Ecological Applications, 00(0), 0000, pp.

000000
0000 by the Ecological Society of America

Unprecedented carbon accumulation in mined soils:

the synergistic effect of resource input and
plant species invasion
LUCAS C. R. SILVA,1,3 RODRIGO S. CORREA,2 TIMOTHY A. DOANE,1 ENGIL I. P. PEREIRA,1
1
AND WILLIAM R. HORWATH
1
Department of Land, Air, and Water Resources, University of California, Biogeochemistry and Nutrient Cycling Laboratory,
One Shields Avenue, Davis, California 95616-8627 USA
2
Department of Ecology, University of Braslia, Campus Darcy Ribeiro, Caixa Postal 04.401, 70.910-970 Brasilia, DF, Brazil

Abstract. Opencast mining causes severe impacts on natural environments, often resulting
in permanent damage to soils and vegetation. In the present study we use a 14-year restoration
chronosequence to investigate how resource input and spontaneous plant colonization
promote the revegetation and reconstruction of mined soils in central Brazil. Using a multi-
proxy approach, combining vegetation surveys with the analysis of plant and soil isotopic
abundances (d13C and d15N) and chemical and physical fractionation of organic matter in soil
proles, we show that (1) after several decades without vegetation cover, resource input
(nutrient-rich biosolids) into exposed regoliths prompted the establishment of a diverse plant
community (.30 species); (2) the synergistic effect of resource input and plant colonization
yielded unprecedented increases in soil carbon, accumulating as chemically stable compounds
in occluded physical fractions and reaching much higher levels than observed in undisturbed
ecosystems; (3) invasive grasses progressively excluded native species, limiting nutrient
availability, but contributing more than 65% of the total accumulated soil organic carbon.
These results show that soilplant feedbacks regulate the amount of available resources,
determining successional trajectories and alternative stable equilibria in degraded areas
undergoing restoration. External inputs promote plant colonization, soil formation and
carbon sequestration, at the cost of excluding native species. The introduction of native woody
species would suppress invasive grasses and increase nutrient availability, bringing the system
closer to its original state. However, it is difcult to predict whether soil carbon levels could be
maintained without the exotic grass cover. We discuss theoretical and practical implications of
these ndings, describing how the combination of resource manipulation and management of
invasive species could be used to optimize restoration strategies, counteracting soil
degradation while maintaining species diversity.
Key words: alternative equilibria; carbon sequestration; central Brazil; land restoration; mines;
resource-ratio theory; soilplant feedback; stable isotope; succession.

INTRODUCTION from the inuence of soils on vegetation patterns (e.g.,

Mining activities represent one the most severe types
of human impacts on natural environments often
resulting in permanent damage to soils and vegetation.
Mined sites offer, however, a unique setting to test and
advance ecological theories and their applications.
Associations between soil properties and vegetation
structure/composition have been shown to modulate
the distribution and performance of ecosystems across
biomes (Leithead et al. 2010, 2012, Silva et al. 2010b,
Silva and Anand 2011, 2012, Ladd et al. 2012, Rossatto
et al. 2012). However, conditions under which the effect
of plants on soils can be unambiguously distinguished
Manuscript received 8 November 2012; revised 16 January
2013; accepted 23 January 2012. Corresponding Editor: R. L.
Sinsabaugh.
3 E-mail: lucascrsilva@gmail.com
Vitousek 2004) are rare in natural ecosystems. For this
reason, fundamental theories such as those that link
succession and the use of resources by plants (e.g., the
resource-ratio theory; Tilman 1985), though extensively
tested in laboratory or microcosms studies, still lack
empirical validation from eld experiments (Miller et al.
2005). Here we contribute to the understanding of how
multiple processes converge to create stable complex
ecosystems, providing an analysis of soilplant feed-
backs following the abrupt reset of interactions
caused by mining disturbance.
Regardless of type or conservation status of an
ecosystem prior to disturbance, topsoil removal exposes
residual substrates, characterized by unconsolidated
non-weathered rocky materials (regoliths) with poor or
no secondary mineral development, that are unsuitable
for plant establishment. Due to depleted soil resources,
vegetation cover remains incipient for decades or

