil
Soil Aggregate
depth Inv_ Pielou_ Simpson_ Inv_ Pielou_ Simpson_
sizes Shannon Shannon
Simpson evenness evenness Simpson evenness evenness

Topsoil >1mm 0.259 0.367 0.096 0.445 0.186 0.252 0.171 0.274

0.5–1mm 0.181 0.807 0.536 0.886 0.254 0.692 0.270 0.714

0.25–0.5mm 0.308 0.934 0.662 0.988 0.380 0.819 0.396 0.840

0.053–
0.818 0.556 0.828 0.478 0.890 0.671 0.906 0.650
0.25mm

< 0.053mm 0.144 0.482 0.211 0.561 0.071 0.367 0.055 0.389
Clay- >1mm 0.436 0.939 0.790 0.860 0.508 0.946 0.524 0.968
layer
0.5–1mm 0.872 0.502 0.774 0.424 0.944 0.617 0.960 0.596
soil
0.25–0.5mm 0.124 0.750 0.479 0.829 0.197 0.635 0.213 0.657

0.053–
0.685 0.689 0.960 0.611 0.758 0.804 0.774 0.782
0.25mm

< 0.053mm 0.019a 0.644 0.373 0.723 0.091 0.529 0.107 0.551
a
The bold ones indicate the significance of the relationship between microbial diversities and aggregate sizes distribution.

(modules) comprising of populations strongly co-occurring (Fig. 4C), rather than those in LS and LF. The results from
with each another (Fig. 4A), and the network structures in LF mantel test showed that soil properties had limited effect on
(Fig. 4B) and ZZ (Fig. 4C) were clustered into merely two soil microbial network, except for OM (r = 0.12, **P < 0.01) and
modules. The networks became more clustered in terms of available K (r = 0.117, **P < 0.01) in LF.
less nodes and more links in microbial networks from LS, LF to Topological roles of the OTUs identified are shown as a Z-P
ZZ (Supplementary Table S1). Average connectivity (avgK) plot (Fig. 5). Generally, the majority of OTUs were peripherals,
and edges were of the highest in the ZZ soils which had the which were mainly linking species within their modules. Some
least nodes. The nodes with higher connectivity are usually of the peripherals from LS and ZZ even barely linked outside
considered as key species, which occupies the central to their own modules (Pi = 0). While the proportion of module
positions of the networks. The phylum of key species was hubs functioned in connecting species within modules were of
significantly different among three sampling sites. Proteobac- the highest in the ZZ networks (1.32%) (Fig. 5C). Gemmati-
teria and Crenerchaeota dominated in LS and LF networks, monadetes Gemm-1 (OTU_30) and Bacteroidetes sapros-
respectively. These two phyla were both important in ZZ pirae (OTU_890) were two module hubs in the ZZ network
network structure. OTUs, belonging to Acidobacteria, had (Fig. 5C). One module hub (OTU_77), belonging to Plancto-
more complex interactions with the other phyla in the ZZ soils mycetes Planctomycetia, was found in the LS network (Fig.
8 Aggregate sizes regulate the microbial community patterns in sandy soil profile

Fig. 4 The co-occurrence ecological network constructed by OTUs in different modules (A: LS; B: LF; C: ZZ; D: surface; E: clay layer). LF:
Langfang City; LS: Lishu County; ZZ: Zhengzhou City. Each node represents an OTU, and the connectivity between two OTUs is indicated by
the edges. Nodes colors signify different phylum. Bigger nodes represent the dominant taxa with a high degree of nodes. The red and green
links indicate positive and negative correlation, respectively.

5A). Network structure of LS showed higher proportion of Crenarchaeota and Proteobacteria close to Thaumarchaeota
connectors linking to the other modules (5.56%) (Fig. 5A). As and Alphaproteobacteria, respectively. Two connectors
for the LS soils, two of the six connector OTUs (OTU_11292 observed in the ZZ network (Fig. 5C) were from Acidobacteria
and OTU_235) were from Actinobacteria that were closely Chloracidobacteria and Firmicutes Bacilli. No network hubs
related to Actinobacteria and MB-A2-108, respectively were detected out in all three networks. No module hubs and
(Fig. 5A). The other four connectors were derived from connectors were found in LF network.
Yifei Sun et al. 9

