FEMS Microbiology Ecology, 96, 2020, fiaa114

doi: 10.1093/femsec/fiaa114
Advance Access Publication Date: 8 June 2020
Research Article

RESEARCH ARTICLE

Soil microbial succession following surface mining is

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governed primarily by deterministic factors
Jennifer L. Kane1 , Ember M. Morrissey1 , Jeffrey G. Skousen1 and Zachary
B. Freedman1,2, *
1
West Virginia University, Division of Plant and Soil Sciences, 1206 Evansdale Drive, Morgantown, WV 26506,
USA and 2 Current Address: University of Wisconsin-Madison, Department of Soil Science, 1525 Observatory
Drive, Madison, WI 53706, USA
∗
Corresponding author: University of Wisconsin-Madison, Department of Soil Science, 1525 Observatory Drive, Madison, WI 53706, USA. Tel: 608 262
2633. E-mail: zfreedman@wisc.edu
One sentence summary: Soil microbial communities were influenced more by environmental factors than by time following surface mining disturbance.
Editor: Petr Baldrian

ABSTRACT
Understanding the successional dynamics governing soil microbial community assembly following disturbance can aid in
developing remediation strategies for disturbed land. However, the influences shaping microbial communities during
succession following soil disturbance remain only partially understood. One example of a severe disturbance to soil is
surface mining for natural resources, which displaces communities and changes the physical and chemical soil
environment. These changes may alter community composition through selective pressure on microbial taxa (i.e.
deterministic processes). Dispersal and ecological drift may also shape communities following disturbance (i.e. stochastic
processes). Here, the relative influence of stochastic and deterministic processes on microbial community succession was
investigated using a chronosequence of reclaimed surface mines ranging from 2–32 years post-reclamation. Sequencing of
bacterial and fungal ribosomal gene amplicons coupled with a linear modeling approach revealed that following mine
reclamation, while bacterial communities are modestly influenced by stochastic factors, the influence of deterministic
factors was ∼7 × greater. Fungal communities were influenced only by deterministic factors. Soil organic matter, texture,
and pH emerged as the most influential environmental factors on both bacterial and fungal communities. Our results
suggest that management of deterministic soil characteristics over a sufficient time period could increase the microbial
diversity and productivity of mine soils.

Keywords: succession; deterministic; stochastic; chronosequence; surface mining

INTRODUCTION ever, the mechanisms governing such patterns among micro-

Ecological succession
Ecological succession can be used as a framework to predict
changes in community composition over time (Black 1965). In
plant communities, ecological succession has been widely stud-
ied (e.g. Grime 1979; Tilman 1988; Morris and Leger 2016); how-
bial communities remain unclear. Current theory in microbial
ecology postulates that selection, diversification, dispersal, and
drift mediate microbial community succession (Vellend 2010;
Nemergut et al. 2013). Still, questions remain regarding the rela-
tive influence of stochastic (e.g. dispersal, ecological drift) and
deterministic (e.g. selection, diversification) processes on soil