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LUCAS C. R. SILVA ET AL.
centuries following this type of degradation and, in such
circumstances, it is necessary to restore edaphic condi-
tions and soil productivity to promote spontaneous
revegetation (Martins et al. 2004, Ehrenfeld et al. 2005,
Reiners et al. 2005, Silva and Correa 2010). Restoration
activities typically involve physical amelioration and
nutrient input in the form of organic matter, which
along with propagule pressure from the regional species
pool are generally expected to trigger autogenic recov-
ery.
As succession progresses the degraded system should
move towards its original state or analogous equilibri-
um, but driven by new edaphic thresholds and plant
community assemblages distinct from those found in the
local ora, alternative stable states may be formed.
Observations of species invasion provide examples of
fast-growing species establishing and dominating areas
where resource cascades appear suddenly and, over time,
consolidate unexpected stable states (Zavaleta et al.
2001, Simard et al. 2007). In the tropics, where light and
water are typically not limiting but soils tend to have low
nutrient capital, the potential effects of invasive species
in determining alternative stable states through the
competitive exclusion of native species is maximized.
Therefore, understanding the transience of invasive
species and their role in altering soil processes and
resource availability is crucial to promote effective
restoration of disturbed tropical ecosystems.
To date, the theoretical question of how to measure
restoration effectiveness and the practical relevance of
anticipating the direction of recovery trajectories have
not been sufciently explored in an integrated manner.
Holistic views, which combine the evaluation of
vegetation-driven impacts on soils, linking nutrient
cycling and productivity, have been proposed to explain
the recovery of degraded ecosystems (Richter and
Markewitz 2001, Anand and Desrochers 2004). How-
ever, analytical frameworks that would allow the joint
interpretation of ecological invasion and restoration are
scarce (Shaw et al. 2010, Gaertner et al. 2012). This is
particularly true for tropical systems, where little
information exists about the effect of plant establish-
ment on soil development. In South America, opencast
mining has resulted in the loss of thousands of hectares
of tropical forest and savanna vegetation (Moran 1993,
Parrotta and Knowles 1999), but restoration efforts
have been limited and long-term evaluations of species
invasion are rare. In Brazilian savannas, despite
empirical support for substrate restoration as an
effective method to increase soil carbon and nutrient
accumulation and promote spontaneous revegetation
(Martins et al. 2004, Silva and Correa 2010, Correa et al.
2012), quantitative approaches for assessing the role of
plant establishment in general, and invasive species in
particular, are yet to be formalized.
In the present study we use analyses of soil carbon
levels, chemically and physically protected pools, and
stable isotope composition of plants and soils, to
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Ecological Applications
Vol. 00, No. 0
investigate the effect of restoration treatments and
spontaneous plant colonization on mined sites of central
Brazil. Based on succession and resource-use theories,
which underlie the fundamental aspects of invasion and
restoration ecology, we hypothesize that: (1) resource
input in severely degraded (mined) sites prompts the
establishment of native and invasive plants originating
from the regional species pool; (2) resource input and
plant establishment have a synergistic effect on soil
restoration; and (3) invasive species, favored by new
edaphic thresholds, dominate sites undergoing restora-
tion, regulating soil development, nutrient availability
and ecosystem productivity. To test these hypotheses,
we link temporal change in plant communities and soil
development through the use of a restoration chronose-
quence, where we quantify the inuence of resource
input and species establishment on soil development and
ecological restoration. Previous studies have used
chronosequences to investigate plant succession (Baer
et al. 2002, Simard et al. 2007) and soil carbon
sequestration (Schlesinger 1990, Richter and Markewitz
2001). This approach has proven particularly useful to
study low-diversity systems of converging successional
trajectories (Walker et al. 2010), such as degraded sites.
Here we build upon this framework, presenting and
interpreting results as a function of time since restora-
tion.
METHODS
Study sites
In central Brazil, mining affects relatively small areas
compared to vegetation clearance for agriculture and
cattle grazing (Klink and Moreira 2002). Nevertheless,
the environmental damage caused by mining activities is
much more severe and longer lasting than the impact
created by agriculture and ruminants. At our study sites
opencast mining activities took place between the late
1960s and early 1970s. All areas were originally covered
by woody savanna vegetation established on Cambisols.
Excavations for gravel extraction left a at surface 46
m below the original soil level, but at all sites the
surrounding areas still have the original vegetation
cover. To stimulate natural revegetation mature indi-
vidual trees were left standing after mining activities, but
despite the continuous propagule pressure no sign of
natural regeneration was observed at the study sites. All
sites are located within the limits of the Brazilian Federal
District (Brasilia-DF; Fig. 1) under homogeneous
climatic and topographic conditions. The local climate
is tropical with well-dened wet and dry seasons, Aw by
Koppens classication. The mean temperature is 22.58C
with annual rainfall ranging from 1200 to 1600 mm and
mostly distributed from November to March (weather
station records of the Instituto Brasiliero de Geograca
e Estatistica [IGBE] reserve, Brasilia-DF). Restoration
activities started several decades after exploitation, but
took place during a different year at each site, providing
a chronosequence of time since restoration.
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 Month 2013 UNPRECEDENTED CARBON ACCUMULATION