3.4 Changes of interaction patterns and key species along soil clay-layer soils (Fig. 6A). Specifically, species in module 7
profile had more complex interactions than the other modules,
where Crenarchaeota occupied an important position and
Microbial taxa were specified into two modules in both top- became more abundant in clay layer along with Proteobac-
teria. The Proteobacteria OTU number in the top-layer
(Fig. 4D) and clay-layer soils (Fig. 4E), with Proteobacteria soils was significantly larger than that of the clay-layers
and Actinobacteria playing a more important role in the top- in module 8 (Fig. 6B). The relative soil microbial abundance
layer soils as compared to that of clay-layer soils. One module was at the similar level for the top- and clay-layer soils
hub (OTU_2478), belonging to Bacteroidetes, was found in across all three sampling sites, except for Crenarchaeota
the clay-layer network (Fig. 5E). New network was recon- which had higher abundance in the top-layer soils
structed by selecting OTUs in both top- and clay-layer soils to (Fig. 6C).
better understand the interaction patterns among different The global properties of empirical network indicated that
phyla between top- and clay-layer soils (Fig. 6). Twelve OTU_11292 (Actinobacteria) and OTU_6 (Proteobacteria)
modules were classified in microbial networks dominated played a dominant role in the microbial interactions in the top-
by a positive correlation (63.7%) between the top- and and clay-layer soils, respectively (Supplementary Table S1),

Fig. 5 Z-P plot exhibiting the distribution of OTUs based on the topological roles (A: LS; B: LF; C: ZZ; D: surface; E: clay layer). LF: Langfang
City; LS: Lishu County; ZZ: Zhengzhou City. Each point represents an OTU. The location of each OTUs determined according to the within-
module connectivity (Zi) and among-module connectivity (Pi). The module hub is defined according to Zi and Pi values (Zi>2.5, Pi£ 0.62).
The four identified module hubs were marked with OTU numbers and its phylum.
10 Aggregate sizes regulate the microbial community patterns in sandy soil profile

Fig. 6 Changes of microbial interaction between surface and clay layer soils. A:Reconstructed network by selecting OTUs in both surface
and clay-layer soils; B: Differences in numbers of OTUs in phylum level; C: Distribution of microbial communities in phylum level between
surface and clay layer soils among three sampling sites. The number of the network signify the module number. LF: Langfang City; LS: Lishu
County; ZZ: Zhengzhou City.

evidenced by the maximum degree and eigenvector centrality. competition between Proteobacteria and Actinobacteria
To better understand the microbial interaction patterns along became stronger across the soil depth, causing decreased
soil profile, two new networks through selecting the first relative abundance. We also restructured network by select-
neighbors of OTU_11292 and OTU_6 in the top- and clay- ing the first-neighbors of Proteobacteria and Actinobacteria to
layer soils were visualized in Fig. 7A and 7B. In the top-layer evaluate whether specific species contributed to such
soil, OTU_11292 and OTU_6 emerged in the same module, competitions (Fig. 7E and 7F). The results showed that
indicating the same ecological niche (Fig. 7A). While Proteobacteria was more closely related with the other
OTU_11292 transferred into the other module away from species (OTU) than Actinobacteria in both top- (Fig. 7E) and
OTU_6 in the clay-layer soils (Fig. 7B), indicating that niche clay-layer soils (Fig. 7F). OTU_30 (Gemmatimonadetes
differentiation occurred in the deeper-layer soils. The recon- Gemm-1) had more complex interaction with OTUs belonging
structed networks by selecting Proteobacteria and Actino- to Crenerchaeota and Bacteroidetes in clay-layer network
bacteria showed that the clay-layer network had more (Fig. 7H), and negatively correlated with OTU_2478 (the
negative correlations (55.37%) as compared with that of the module hub that enhanced the connectivity within module)
top-layer soils (37.3%) (Fig. 7C and 7D). It indicated that the and a new species OTU_75.
Yifei Sun et al. 11

Fig. 7 The reconstruct network by selecting the first- neighbor of OUT_6 and OUT_11292 in surface (A) and clay-layer soils (B). Microbial
network of surface and clay-layer soils based on the interaction between Proteobacteria and Actinobacteria (C–D). Restructured network by
selecting the first neighbor of Proteobacteria and Actinobacteria, respectively (E–F) and OTU_30 (G–H) from surface and clay-layer soil
networks, respectively.