Received: 1 April 2020; Accepted: 5 June 2020


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1
 2 FEMS Microbiology Ecology, 2020, Vol. 96, No. 11
microbial communities across environmental and temporal gra-
dients (Nemergut et al. 2013; Rocca et al. 2019). To date, investi-
gations of microbial community assembly mainly explore suc-
cession after natural disturbances like glacial retreat (Jumppo-
nen 2003; Cline and Zak 2014; Freedman and Zak 2015a), fire
(Hart et al. 2005; Gassibe et al. 2011; Ferrenberg et al. 2013), or vol-
canic eruption (Ibekwe et al. 2007; Zeglin et al. 2016). Fewer stud-
ies on microbial community assembly focus on anthropogenic
disturbances like land use change (Lauber et al. 2008; Jesus et al.
2009; Cline et al., 2017) or physical disturbances like tillage (Lup-
wayi, Rice and Clayton 1998; Calderón et al. 2000). Understanding
microbial succession following disturbance is vital, as the soil
microbial community contributes to key ecosystem services (e.g.
nutrient cycling, mutualisms with plant hosts) and may con-
tribute to the future ecological success of a previously disturbed
environment (Harris, 2003; Fierer et al. 2012).
During succession, selection imposed by deterministic fac-
tors (e.g. changes in soil chemical and physical properties)
affects the presence, absence, and relative abundance of micro-
bial taxa within communities as species unable to acclimate are
lost and those suited to the changing environment become dom-
inant (Keddy 1992; Girvan et al. 2003; Lauber et al. 2008). Deter-
ministic factors driving microbial community composition may
include soil pH which is influential at the global scale (Fierer and
Jackson 2006; Lauber et al. 2009; Rousk et al. 2010), and other
factors such as soil moisture, total carbon (C), total nitrogen
(N), and C:N ratio (Högberg, Högberg and Myrold 2007; Lauber
et al. 2008; Freedman and Zak 2015a; Romanowicz et al. 2016). In
contrast, succession can be influenced by stochastic processes
which occur randomly. These include dispersal (e.g. by wind and
water) and ecological drift (Nemergut et al. 2013; Vellend 2010).
Further, stochastic and deterministic processes can interact to
influence microbial community composition in soil (Caruso et al.
2011; Ferrenberg et al. 2013; Cline and Zak 2014; Freedman and
Zak 2015a). While soil microbial community assembly in anthro-
pogenically disturbed soils is not widely studied, deterministic
factors such as bulk density, water-holding capacity, pH, and
plant species can be influential in shaping microbial commu-
nities following physical, anthropogenic disturbance of the soil
profile (Calderón et al. 2000; da C Jesus et al. 2009; Dimitriu and
Grayston 2010; Peschel et al. 2015; Li, Liu and Chen 2016). Under-
standing the deterministic factors that influence microbial com-
munity succession following anthropogenic disturbance may
enable more timely and effective soil reclamation strategies.
Surface mining and microbial community assembly
One particularly severe anthropogenic disturbance to soil is sur-
face mining for natural resources (e.g. coal, iron ore). In the
United States, upwards of 2.5 million hectares of land have
been surface mined over the past 100 years (Emerson, Skousen
and Ziemkiewicz 2009). The goal of many post-mining land
uses is economically and/or ecologically beneficial for above-
ground productivity (e.g. agriculture, forestry), which relies on
soil microbial function for resources but also contributes to soil
restoration by the addition of litter and root exudates (Fig. 1;
Skousen and Zipper 2014; Macdonald et al. 2015). During the pro-
cess of surface mining, vegetation and soil is removed from the
landscape to access the coal seam and the topsoil is stored for
later use in the reclamation process (Skousen and Zipper 2014).
This results in a loss of soil structure and vegetative cover as
well as spatial displacement of the established microbial com-
munities. Further, despite the storage and use of native top-
soil to reclaim sites, mine soils often exhibit adverse chemical
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Figure 1. Conceptual model of mining disturbance impacts on soil microbial
communities. Here, deterministic (e.g. soil chemical and physical properties) and
stochastic (e.g. time) processes interact during succession and influence post-
mining soil health and aboveground productivity.
and physical properties for years after reclamation (e.g. low soil
organic matter content, high bulk density; Emerson, Skousen
and Ziemkiewicz 2009). Since dominant vegetation influences
belowground communities through the addition of organic mat-
ter to soil (e.g. litter addition, root exudation) and other means,
successful establishment of vegetation is important to achieve
whole-ecosystem improvements post-mining. In particular, the
interplay between organic matter inputs from aboveground veg-
etation and its decomposition by the microbial community reg-
ulates in part the restoration of key soil features, such as accu-
mulation of nutrients (Veselá, Mudrák and Frouz 2018). Soil
microbial communities may recover over time post-reclamation,
affecting the overall success of the post-mining land use in
terms of aboveground productivity and soil fertility, for instance
by obtaining and storing N as biomass (Józefowska et al. 2017).
In this way, the nature of surface mining makes reclaimed mine
soils a novel system to investigate soil microbial community
succession.
To date, there has been some insight into microbial commu-
nity succession following surface mining (Banning et al. 2008;
Dimitriu et al. 2010; Li, Liu and Chen 2016; Sun et al. 2017). For
example, it has been established that physical disturbances such
as mining, tillage, and land use change (e.g. from forest to grass-
land) can cause declines in ecologically important soil physical
and chemical parameters (e.g. pH, C: N) as well as reductions
in microbial activity, biomass, and diversity, which is sugges-
tive of deterministic selection (Calderón et al. 2000; Banning et al.
2008; da C Jesus et al. 2009; Dimitriu et al. 2010; Li, Liu and Chen
2016; Sun et al. 2017). Stochastic processes also shape micro-
bial community succession following disturbance, for example,
through barriers to dispersal or genetic bottlenecks (Verbruggen
et al. 2012; Schmidt et al. 2014; Sun et al. 2017). However, the rela-
tive influence of stochastic and deterministic factors on micro-
bial communities during succession following surface mining
remains unexplored.
Objective and hypotheses
Here, we assessed the relative influence of deterministic and
stochastic processes on soil microbial communities across a
chronosequence of reclaimed mine sites in West Virginia (WV),
USA. Our hypotheses were: 1) The relatively stressful conditions
imposed on soil microbial communities following mining and