FIG. 1. Location of mined sites in the Brazilian Federal District. The ve dark gray circles and ovals show mined areas selected
for the present study. Years associated with sites represent the year when restoration activities took place at that site. Substrate
restoration was identical at all sites, with a single intervention composed of decompaction and incorporation of nutrient-rich
biosolids. At all sites, samples were taken ;6 months after soil treatment at site 2011. Along with untreated exposed regoliths (time
zero), the study sites represent a 14-year restoration chronosequence.

Substrate treatment and restoration chronosequence
Mined substrates are typically compacted and deplet-
ed in organic matter, nutrients, and microorganisms. It
has been previously shown that substrate decompaction
alone is not sufcient to promote colonization of plant
species (Martins et al. 2004, Silva and Correa 2008). For
this reason, along with mechanical subsoiling (loos-
ening of compacted subsoils) to 20 cm depth, restoration
activities included the incorporation of biosolids (tertia-
ry-treatment domestic sewage sludge; 100 dry Mg/ha) as
a common source of organic matter (;50% carbon) and
nutrients (N, 5.5%; P, 2%; K, 0.2%; Ca, 2.5%; Mg, 0.5%;
S, 0.5%). The biosolids were added before the mechan-
ical subsoiling, which then resulted in a homogeneous
incorporation and distribution of the applied material
through the 020 cm layer. The biosolids originated
from the same local treatment plants (Companhia de
Saneamento Ambiental do Distrito Federal, Brasilia-
DF) following identical treatment and stabilization
protocols. Detailed description of treatment/stabiliza-
tion and chemical composition can be found in Correa
et al. (2012).
Biosolid incorporation into exposed regoliths took
place in different years, namely 1997, 2002, 2005, 2008,
and 2011 (Fig. 1). Our site selection was such that each
of these years corresponds to a given study site. Since
our nal measurements (performed simultaneously
across sites) took place approximately six months after
soil restoration in the 2011 site, we were able to
establish a chronosequence as follows: 0.5, 3, 6, 9, and
14 years since restoration. For control purposes, we
also characterized untreated substrates, adding a time 0
(average of all sites) since restoration to the chronose-
quence. Undisturbed soils and vegetation in the vicinity
of each site served as reference for pre-disturbance
conditions.
Sampling design
Our sampling design was determined using an a priori
test of statistical power, anticipating three potential
predictors: time since restoration, soil depth, and
vegetation cover. We expected an intermediate effect
of time (site), measurable with moderate statistical
power of 0.8 (Park 2010). To meet these conditions
our design consisted of 15 randomly distributed
sampling points within 1 ha at each site. at all sites we
conducted a vegetation census of the woody and
herbaceous layer and collected soil samples at 5-cm