3.5 Microbial function prediction between the top- and clay- 4 Discussion
layer soils

FAPROTAX and PICRUSt were adopted to predict ecological In this study, the changes of environmental properties,
and biological functions to the bacteria OTUs. A total of 53 microbial community composition and functional networks
categories were linked to the bacterial communities according between top- and clay-layer soils were mainly driven by niche
to the FAPROTAX with the dominant functional groups (>1%) differentiation typically in available nutrient resources and
being assigned as nitrification, aerobic ammonia oxidation, other environmental characteristics (Smith et al., 2014).
chemoheterotrophy, aerobic chemoheterotrophy and fermen- Significant correlations between TN and TP and aggregates
tation (Supplementary Fig. S3A). KEGG showed (Supple- observed in this study also confirmed that different distribu-
mentary Fig. S3B) that the majority of predicted protein tions of soil aggregates along soil profile directly contributed to
sequences was associated with the functions involved in manipulating the processes of nutrient movements. The TP
metabolism (44.34%–44.93%), genetic information proces- significantly declined from top- to clay-layer soils in all three
sing (16.23%–16.90%), environmental information processing sampling sites, and the OM strongly decreased from the top-
(12.86%–13.57%), organismal systems (7.40%–7.72%) and to clay-layer soils sampled in LS and LF. The relative
cellular processes (3.23%–3.83%). abundance of microbial community in phylum level was highly
12 Aggregate sizes regulate the microbial community patterns in sandy soil profile

related to aggregates proportion in the clay-layer soils, rather microbial community patterns (Bending et al., 2004). Aggre-
than that of the top-layer soils in this study, likely due to the gate fraction with different sizes formed different pore
differences in available nutrients between the top- and clay- networks through soil profile and induced difference in nutrient
layer soils. The clearly different soil aggregates distributions availability and water retention at soil depth. Petersen et al.
diversified the environmental characteristics along soil profile (2016) found that pore-size distribution systematically affects
and thereby, variable resources availability and niches soil water retention. Microbial communities were shaped
differentiation for hosting the large variety of microbial partly by different resources and environmental conditions
communities (Bending et al., 2004). within different aggregate sizes. The resources composition
In addition, aggregate distribution directly contributed to and availability in microaggregates were considered to be
manipulating the processes of water evaporation, pore-size more favorable for microbes to utilize than those in macro-
configuration in soil profile, which conspired to determine aggregates, even though the later ones often had higher
resources and physical niches availability to the residing resource content (Mummey et al., 2006; Davinic et al., 2012).
microbial populations. The top-layer soils often experienced Studies revealed that soil OM in microaggregates with higher
more frequently varying water configuration as compared with OC and N concentrations may have higher turnover rate and
the relatively stable ones in the deeper-layer (clay-layer) soils. thereby, having a stronger selectivity for microbial community
This might induce changes of resources availability and other pattern as compared with macroaggregates (Cheng et al.,
environmental characteristics (to the hosting microbes) 2011; Davinic et al., 2012; Nie et al., 2014). The results
across soil profile, hosting distinctly functioning microbial observed that the alpha-diversity of soil microbial commu-
communities. Soil water content might be partially responsible nities was obviously correlated with microaggregates propor-
for the differentiation of microbial communities with depth in tion ( < 0.053 mm) in clay-layer soils, evidencing that
sandy soil. Studies had showed that variability in soil moisture resources availability of microaggregates was a driving factor
could influence both bacterial and fungal community compo- regulating microbial community patterns.
sitions (Brockett et al., 2012; Li et al., 2017). Our results The results of microbial networks analysis provided some
showed that the dominant phyla in microbial networks differed fresh information about the differences in microbial community
between the top- and clay-layer soils, with a negative interactions by evaluating distribution patterns of functional
correlation being stronger in the clay-layer soils, which was groups among different niche. The network complexity
most likely attributed to the OTU_30 that belongs to increased with soil water content, suggesting that favorable
Gemmatimonadetes Gemm-1. Gemmatimonadetes was water content should support more stable and complex
often inversely correlated to moisture (Bowen et al., 2012), microbial network structure. Wang et al. (2018) also observed
indicating that microbial groups specialized for deeper soils that microbial networks became more complex as the
had a high tolerance for moisture stress and the variation of precipitation increases. Acidobacteria and Bacteroidetes
microbial networks were associated with the water content were more abundant in ZZ, supporting that these two phyla
between the top- and clay-layer soils. While the soil water became more dominant at increased precipitation (Cho and
content did not vary appreciably or the variability was much Jang, 2014; Zhang et al., 2016; Li et al., 2017;).
higher at the top-layer soils than those of deeper-layer ones
(Supplementary Fig. S1), suggesting that the moisture within
aggregates to be a driving force regulating microbial commu- 5 Conclusion
nity compositions and functions. Therefore, it is confident that
microenvironment induced by soil aggregates distribution To our knowledge, few studies investigated the influence of
profoundly affects the compositions and interactions of soil aggregates on microbial communities in sandy soil profile.
microbial communities, with the resources availability being Microbial interactions and key species significantly changed
of a key factor responsible for the changes of microbial between top- and clay-layer soils due to the more intense
communities through soil profile. competition in clay-layer soils, thereby inducing the niche
Aggregates comprise the microenvironment where the differentiation through soil profile. Small-size aggregates
physical properties (water holding capacity, structural stability) typically in deeper clay-layer soils, relative to macroaggre-
and chemical properties (cation exchange capacity, organic gates from the top-layers, play a more important role in
matter content and quality) were different among those of determining the microbial community abundance and struc-
different sizes. Microaggregates (0.053 – 0.25 mm) were more ture. The combined analysis of soil structure, microbial
abundant in clay-layer soils than those top-layer ones, and community composition and microbial networks provided a
microbial relative abundance was tightly related to the comprehensive insight in evaluating the influencing factors on
aggregate proportions typically in the clay-layer soils yet the variations of soil microbial community in sandy soil profile.
top-layer ones. It suggested that the microaggregates to be Nevertheless, studies on nutrients and microbial community
more beneficial to microbial growth. Difference in soil OM structure within different size aggregates are needed to better
quality within aggregate fractions diversified substrates understand the mechanisms of niche differentiation in soil
availability in situ, which directly contributed to the nature of profile under long-term agricultural practice.
Yifei Sun et al. 13