reclamation would elicit strong deterministic influences on suc-
cession while stochastic processes would play a smaller role. 2)
With time since reclamation, soil physical, chemical, and biolog-
ical properties would tend towards that of an undisturbed refer-
ence site.
METHODS
Study site description
To investigate soil microbial community succession following
surface mine land reclamation, four reclaimed mine sites in
Monongalia County, WV were selected, two of which (Mylan Park
and New Hill) have been previously studied and their soil and
vegetation described within the last decade (Chaudhuri et al.
2011; Chaudhuri et al. 2012; Chaudhuri et al. 2013; Chaudhuri
et al. 2015). These four sites were owned by the same com-
pany and were mined and reclaimed in the same manner, dif-
fering only in the time since mining and reclamation occurred
(Table 1). The sites comprise a chronosequence, including four
sites which were sampled 2, 10, 15, and 32 years post-mining
and reclamation, respectively. The four sites that comprised the
chronosequence were mined using contour mining methods.
After coal removal, sites were reclaimed by backfilling the over-
burden and regrading the land to the approximate original con-
tour. Topsoil was then compacted with bulldozers to a depth
of approximately 20 cm in accordance with the Surface Mining
Control and Reclamation Act of 1977. The sites were then reveg-
etated with mixed grass and leguminous species such as orchard
grass (Dactylis glomerata), clover (e.g. Trifolium spp.), birdsfoot tre-
foil (Lotus corniculatus), and fescue grasses (Festuca arundinacea;
Chaudhuri et al. 2013). Due to their close geographical proximity,
the sites experience similar climactic conditions and are of sim-
ilar geology (sandstone, shale, siltstone, and limestone), hence
the sites across the chronosequence have experienced similar
soil forming conditions (Chaudhuri et al. 2013).
Sampling and experimental design
Sites were sampled in September of 2017, a time during which
warm temperatures and high moisture favor microbial activ-
ity. According to the topographic relief of each site, two 50 ×
50 m plots were defined in lowland and highland regions at
each of the four chronosequence sites (i.e. eight plots total across
the chronosequence). While the topographic relief is not major
(<10 m difference in elevation between highland and lowland
plots), it was confirmed by gravimetric water content measure-
ments that soil moisture content is greater in lowland plots than
in the highland plots, which may have implications for micro-
bial community succession (Cong et al. 2015; Stark and Firestone
1995; Suseela et al. 2012). From both the lowland and highland
plot at each site, five random soil samples were taken to a 10-
cm depth using a shovel, homogenized by hand, and kept on ice
until return to the lab, where they were passed through a 2 mm
sieve. Subsamples were kept at -80åC for DNA analysis and the
remainder was kept at 4åC for biological and chemical analyses.
From each homogenized sample (i.e. five samples taken in the
field and mixed), three subsamples were analyzed to account for
the high variability in microbial communities. To serve as a ref-
erence, highland and lowland plots were established at the West
Virginia University (WVU) Organic Farm (39å38’48’ N, 79å56’14’
W) on an undisturbed perennial pasture and were sampled in
September of 2018 in a similar manner to the chronosequence
sites. This site was chosen due to its close geographic proximity
Kane et al. 3
to the chronosequence sites, as well as its similar management
practice (i.e. grass-legume pasture). In all, three replicate sam-
ples from both highland and lowland plots were analyzed from
each of the four chronosequence sites and the reference site,
resulting in 30 total samples.
Soil physical and chemical analyses
Soil physical and chemical parameters that have been deter-
mined to influence soil microbial activity and diversity were
assessed (Fierer, Bradford and Jackson 2007; Freedman and Zak
2015a; Romanowicz et al. 2016). Gravimetric soil moisture was
determined by drying 10 g of fresh soil at 105åC for 24 hours
(Black 1965). Soil pH was measured using a 1:5 soil: CaCl2 sus-
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pension of air dried soil (Rayment and Higginson 1992) on an
Accumet AE150 pH meter (Fisher Scientific, Hampton, NH, USA).
Organic matter content was determined by loss-on-ignition,
whereby dried soil from moisture analysis was combusted at
500åC for 6 hours (Ball 1964). Percent clay was estimated using
the Texture-by-Feel method (Thien 1979). Total C and total N
were determined by combustion of ∼250 mg of soil on a vario
MAX cube (Elementar, Landenselbold, Germany). The bioavail-
able carbon pool was estimated according to the Permangenate
Oxidizable Carbon Method (Weil et al. 2003). Soil N and P were
extracted from bulk soil in 1 M KCl and Modified Morgan extract
respectively by shaking at 100 RPM for 90 minutes followed by
filtration through a Whatman #42 filter (Whatman, Maidstone,
UK). Extracts were stored at -20åC until analysis using an AQ300
Discrete Chemical Analyzer (SEAL Analytical, Mequon, WI, USA)
following U.S. Environmental Protection Agency (EPA) methods
353.2 for nitrate and nitrite, 350.1 for ammonium, and 365.1 for
phosphate. Total organic N was estimated by subtracting the
inorganic N (sum of nitrate, nitrite, and ammonium) concentra-
tion from total N content.
Soil microbial biomass
Microbial biomass was estimated by substrate induced respi-
ration (Anderson and Domsch 1978) whereby 7 g (dry-weight
equivalent) of soil were incubated with 7 mL of 1.2% yeast extract
and CO2 levels were measured using a LI-COR 6400XT fitted with
a Trace Gas Sampler (LI-COR Biosciences, Lincoln, NE, USA) at 0,
2, and 4 hours. Carbon respired hour−1 gram−1 soil was calcu-
lated and used as a proxy for microbial biomass (sensu Fierer,
Schimel and Holden 2003).
DNA extraction and sequencing
DNA was extracted from 0.5 g of homogenized soil using a
DNeasy PowerLyzer PowerSoil Kit (Qiagen, Hilden, Germany)
following the manufacturer’s instructions. DNA was quality
checked and quantified using NanoDrop and Qubit, respec-