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LUCAS C. R. SILVA ET AL.
increments to a depth of 20 cm, spanning the depth of
disturbed regolith. Samples were acquired by vertically
pressing 100-cm3 tubes into each 5-cm layer of
sequentially dug pits. Tubes were inserted so that
pressure was only exerted in the tube walls, thereby
preventing compaction.
Determining SOC levels
After collection, soil samples were sieved (2 mm) and
air-dried. Soil organic carbon (SOC) content was
determined by dry combustion gas chromatography
(GC). Soil density was used in the calculation of total
SOC, which was then expressed as a fraction of soil
volume at each depth. Accumulation of SOC over time
is shown as average values of all replicates collected at
the different study sites.
Multi-proxy analysis
Further analyses were performed to quantify changes
associated with the effects of restoration activities,
natural regeneration, and invasive species on SOC
pools. We used three independent approaches. First,
we characterized the isotopic composition of SOC as a
proxy for the determination of the relative contribution
of C3 and C4 plants to soil carbon pools. Second, we
determined the chemical composition of SOC as a proxy
for recalcitrance and dynamics of organic compounds.
Third, we measured SOC in distinct soil physical
fractions to determine the fate of organic matter applied
during substrate restoration and materials derived from
plant establishment. Though rarely used in combination,
similar approaches have been employed to investigate
ecotone (Silva et al. 2008, 2010a), patch dynamics (Silva
et al. 2010b, Silva and Anand 2011), and SOC
stabilization and accumulation (Doane et al. 2003,
Luo et al. 2006) in tropical and temperate ecosystems.
These approaches offer an integrated picture of the
effect of plant and soil associations, critical to test the
hypotheses described in the Introduction. Details of each
method are described as follows.
Isotopic composition.The isotopic composition
(d13C) of organic matter applied during substrate
treatment, colonizing plants, and resulting SOC was
determined by dry-combustion gas chromatography
coupled with continuous-ow isotopic-ratio mass spec-
trometry (GC-IRMS 20-20/ANCA-NT; Europa, Crewe,
UK). The average d13C value of the applied biosolids
was determined to be 27.1% 6 0.3% (mean 6 SE),
which falls within the range typical of carbon from C3
plants. The biomass of the invasive grasses was 12.2%
6 0.2%, which is a typical value for C4 species. These
signatures represent average values of ve homogenized
composite samples. Based on these distinct isotopic
values a two-end member mixing model (Lloyd et al.
2008) was used to quantify the effect of applied biosolids
(combined with C3-originated biomass) and invasive C4
grasses, as distinct sources of SOC. We used the
following equation to partition carbon derived from
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C4 grasses from the combined effect of the applied
organic matter and carbon derived from C3 plants:
C4 Ct dt  d3 =d4  d3 1
where Ct C3 C4 and represents the total amount of
carbon in the soil, C3 is the amount of carbon derived
from C3 plants, C4 is the amount of carbon derived
from C4 plants, dt is the d13C value of the Ct carbon, d3
is the d13C value of the C3 plant carbon, and d4 is the
d13C value of the C4 soil carbon. Results of C4
contribution were expressed as the relative (percent-
age) portion of total SOC. The SOC content of the
exposed regoliths, before treatment, was considered
negligible. Other sources of variation, such as changes
in plant d13C in response to elevated atmospheric CO2
or across altitudinal ranges (Silva and Horwath 2013),
are not relevant at the scale studied here. These effects
would be much smaller than differences between C3
and C4 species (Silva et al. 2010b, 2011, Silva and
Anand 2011) and, therefore, were not considered in our
analysis.
Chemical fractions.To distinguish between stable
organic compounds and transient soil carbon pools, we
applied classic chemical fractionation for isolation of
humin, humic acid, and fulvic acid fractions. These
measurements are based on differences in solubility in
alkaline and acid solutions and closely followed
International Humic Substance Society (IHSS) recom-
mended extraction procedure. A preliminary rinse with
0.1 mol/L HCl was used to remove any carbonates, as
well as light material and debris, such as pieces of
undecomposed plant material and rootlets (Doane and
Horwath 2003). This material was decanted following
centrifugation for 10 min at 9 G (;88.3 m/s). After this
pretreatment, soils were shaken with 0.4 mol/L NaOH
under N2 overnight, centrifuged, and the extract ltered
through glass wool to ensure removal of any remaining
debris. The humic acid fraction was precipitated by
acidifying to pH , 2, while the fulvic acid fraction
remained in solution. Extractions were repeated and
combined until no more organic matter was present in
the extractant. The fulvic and humic acid fractions were
then separated by centrifugation, the dissolved organic
carbon determined by UV/persulfate oxidation (Phoenix
8000; Teledyner-Tekmar, Mamson, Ohio, USA), and
results expressed as mass of each fraction in relation to
soil mass. The remaining organic matter not directly
extractable with alkali is referred to as humin (Doane
et al. 2003).
Physical fractions.Soil samples were separated in
four aggregate size fractions by wet sieving according to
a modied method of Elliott (1986). Two sieves were
used to separate soil into light fraction (oating residue),
macroaggregates (.250 lm), microaggregates (53250
lm), and mineral particles (silt and clay; ,53 lm). A 70-
g subsample of soil was evenly spread over the 250-lm
sieve and submerged in 1 cm of deionized water at room
temperature for 5 min. The soil was subsequently sieved
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 Month 2013 UNPRECEDENTED CARBON ACCUMULATION

FIG. 2. Study sites and total number of C3 and C4 species that spontaneously colonized mined areas after incorporation of
organic matter (OM; biosolids as tertiary-treatment domestic sewage sludge). As part of the management plan, mature trees were
left standing after mining activities took place, to stimulate natural revegetation via seed dispersal. However, vegetation cover was
absent in all sites for several decades after abandonment and natural colonization only happened after substrate treatment. The
restoration chronosequence matches the year of substrate treatment as described in Fig. 1. A complete list of species at each site is
presented in Appendix A. Photo credit: Rodrigo S. Correa.