Conflict of interest Reeder, J., Sevinsky, J.R., Turnbaugh, P.J., Walters, W.A.,
Widmann, J., Yatsunenko, T., Zaneveld, J., Knight, R., 2010.
The authors declare no conflict of interest. QIIME allows analysis of high–throughput community sequencing
data. Nature Methods 7, 335–336.
Acknowledgments Castellazzi, M.S., Brookes, P.C., Jenkinson, D.S., 2004. Distribution
of microbial biomass down soil profiles under regenerating
The authors acknowledge the financial supports of the National woodland. Soil Biology & Biochemistry 36, 1485–1489.
Basic Research Program of China (Grant no. 2016YFD0200306), Cheng, X., Luo, Y., Xu, X., Sherry, R., Zhang, Q., 2011. Soil organic
the National Natural Science Foundation of China (Grant no. matter dynamics in a North America tallgrass prairie after 9 yr of
41877412), and the Scholarship of the ‘National 1000 (Young) experimental warming. Biogeosciences 8, 1487–1498.
Talents Program’ of China. Cho, B.C., Jang, G.I., 2014. Active and diverse rainwater bacteria
collected at an inland site in spring and summer 2011. Atmospheric
Electronic supplementary material Environment 94, 409–416.
Davinic, M., Fultz, L.M., Acosta-Martinez, V., Calderόn, F.J., Cox, S.
Supplementary material is available in the online version of this B., Dowd, S.E., Allen, V.G., ZakJ.C., Moore-KuceraJ., 2012.
article at https://doi.org/10.1007/s42832-021-0095-1 and is Pyrosequencing and mid–infrared spectroscopy reveal distinct
accessible for authorized users. aggregate stratification of soil bacterial communities and organic
matter composition. Soil Biology & Biochemistry 46, 63–72.
Deng, Y., He, Z., Xiong, J., Yu, H., Xu, M., Hobbie, S.E., Reich, P.B.,
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