tively (ThermoFisher Scientific, Waltham, MA). Bacterial 16S
rRNA genes were amplified using barcoded primers 515f (Parada,
Needham and Fuhrman 2016) and 806r (Apprill et al. 2015).
Fungal internal transcribed spacer (ITS) region amplicons were
amplified using primers ITS1f and ITS2 (White et al. 1990;
Gardes and Bruns, 2003). All PCR amplifications were per-
formed according to Earth Microbiome Project standard pro-
tocols (Thompson et al. 2017). Libraries were built from the
resulting amplicons and sequenced at the University of Min-
nesota Genomics Center using the MiSeq platform (V3 chem-
istry; Illumina, San Diego, CA, USA) and a dual-indexed approach
(Gohl et al. 2016). Amplicon sequences have been deposited
 4 FEMS Microbiology Ecology, 2020, Vol. 96, No. 11
Table 1. Reclaimed mine sites that comprise the reclamation chronosequence. More detailed site descriptions can be found in Chaudhuri et al.
(2013).
Shafer
Years since reclamation 2 10
Latitude 39◦ 38’ 26” N
Longitude 80◦ 01’ 59” W
Altitude (m) 390.63
Dominant soil series‡ mesic Typic
Udorthents
Soil texture class Clay Loam
Coal seam
Overburden type
Mining Method
Reclamation method Backfilling followed by application of 20 cm of topsoil and grading
Post-reclamation land type
10-year average annual precip. (mm)†
10-year average annual temp. (åC)†
†
Source: NOAA National Centers for Environmental Information
‡
Source: USDA NRCS Soil Survey
to the National Center for Biotechnology Information (NCBI)
Sequence Read Archive (SRA) repository under accession #PRJNA
529237.
DNA sequence processing and quality control
Forward and reverse reads were merged by alignment to yield
a single sequence in USEARCH (Edgar 2010). Prior to align-
ment, reads which had a quality score of less than 25 were
truncated and sequences with more than 10 mismatches in
the alignment were removed from the data set. Operational
Taxonomic Units (OTUs) were picked using QIIME (Version
1.9.1; Caporaso et al. 2010) at the 97% similarity level using
the open-reference method with default parameters. OTUs
that occurred less than five times across the dataset were
removed. Bacterial taxonomy was assigned using the UCLUST
algorithm (Edgar 2010) against the Green Genes database (Ver-
sion 13.8; McDonald et al. 2012). Fungal taxonomy was assigned
by the Ribosomal Database Project Naı̈ve Bayesian Classifier
(Wang et al. 2007) using the UNITE fungal ITS trainset (Nils-
son et al. 2019; Abarenkov et al. 2010). Fungal OTUs with tax-
onomic assignments to the genus level were then assigned
to functional guilds using FUNGuild (Nguyen et al. 2016). Prior
to analysis, bacterial and fungal sequences were rarefied to
5000 and 1300 sequences, respectively. For each site within
the chronosequence, bacterial and fungal richness was cal-
culated using the Chao1 metric (Chao 1984) and β-diversity
was estimated using Bray–Curtis dissimilarity (Bray and Curtis
1957).
Statistical analyses
All statistical analyses were performed in R (Version 3.4.1) using
the Vegan package (Oksanen et al. 2019) or Primer (PrimerE, Ver-
sion 7); an α level of 0.05 was accepted as significant. Residu-
als of the environmental variables were checked for normality
using the Shapiro-Wilk Normality Test (Shapiro and Wilk 1965).
Prior to analysis, non-normal data were normalized using arc-
sine, square root, and natural logarithm transformations where
appropriate.
New Hill Metz Mylan Park
15 32
39◦ 40’ 24” N 39◦ 38’ 48” N 39◦ 38’ 26” N
80◦ 03’ 13” W 80◦ 01’ 32” W 80◦ 01’ 59” W
371.44 395.55 390.00
mesic Ultic mesic Typic mesic Typic
Hapludalfs Udorthents Udorthents
Silty Clay Loam Silty Clay Loam Silty Clay Loam
Waynesburg
Sandstone (70%–80%), shale (20%–30%)
Contour
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Mixed Grass and Legume Pasture
1189
11
To determine whether soil physical, chemical, and biological
characteristics increased or decreased over time after reclama-
tion, linear regressions were used (Kenney and Keeping 1954).
To determine if microbial community composition varied sig-
nificantly across site and topographic relief (i.e. highland or low-
land), two-way Permutational Multivariate Analysis of Variance
(PerMANOVA; Anderson 2001) was performed on Bray-Curtis dis-
similarity matrices of bacterial and fungal OTU relative abun-
dances with chronosequence site and topographic relief (i.e.
highland or lowland) as well as their interaction as factors. If
significant interactions were observed between factors, pairwise
PerMANOVA was performed to determine differences between
individual factor means across the chronosequence.
To determine the relative influence of stochastic (time since
reclamation, space between sites) and deterministic (soil chem-
ical and physical properties) on variation in microbial commu-
nity composition across the chronosequence, Distance Based
Linear Modeling (DistLM; Legendre and Legendre 1998) was
implemented. Here, the adjusted-R2 selection criterion was
used, which increases only if a given factor improves a model
more than would be expected by chance. Prior to model building,
environmental variables were first tested for collinearity using
Draftsman Plots, with significant collinearity defined as R2 >0.90
(Hair Jr. et al. 2004). If variables emerged as collinear, one was
selected for use in subsequent DistLM model building proce-
dures. Marginal DistLM was initially performed to determine the
relative influence of each factor on variation in microbial com-
munity taxonomic structure considered alone, not accounting
for the influence of other factors. Only significant (P<0.05) fac-
tors in the Marginal DistLM were included in subsequent mod-
els. To determine if the variation in microbial community com-
position explained by time and space (i.e. stochastic processes)
was distinct or shared by other factors, Conditional DistLM was
performed in a step-wise fashion with time (chronosequence
site age) and space (site latitude and longitude) added last, after
the variation attributable to all other significant factors had been
accounted for. Lastly, ‘best’ model selection was used to deter-
mine combination of factors that together accounted for the
greatest variation in community composition as indicated by the
adjusted-R2 criterion.