for 2 min by moving the sieve up and down with 50
repetitions. Macroaggregates remaining on the sieve
were backwashed into a pre-weighed pan and oating
residue was aspirated off and transferred to a pre-
weighed tin for isolation of the light fraction. Soil and
water that passed through the sieve was transferred to
the 53-lm sieve and the sieving procedure was repeated.
All water and mineral particles passing through the 53-
lm sieve were transferred to a nal pan. The residue and
aggregate fractions were oven-dried at 608C and
weighed. Total C in the soil fractions was determined
on 1020 mg subsamples by dry-combustion gas
chromatography.
Statistical analysis
Analyses of variance, followed by post hoc Tukeys
honestly signicant difference (HSD) tests, were per-
formed to compare differences across sites, soil depth,
and/or chemical and physical fractions. When appro-
priate, least-squares regression analysis was used to
investigate the effect of resource input or species
colonization as a function of time since restoration.
All statistical analyses were performed using the SAS-
created JMP statistical software (SAS Institute 2012).
RESULTS
Species colonization and accumulation of SOC

Soil nutrients and leaf d15N
For the same soil samples described above we
characterized total nitrogen (N), using the Kjeldahl
method, and extractable phosphorus (P), using Melich
extractions (Rashid and Memon 1996). Using the same
methods described for carbon isotopic analysis (GC-
IRMS 20-20/ANCA-NT, Europa, Crewe, UK), we
determined leaf d15N in three composite samples of
Brachiaria sp. (dominant invasive species) at each study
site.
After decades of absent vegetation cover, substrate
restoration activities prompted the colonization of
native and invasive plant species. However, despite
the establishment of several woody species, herbaceous
vegetation dominated all sites. Several native C4 grasses
can be found in this region, but at the study sites only
exotic (C4 ) grasses of the genera Brachiaria and Melinis
were found. In Fig. 2 we show images of each study
site, including the number of species (C3 herbs, C3
woody, and C4 invasive) found at each site after
restoration. The oristic census shows that invasive C4

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FIG. 3. (A) Average values of total soil organic carbon, expressed as percentage of soil volume, at different soil depths across a
chronosequence of mined sites undergoing restoration. Although the incorporation of biosolids into exposed regoliths took placed
in different years (Fig. 1), our nal measurements all took place about six months after soil restoration at the 2011 site, so we were
able to establish the restoration chronosequence in (a), and added a time 0 (control) for untreated substrates. The light-gray arrow
shows maximum (05 cm depth) carbon levels found in local savannas (original ecosystem at all sites) prior to disturbance. The
dark-gray arrow represents maximum carbon levels found in neighboring evergreen forests established on well-drained Cambisols
(Silva et al. 2008, 2010a). The black arrow shows maximum carbon levels found in riparian forests on seasonally ooded
(hydromorphic) soils also in the same region (Silva et al. 2008). (B) Actual soil organic C values across sites and depths. The
uppercase letters show signicant differences between sites/years (comparison of each studied depth across all sites), as determined
by ANOVA and Tukeys HSD post hoc test (P , 0.05).

grasses represent more than 95% of the total herba-
ceous cover 14 years after treatment, whereas the cover
of herbaceous dicots, small trees, and shrubs remained
inexpressive. A complete list of species is presented in
Appendix A.
The combination of organic matter application and
plant colonization led to a substantial change in soil
organic carbon (SOC) from time 0 to 0.5 year after
restoration, increasing SOC from 0.3% to pre-distur-
bance levels (.2%). However, the greatest increase in
SOC occurred gradually following vegetation establish-
ment (Fig. 3A). The highest SOC values (.9% in the 0
5cm depth), measured between years 3 and 9 after
restoration (Fig. 3B), are comparable to levels found in
hydromorphic soils and about twice as high as measured
in undisturbed primary forests established on Cambisols
in the same region. The highest values of SOC
correspond to the period of greatest species diversity
(Fig. 2) and, in spite of a decrease by year 14 when exotic
grasses became dominant, levels of SOC remained much
higher than pre-disturbance levels.
Invasive species as the main source of SOC
A two-end member mixing model, based on d13C
values of the incorporated residue and the more 13C-
enriched C4-originated biomass, allowed us to quantify
the relative contribution of C3- and C4-derived biomass
to total SOC. In Fig. 4 we show the average outcome of
these calculations at each site. Prior to restoration
activities less than 5% of the soil carbon content
originated from C4 plants. After restoration a mono-
tonic increase in C4 contribution was observed (R 2
0.96; P , 0.01) with no signicant differences (P 0.31)
between soil depths. At years 6 and 9, over 40% of total
soil carbon was originated from exotic C4 plants. By
year 14, C4-derived carbon constituted more than 65%
of the total SOC, and only about 35% of the
accumulated carbon could have originated from bio-
solids and native C3 plants together.
SOC chemical and physical fractions
The results of chemical (Fig. 5A) and physical (Fig.
5B) fractions show that as SOC levels increased both the
stability and fate of soil carbon were subject to

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 Month 2013 UNPRECEDENTED CARBON ACCUMULATION

carbon source (Fig. 4), changes in chemical fractions

FIG. 4. The relative contribution of C4 grasses to total soil
carbon across a chronosequence of mined sites undergoing
restoration. Floristic surveys show that invasive grasses of the
genera Brachiaria and Melinis dominate the herbaceous layer of
all sites. Native C4 grasses occurred in surrounding areas, but in
mined sites they were rare or absent. The line represents a
signicant relationship between time since restoration and
contribution of C4-derived carbon to total soil pools (P , 0.01).
No signicant differences in the source of carbon were found
across soil depths (P 0.31). Error bars represent SDs of mean
values (15 proles) at each site.
signicant variation. A monotonic increase in the humic
(recalcitrant) fraction is observed over time, while fulvic
acid content peaked by year 3, decreasing progressively
in the following years. Combined with SOC accumula-
tion (Fig. 3) and change in the relative contribution of
reect a fast turnover of the applied biosolids and
stabilization of plant-derived compounds. A propor-
tional decrease in the humin fraction (;10%) resulted
from new inputs from plant residues. However, when
total increases in SOC are taken into account (Fig.
3A, B) the absolute content of humin also increased.
The analysis of physical fractions shows a continuous
accumulation of carbon in stable microaggregates. The
relative accumulation of SOC as macroaggregates
decreased between years 3 and 6 after restoration, as a
result of microaggregate formation and increases in light
fraction. Coinciding with changes in chemical fractions,
physical fractions reect fast turnover of biosolids and
fresh residues, which stabilized and accumulated as
occluded SOC. A decrease in the relative accumulation
of carbon in the silt and clay size fraction is also
observed as a result of new carbon inputs. When total
SOC is taken into account, however, absolute values of
SOC in this fraction increase. These results were
consistent across depths, with no signicant interactions
between the proportion of either chemical or physical
fraction and soil layer (P 0.26). For simplicity, only
average values are presented.
Soil nutrient and leaf d15N
Soil C to N ratios increased in all physical fractions
from the application of biosolids onward (Appendix B),
indicating increasing nutrient constraint associated with
the dominance of invasive species. Average soil C to P
ratios showed a similar increasing trend (Appendix C),
matching the growing contribution of invasive grasses to
SOC (Fig. 4). Leaf d15N measured in invasive grasses

FIG. 5. (A) Chemical and (B) physical fractions of soil organic carbon averaged at each site across all soil depths. Stacked bars
represent the relative contribution of each fraction to total carbon pools. No signicant interactions were found between the
fraction and soil layer (P 0.26). Uppercase letters show signicant differences between sites/years (comparison of each fraction
across sites) as determined by ANOVA and Tukeys HSD post hoc test (P , 0.05).