Figure 2. Soil oxidizable C, phosphate, organic N, and inorganic N concentrations
over time since reclamation and at the reference grassland site in highland (dot-
ted line) and lowland (solid line) soils. Error bars represent +/- SE, n = 3.
RESULTS
Soil chemical properties
In the lowland soils, several soil properties increased with
time since reclamation, including phosphate content (+1168%),
organic N content (+3163%), inorganic N content (+306%), total
C content (+181%), oxidizable C content (+88%), organic mat-
ter content (+38%), and moisture content (+24%; P<0.05; Fig. 2;
Tables 1 and 2, Supporting Information). Carbon to nitrogen ratio
(C: N) significantly decreased in lowland soils (-37%, P<0.05). In
the highland soils, soil properties increasing over time included
organic N content (+807%), phosphate content (+406%), inor-
ganic N content (+444%), organic matter content (+38%), pH
(+16%), and moisture content (+10%; P<0.05). Like in the low-
land soils, there was a significant decrease in C:N in high-
land soils (-52%, P<0.05). Over time, inorganic N content tended
toward that of the reference site in both the highland and low-
land soils. There were no other clear tendencies of soil chemical
properties to mirror those of the reference site over time.
Microbial community succession over time since
surface mine reclamation
Microbial biomass and richness
In the lowland soils, there was no change in microbial biomass
over time (Figure 1, Tables 2 and 3, Supporting Information).
In the highland soils, microbial biomass increased over time
(+35%; P<0.05). Microbial biomass did not clearly tend toward
that of the reference site over time, with the biomass of the
32-year site varying by 31% and 23% from the reference site
for lowland and highland soils, respectively. Bacterial richness
decreased significantly over time in highland soils (-30%; P<0.05;
Fig. 3; Table 2, Supporting Information), but not lowland soils.
Clear tendencies toward the reference site did not emerge for
bacterial richness, with % differences between the 32-year site
and the reference site of 45% and 50% for highland and lowland
soils, respectively. There was no significant response of fungal
richness to time in either highland or lowland soils. For both the
lowland and highland soils, there were not clear tendencies in
microbial biomass and richness toward the reference site over
time.
Microbial community composition and taxonomy
Principle coordinate analyses calculated from Bray-Curtis dis-
similarity between sites and topographic relief (i.e. highland or
Kane et al. 5
lowland) show that bacterial and fungal communities were gen-
erally distinct across the chronosequence as well as from the
reference site, however the degree of difference varied by site
and topography as a significant two-way effect of Site × Topog-
raphy (P<0.05; Fig. 4; Tables 4 and 5, Supporting Information).
Additionally, the changes in community composition were rela-
tively progressive over time (Fig. 4); sites with similar ages (i.e.
2 and 10 years) were more alike than sites with distant ages
(i.e. 2 and 32 years). Despite this, there was no clear tendency
toward the community composition of the reference site for
either bacterial or fungal communities. Though both were sta-
tistically significant, time since reclamation (site differences)
was much more influential than topography in shaping bac-
terial and fungal community composition (Table 4, Supporting
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Information).
Across the chronosequence, bacterial communities were
dominated by members of the phyla Proteobacteria (23%-30%
of 16S rRNA genes), Actinobacteria (6%-22%), and Acidobac-
teria (10%-22%). In the lowland soils, there were increases in
members of the classes Delta- (+39%) and Gammaproteobac-
teria (+29%; Figure S2 and Table 2, Supporting Information),
and decreases in the class Alphaproteobacteria (-51%) over
time since reclamation. Of the other highly abundant taxa
in the lowland soils, Acidobacteria and Actinobacteria, nei-
ther changed in relative abundance over time (Figure 3 and
Table 2, Supporting Information). The relative abundance of
Alpha- and Deltaproteobacteria, but not Beta- or Gammapro-
teobacteria, tended toward that of the reference site (Figure
2, Supporting Information). In the highland soils, there were
increases over time since reclamation in the relative abundance
of members of the classes Delta- (+46%) and Gammaproteobac-
teria (+29%) and decreases in relative abundance of the class
Alphaproteobacteria (-50%; P<0.05; Figure 2 and Table 2, Sup-
porting Information), responses similar to that of the highland
plots. Further, the relative abundance of phylum Acidobacte-
ria increased (+29%) and relative abundance of phylum Acti-
nobacteria decreased (-30%; P<0.05; Figure 3 and Table 2, Sup-
porting Information). Similar to the lowland soils, the relative
abundance of Alpha- and Deltaproteobacteria, but not Beta-
or Gammaproteobacteria, tended toward the reference site in
highland soils.
Across the chronosequence, fungal communities were
dominated by members of the phyla Ascomycota (26%-
65% of ITS region sequences) and Basidiomycota (18%-66%).
When fungal OTUs were assigned to functional guilds at
the genus level using FUNGuild, saprotrophic fungi were
most dominant, ranging from 30%-79% relative abundance
across the chronosequence. Other abundant functional guilds
were arbuscular mycorrhizae (0%-14%) and ectomycorrhizae
(0%-11%).
In the lowland soils, members of the Ascomycota increased
in relative abundance (+20%; P<0.05; Figure 4 and Table 2, Sup-
porting Information) whereas members of the Basidiomycota
did not change in relative abundance over time since recla-
mation. There was a tendency of Ascomycotal relative abun-
dance toward the reference site, with the relative abundance
at 32-year site varying from the reference site by only 7%.
When considered as functional guilds, there were no signif-
icant changes in abundance of saprotrophic fungi, ectomy-
corrhizae nor arbuscular mycorrhizae in lowland soils (Fig-
ure 5 and Table 2, Supporting Information). In the highland
soils, there were no significant changes over time in the
abundance of Ascomycota, Basidomycota nor any functional
guild.
 6 FEMS Microbiology Ecology, 2020, Vol. 96, No. 11
Figure 3. Bacterial and fungal richness with time since reclamation over time since reclamation and at the reference grassland site in highland (dotted line) and
lowland (solid line) soils. Error bars represent +/- SE, n = 3.
Figure 4. Principle coordinate analysis of Bray-Curtis dissimilarity in bacterial and fungal community composition between chronosequence sites and the reference
site. PerMANOVA results including pairwise significance are shown in Tables S4 and S5 (Supporting Information).
The influence of stochastic and deterministic processes on microbial
community succession
For bacterial communities, all chemical and physical soil char-
acteristics with the exception of organic N and C:N accounted
for a significant proportion of β-diversity across the chronose-
quence when considered independently (Marginal DistLM,
P<0.05; Table 2). Among stochastic factors, time accounted for
a significant proportion of β-diversity (Marginal DistLM, P<0.05)
whereas longitudinal distance did not emerge as a significant
predictive variable. Total C (21%), pH, organic matter content,
clay content and time since reclamation (25% each) accounted
for the greatest proportions of bacterial β-diversity when consid-
ered alone, not accounting for variance explained by other fac-
tors (P<0.05). When factors were considered together, the ‘best’
model included all measured factors except organic N, C: N, and
longitude and accounted for 44% of bacterial dissimilarity across
the chronosequence (Table 2).
For fungal communities, all chemical and physical soil char-
acteristics except organic N accounted for a significant propor-
tion of the β-diversity across the chronosequence when consid-
ered independently (Marginal DistLM, P<0.05; Table 3). Stochas-
tic factors (time, space) also accounted for fungal β-diversity
when considered alone, without other variables accounted for.
Organic matter (16%), pH (17%), clay content (19%) and time
since reclamation (19%) independently explained the most vari-
ation in fungal β-diversity (P<0.05). When deterministic fac-
tors were considered together the ‘best’ model included all
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factors except organic N and C: N. This model accounted
for 51% of fungal dissimilarity across the chronosequence
(Table 3).
Lastly, to determine whether stochastic processes shaped
bacterial and fungal succession once all deterministic factors
were accounted for, a conditional DistLM was performed with
time and longitudinal distance added to the model last. For bac-
terial communities, once all variation attributable to determin-
istic (environmental) factors was accounted for, time explained
an additional 4% of the remaining variance (Table 2, conditional
DistLM, P<0.05). For fungal communities, stochastic processes
did not explain any additional variation (Table 3, conditional Dis-
tLM).
DISCUSSION
In this study, we show that soil microbial communities exhibit
successional patterns across a ∼30-year mine land reclamation
chronosequence by surveying soil chemical and physical charac-
teristics, microbial biomass, diversity, and community compo-
sition, then modeling relationships between microbial commu-
nity composition and soil parameters using distance-based lin-
ear modeling. For bacterial communities, both deterministic (e.g.
pH, texture, soil C, N) and stochastic (e.g. dispersal and drift) fac-
tors exhibited distinct influences on the post-disturbance suc-
cession of these communities, but deterministic factors together
 Kane et al. 7