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LUCAS C. R. SILVA ET AL.
also points to increasing efciency and progressive
nutrient limitation. Time 0.5 had the most enriched
values (5.7%) indicating that most of the N used by
plants at that stage came from enriched biosolid sources
(8.1% 6 0.7%). The establishment of a diverse plant
community (including N-xers) between years 3 and 6
led to lower leaf d15N (,4%), which became progres-
sively more enriched as invasive excluded native species
(Appendix D).
DISCUSSION
Unprecedented accumulation of SOC
Conrming hypothesis (1), our results show that
substrate restoration prompted the natural establish-
ment of vegetation cover for the rst time since mining
activities took place in the 1960s (Fig. 2). Organic matter
application enhanced soil organic carbon (SOC) to pre-
disturbance levels, but the synergistic effect of resource
input (organic matter and nutrients) and plant coloni-
zation further promoted soil development and SOC
accumulation (Fig. 3), conrming hypothesis (2). After
14 years, neither vegetation structure nor composition
resembled those of the original woody savanna that
dominates the surrounding landscape in the Federal
District of central Brazil. Instead, exotic grasses
established a oristically and structurally simpler
physiognomy. This was characterized by vegetation
surveys and shifts in SOC isotopic composition (Figs.
2 and 4), which show that invasive grasses progressively
dominated recovering sites, representing the most
important source of SOC. This conrms hypothesis
(3), as it shows that new edaphic thresholds determined
by enhanced resource availability favored invasive
species, which now control the systems productivity.
Restoration techniques that rely solely on subsoiling
are typically not sufcient to promote plant colonization
(Martins et al. 2004, Silva and Correa 2008), but here
the incorporation of nutrient-rich biosolids (tertiary-
treatment domestic sewage sludge) immediately prompt-
ed revegetation and soil formation. Soil carbon has been
recognized as the most useful index to assess restoration
success and the inuence of colonizing species in
repairing ecosystem function (Baer et al. 2002, Banning
et al. 2008, De Deyn et al. 2008, Berthrong et al. 2012).
Surprisingly, only a few years after treatment, SOC in
mined areas was higher than what had accumulated over
thousands of years in primary forests of the same region
(Silva et al. 2008, 2010a). Soil carbon pools represent the
balance between input via primary productivity, and
output via decomposition, charring or burning, volatil-
ization, and leaching of compounds (Amundson 2001).
The maximal potential of soils to sequester carbon is
determined by intrinsic abiotic variables, but carbon
dynamics can also be driven by biotic factors (De Deyn
et al. 2008). In our present study no differences in
topography, mineralogy, soil texture, or pH, which
could perhaps explain accumulation by adsorption
processes (Hassink 1997, Rasmussen et al. 2007), were
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found. Soil development has been driven by species
colonization, but stabilization mechanisms may also be
important to explain SOC accumulation.
Potential mechanisms
Primary producers affect SOC stabilization, through
changes in predominant traits (e.g., C to N ratio, lignin
to N ratio, and so forth) (De Deyn et al. 2008, Dumig et
al. 2009, Berthrong et al. 2012). Therefore, changes in
community composition could affect patterns of SOC
accumulation (Reiners et al. 2005, Silva and Anand
2011). Indeed high productivity and slow decomposi-
tion, typical of grasses, can increase SOC. However, in
areas where the same invasive grass species are
cultivated for pasture under ideal edaphic conditions
and similar climates, levels of SOC are typically much
lower than what we measured here (e.g., ,2%; Tarre et
al. 2001).
True accumulation of organic matter (as opposed to
the temporary presence of organic material) combines
the decomposition of inputs, assimilation by microbes,
and accumulation of microbial and plant residues as
stable compounds (Collins et al. 1997). At our sites
much of the increase in SOC occurred in the humic acid
fraction, showing continuous transformation of organic
matter and accumulation in a form distinct from the
inputs. Since chemical fractions, depending on the
environment, are all dynamic and heterogeneous to
some degree, it may be simplistic to state that one
fraction is more stable than another. However, many
studies have shown that different fractions do in fact
have different roles in carbon accumulation, with the
humic fraction representing compounds with greater
stability (e.g., Doane et al. 2003).
The balance between carbon input and turnover also
depends on the interplay of stochastic processes and
biochemical kinetics determined by the variability of
decomposers (Forney and Rothman 2012). In degraded
soils decomposition may be limited by the absence of
decomposers, or nutrients, rather than by the carbon
source itself (Hassink 1995). Here, fast mineralization of
the applied biosolids was likely promoted by their own
heterotrophic decomposers (Correa et al. 2012). How-
ever, the development of a new microbial community
probably triggered further transformation of primary
minerals and coating of organic compounds.
The effects of microbes on composition and recalci-
trance of SOC, have been shown to be as important as
climatic and edaphic factors (Throckmorton et al. 2012).
Although the processes that lead to formation of soils
are fundamentally the same in all climate zones, the
intensities of biological transformations are consider-
ably greater in the tropics, where cambisols can form in
a few years (Rashid and Memon 1996). As with
chemical fractionation, our observations of physical
fractions provide evidence for this process, with
occluded SOC representing the most important repos-
itory of plant-derived carbon as soil develops. After the