Table 2. Bacterial taxonomic variation accounted for by each soil chemical/physical property and time since reclamation as determined by
marginal, conditional, and ‘best’ DistLM.

Marginal Conditional

Variance explained

Variance
Variable Pseudo-F explained Adjusted R2 Pseudo-F Proportion Cumulative

Altitude† 4.47∗ 0.17 0.13 4.47∗ 0.17 0.17

Moisture† 3.98∗ 0.15 0.21 3.15∗ 0.11 0.28
pH† 7.20∗ 0.25 0.31 4.07∗ 0.12 0.40
Organic matter† 7.45∗ 0.25 0.36 2.55∗ 0.07 0.47
Oxidizable C† 4.79∗ 0.18 0.67 1.20 0.03 0.5
Organic N 1.21 0.05 – – – –

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Inorganic N† 1.94∗ 0.08 0.38 1.50∗ 0.04 0.54
Phosphate† 4.29∗ 0.16 0.40 1.35 0.04 0.58
C:N 1.26 0.05 – – – –
% Clay† 7.25∗ 0.25 0.40 1.19 0.03 0.61
Total C† 5.97∗ 0.21 0.41 1.37 0.03 0.65
Longitude 1.87 0.08 – – – –
Time† 7.59∗ 0.25 0.44 1.60∗ 0.04 0.69

∗
P < 0.05
†
Included in overall best solution; adjusted R2 = 0.44

Table 3. Fungal taxonomic variation accounted for by each soil chemical/physical property and time since reclamation as determined by
marginal, vonditional, and ‘best’ DistLM.

Marginal Conditional

Variance explained

Variance
Variable Pseudo-F explained Adjusted R2 Pseudo-F Proportion Cumulative

Altitude† 3.82∗ 0.15 0.11 3.82∗ 0.15 0.15

Moisture† 2.59∗ 0.11 0.17 2.60∗ 0.06 0.24
pH† 4.58∗ 0.17 0.27 3.81∗ 0.12 0.36
Organic matter† 4.13∗ 0.16 0.32 2.70∗ 0.08 0.44
Oxidizable C† 2.92∗ 0.12 0.34 1.54 0.04 0.48
Organic N 1.56 0.07 – – – –
Inorganic N† 2.70∗ 0.11 0.40 2.63∗ 0.07 0.55
Phosphate† 3.75∗ 0.15 0.41 1.38 0.04 0.59
C:N 2.35∗ 0.10 0.43 1.47 0.04 0.63
% Clay† 5.18∗ 0.19 0.49 1.97∗ 0.05 0.67
Total C† 3.50∗ 0.14 0.47 1.18 0.03 0.70
Longitude† 3.44∗ 0.14 0.49 1.71 0.04 0.74
Time 5.18∗ 0.19 0.49 – 0.00 0.74

∗
P < 0.05
†
Included in overall best solution; adjusted R2 = 0.51

accounted for ∼7 times more variation. Fungal community vari-
ation was only explained by deterministic (i.e. environmental)
factors.
Deterministic factors strongly influence microbial
community succession following surface mine
reclamation
Both bacterial and fungal community composition gradually
changed across the chronosequence, with the 2-year sites being
more taxonomically similar to the 10- and 15- year sites than
to the 32-year site for both bacteria and fungi (Fig. 4). This
is similar to trends in another mine chronosequence, where
fungal, but not bacterial, communities were grouped within
chronosequence site, and these trends were related to soil
chemical and physical (i.e. deterministic) properties of the sites
(Sun et al. 2017). Our data also shows that successional pro-
cesses are occurring over time, and the DistLM results pro-
vide statistical evidence that these processes are driven primar-
ily by deterministic influence (i.e. soil chemical and physical
properties).
Soil pH emerged as highly influential in constraining the tax-
onomic composition of both bacterial and fungal communities
across the chronosequence (Tables 2 and 3). Following surface
mining disturbance, soil pH can decrease when acidic material
is exposed during the process of mining, and amendments to the
soil (e.g. lime) are often applied to mitigate the effects of acidic
soil on the productivity of the site (Skousen and Zipper 2014).
Across the chronosequence, as expected, pH increased over time
in highland soils as well as the lowland soils, though this was
 8 FEMS Microbiology Ecology, 2020, Vol. 96, No. 11
only significant in highland soils (P<0.05). The strong influence
of pH on microbial community composition is well documented
(e.g. Fierer and Jackson 2006; Lauber et al. 2009; Rousk et al. 2010).
Moreover, pH is tightly linked to other deterministic soil prop-
erties through its chemical influence on nutrient availability,
metal solubility, and organic matter dynamics (Ashman and Puri
2002). Further, the influence pH alone on microbial communi-
ties may be attributable to a requirement of microbial taxa for
a specific cellular pH optimum, therefore eliciting a determinis-
tic influence (Rousk et al. 2010). From these data, it is apparent
that the management of pH is essential during the restoration
of mined lands.
Though explaining less variation across sites than pH, soil
clay content also emerged as influential in shaping both bac-
terial and fungal communities across the chronosequence. Soil
texture is important in determining microbial community com-
position (Girvan et al. 2003; Lauber et al. 2008), and like pH, it
may function as a keystone variable influencing other deter-
ministic soil factors such as water dynamics and nutrient con-
tent (Ashman and Puri 2002; Ghezzehei et al. 2019). The sub-
sequent influence of soil water dynamics (e.g. connectivity of
soil aggregates by flow of water) mediates to the diffusion of
substrates which can elicit selective pressure on microbial taxa
through resource constraints or surpluses (Carson et al. 2010).
This highlights that, like pH, soil texture is tightly linked to many
other deterministic factors which shape microbial communities
following disturbance, and therefore can be leveraged during
restoration.
Increases in total and oxidizable C over time indicate
increased storage of C in these soils following reclamation as
well as increased C availability for the microbial community
(Fig. 2; Tables 1 and 2, Supporting Information). Though varying
between bacteria and fungi and among models, both factors did
emerge as important in explaining taxonomic variation between
sites in the Marginal DistLM (Tables 2 and 3). The quantity and
chemical complexity of C compounds can shape microbial com-
munity composition through imposing selection for taxa with
a genomic capacity to metabolize the available C substrates
(Eilers et al. 2010; Li et al. 2012). Further, soil microorganisms
can contribute to the accumulation of stable soil organic mat-
ter (e.g. through the generation of necromass which can sorb
to soil minerals (Cotrufo, Wallenstein and Boot 2013)). Together
with the apparent increase in organic matter content across
the reclamation chronosequence interrogated in this study, the
increases in soil C and N contents suggest that microbially-
mediated soil organic matter cycling may be recovering post-
mining. Soil organic matter is important for overall ecosys-
tem function; for instance, it fosters favorable soil structure,
water dynamics, and nutrient storage (Brady and Weil 2008).
This result highlights the potential importance of soil microor-
ganisms in replenishing organic matter stocks after mining
disturbance.
Inorganic N, organic N, and phosphate contents all increased
across the chronosequence. This is consistent with findings
that, in mined lands reclaimed to grasslands, both C and N
increased over time (Čı́žková et al. 2018). Nitrogen limitation is
widely known to deterministically shape microbial communi-
ties (Hu et al. 2001; Craine, Morrow and Fierer 2007; LeBauer and
Treseder 2008). For example, the addition of N influences micro-
bial community composition and decreases microbial extracel-
lular enzyme activity, which often results in increased soil C
storage (Peacock 2001; Ramirez, Craine and Fierer 2012; Freed-
man et al. 2016; Zak et al. 2019). Recently, P limitation has
emerged as a limiting nutrient in some temperate ecosystems
(DeForest et al. 2012), and in this study, phosphate content
increased over time since reclamation and was significant in
explaining taxonomic variation for both bacteria and fungi in all
models (Tables 2 and 3). Since surface mining exposes previously
unweathered rock to biotic and abiotic influence, soil P may
increase as soil mineral weathering proceeds post-reclamation.
Changes in microbial community composition and increases in
microbial biomass have been recorded post-phosphorous addi-
tion (Liu et al. 2012; Carrino-Kyker et al. 2016), and elevated P can
also reduce microbial decomposition of soil C (DeForest 2019),
further highlighting the key deterministic role of nutrient avail-
ability in shaping microbial communities.
Taken together, these results not only support the hypothesis
that soil nutrient content would increase over time post-mining,
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but that this deterministically shapes the succession of micro-
bial communities. However, it was expected that soil chemical
content would tend toward that of the undisturbed reference
site, and our results did not align with this expectation. The ref-
erence site was a previously established managed pasture and is
composed of varying plant species and experience tillage, har-
vesting, and grazing. This historical difference in management
strategy (e.g. periodic tillage and aboveground forage harvest-
ing) may explain the lack of coherence with the chronosequence
ecosystem trajectory.
Stochastic factors explain a distinct and significant
amount of variation in bacterial but not fungal
communities
Though deterministic factors clearly emerged as dominant in
shaping both bacterial and fungal communities, time explained
distinct and significant variation in bacterial, but not fungal,
community composition as determined by Conditional DistLM
(Tables 2 and 3). The difference in response between bacterial
and fungal communities is supported by other studies showing
that the two communities respond differentially during succes-
sion (Brown and Jumpponen 2014; Sun et al. 2017). The small
but significant concomitant role of stochastic factors in shap-
ing bacterial communities aligns with other studies showing
that both stochastic and deterministic factors shape bacterial
communities following disturbance (Dumbrell et al. 2010; Caruso
et al. 2011; Ferrenberg et al. 2013; Freedman et al. 2015). While,
contrary to our results, others have observed fungi to exhibit
stochastic successional patterns (Brown and Jumpponen 2014;
Cline and Zak 2014), it has also been shown that fungi sub-
ject to a severe pH gradient exhibited dominantly deterministic
successional patterns (Dumbrell et al. 2010), a factor which also
was influential to fungal communities in our study. Notably, the
observation of dominant stochastic influences on fungal suc-
cession is found in much longer term studies, such as glacial
chronosequences that represent more than double that which
our chronosequence encompasses (i.e. 70 years (Brown and
Jumpponen 2014), ∼14 000 years (Cline and Zak 2014)). Owing
to their shorter generation times (Rousk and Bååth 2011), bac-
teria may be more strongly impacted by stochastic factors (i.e.
genetic drift) in the short term, like our chronosequence rep-
resents (∼30 years). Further, fungal spores may persist as dor-
mant for long periods of time, awaiting favorable environmental
conditions to emerge (Deacon 2006). Hence, more time may be
necessary for stochastic influences to affect soil fungal commu-
nities, as has been resolved in the noted longer-term studies.
This suggests that, while stochastic factors may shape fungal
communities following surface mining, a longer time sequence

would be needed to resolve these effects. As an ecosystem devel-
ops over time, it is plausible that communities may be more sus-
ceptible to a stochastic influence like dispersal limitation (e.g.
as soil structure and hydrologic dynamics develop). Hence, our
relatively short chronosequence may not capture the extent of
stochastic effects on these communities which may fluctuate in
intensity with time post-disturbance.
The relative influence of stochastic and deterministic pro-
cesses can shift over time within a system; for example, stochas-
ticity may primarily shape communities in early succession, fol-
lowed by deterministic influences shaping communities later in
successional time (Ferrenberg et al. 2013; Brown and Jumpponen
2014; Dini-Andreote et al. 2015). Environment type and extremity
are also noted as a driver of this interplay. Increased extremity of
environmental conditions is linked to increasingly deterministic
influence on succession (Stegen et al. 2012). In the case of sur-
face mining, many abiotic factors are suddenly and drastically
altered (Skousen and Zipper 2014), rendering the emergence of
deterministic factors as dominant unsurprising. Our study sup-
ports that, while it is useful to consider broad scale ecological
patterns, it is plausible to expect results to vary based on the
many differing factors that comprise local conditions.
It is possible that a proportion of community variance
attributable to stochastic processes in our models may represent
unmeasured confounding environmental variation across the
chronosequence. In this study, environmental factors were mea-
sured that have been well documented as deterministic drivers
of soil microbial community composition (Fierer, Bradford and
Jackson 2007; Högberg, Högberg and Myrold 2007; Lauber et al.
2008; Freedman et al. 2015; Romanowicz et al. 2016) in order to
account for as much deterministic influence on succession as
possible. Additionally, these sites were chosen because they har-
bor minimal heterogeneity between them in plant communi-
ties, climate, and management regime, further minimizing con-
founding variation which could contribute to community dis-
similarity. Still, only ∼70% of variation was explained once all
factors were added to the Conditional DistLM (Tables 2 and 3).
It is plausible that microbe-microbe interactions such as com-
petition, predation, mutualism, and order-of-colonization may
account for a portion of unexplained variation (Jiang and Patel
2008; Vannette and Fukami 2013; Freedman and Zak 2015b;
Fukami 2015), which were not quantified in this study.
Microbial richness and biomass responded variably
over time following reclamation
It was expected that microbial biomass and richness would
increase over time as has been shown in other studies of micro-
bial community succession following disturbance (Brown and
Jumpponen 2014; Harris 2003; Freedman and Zak 2015a; Li, Liu
and Chen 2016). Biomass increased over time in the highland
plots, but in the lowland plots, biomass initially decreased from
the 2- to 10-year observations followed by an increase at the
15-year site and a return to levels similar to the 2-year site.
Since soils in this study were physically disturbed during mining
(Skousen and Zipper 2014), successional dynamics may mirror
a disturbance like tillage, whereby communities are displaced
but not necessarily reduced in population size, as opposed to a
severe disturbance that would result in a drastically decreased
population at time zero (e.g. volcanic eruption; Zeglin et al. 2016).
The fluctuation of biomass in the lowland plots over time may
reflect deterministic influence, as it resembles the change in
organic N content over time (Fig. 2). Indeed, the response of
Kane et al. 9
microbial biomass to a physical disturbance like tillage is vari-
able, depending on deterministic soil properties (Calderón et al.
2000; Anderson et al. 2017).
CONCLUSIONS
The successional trajectory of microbial communities follow-
ing mine reclamation is influenced by both deterministic and
stochastic processes. Though stochastic processes did emerge
as distinct for bacterial communities, the influence of deter-
ministic factors was ∼7 times greater, and fungal communi-
ties were influenced only by deterministic factors. Though there
were not clear trends in microbial biomass or diversity, there
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were shifts in community composition and increases in soil
nutrient content. However, they did not clearly tend toward the
reference site, suggesting that reclaimed mine soils may not
come to resemble undisturbed ecosystems for many decades
post reclamation. Taken together, these results indicate that
reclaimed mine soils harbor dynamic microbial communities
which are dominantly shaped by soil chemical and physical
properties following reclamation. These deterministic factors
regulate changes in relative abundance of microbial groups over
time post-disturbance. By managing key soil properties, it may
be possible to accelerate microbial succession to develop com-
munities which fit the ecological needs of the desired post-
mining land use.
SUPPLEMENTARY DATA
Supplementary data are available at FEMSEC online.
ACKNOWLEDGMENTS
We thank Brianna Mayfield and Gregory Martin for their help
with field work and insight into this project. We also thank two
anonymous reviewers for their constructive feedback on this
manuscript. Funding for this project was provided by the United
States Department of Agriculture National Institute for Food
and Agriculture (Accession No. 1018856, Project No. WVA00921)
as well as USDA HATCH (Accession No. 1011670, Project No.
WVA00695 and Accession No. 1010898, Project No. WVA00688)
and the WVU Faculty Senate Grant program.
Conflicts of Interest. None declared.
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