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 Month 2013 UNPRECEDENTED CARBON ACCUMULATION
incorporation of fresh residue, microorganisms produce
mucilages that result in the formation of macroaggre-
gates, which after further decomposition form the stable
core of microaggregates (Six et al. 2004). Microaggre-
gates take longer than macroaggregates to decompose
(Forney and Rothman 2012) and, therefore, they persist
and accumulate over time despite the relatively faster
formation of the latter (Fig. 5).
In summary, simultaneous shifts in chemical and
physical fractions that we observed show that increases
in SOC are due to the accumulation of stable carbon and
do not simply represent a transient effect of dynamic
pools. Furthermore, these shifts indicate that post-
depositional processes exert long-lasting effects on
SOC. Associations of organic compounds with short
range order minerals, such as aluminosilicates (allo-
phane and imogolite) and iron oxyhydroxides (ferrihy-
drite), stabilize carbon in deep soils (Rasmussen et al.
2007) and exposed parent materials (Baldock and
Skjemstad 2000). Short range order mineralsones that
lack well-dened arrangement of atoms in their crystals,
commonly described as amorphous material, with high
surface area and high adsorption and ion-exchange
capacities decrease carbon bioavailability (Negre et al.
2002), and primary clays, more abundant in regoliths
than in topsoils, protect organic particles from decom-
position resulting in SOC accumulation (Hassink 1997).
Accumulation of SOC could thus be more related with
organo-mineral associations than with the source of
organic matter. Molecular analyses could be used in
future studies to characterize these associations and
reveal specic mechanisms at play in SOC stabilization
in developing soils.
Theoretical implications
In the hierarchical analysis of ecosystem resilience,
soil formation is considered a slow process, as opposed
to the relatively faster establishment of stable plant
communities (Gunderson and Holling 2002). However,
fast and slow processes interact across various scales of
space and time, determining recovery trajectories. Here
we show that resource input moves degraded systems
from a low-energy (bare soil) stable state to a dynamic
condition where successional forces can actuate. Steep
SOC accumulation (0 to 6 years) followed by a slight
decline (9 to 14 years) match changes in species diversity,
where the shape of SOC curves resembles those
predicted for diversity-dependent productivity in natural
forests and grasslands (Reich et al. 2012, Tilman et al.
2012). This suggests that community-level feedbacks
play a key role in driving soil formation, not only in
natural ecosystems (Ehrenfeld et al. 2005) but also in
degraded areas. Interestingly, soil restoration appears to
be occurring at faster rates than community reorgani-
zation in our sites, reecting the effect of resource input
and exotic species invasion. These ndings reinforce the
notion that soil development and competition for
resources are interdependent processes (Casper and
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Castelli 2007) and, as such, their importance in
(re)structuring plant communities cannot be judged in
isolation from each other.
In the context of succession, resource-ratio theory
(Tilman 1985) predicts that resource supply determines
whether competing species can coexist and, if not, which
species will exclude others. It also predicts that the
highest diversity of competing species should occur at
intermediate resource availability. Well over a thousand
research articles have cited this theory since its
publication, but only a few tested its predictions (until
2005 only six tested the rst and none the second
prediction mentioned above; Miller et al. 2005). The
conrmation of the hypotheses posed here provides
critical support to both these predictions. As we have
shown, a pulse of resource availability allowed plant
colonization, but favored exotic grasses at the expense of
native species. Moreover, maximum diversity was
reached (year 6) at an intermediate ratio of resource
availability, after the peak of biosolid mineralization
(year 3), but before nutrient sequestration in the
biomass of invasive species reached critical levels (year
9) (Appendices B and C). These ndings advance our
understanding of how soilplant feedbacks regulate the
stability of ecosystems, but also have important
practical implications.
Implications for conservation and management
Through impacts associated with differential re-
source-use strategies, exotic species alter community
conguration and function in various ecosystems
(Knops et al. 2002, DAntonio and Meyerson 2002,
Gurvich et al. 2005, Gaertner et al. 2012). In Brazilian
savannas, exotic grasses affect early growth and survival
of native trees, hindering seedling development due to
shading and competition for nutrients (Hoffmann and
Haridasan 2008). Native savanna trees demand more
nutrients than invasive grasses to establish, thus,
nutrient sequestration in the biomass of grasses can
limit the success of seedlings in invaded plots. The
combination of disturbance regime and resource avail-
ability shape the distribution of grasslands, savannas,
and forests in central Brazil (Hoffmann et al. 2012).
However, the stability of these ecosystems over the
geological past (Silva et al. 2008, 2010a) suggest that
more complex vegetation forms will eventually develop
in recovering areas.
At our sites, soil C to N and C to P ratios (Appendices
B and C) and d15N in the leaves of invasive grasses
(Appendix D) increased consistently over time. This
reects increasing connectedness and efciency, as
nutrient cycling between soils and plant biomass become
tightly linked, with invasive grasses controlling resource
availability. Changes in litter quality and additional
resource input from colonizing trees could promote the
establishment of other species and sustain more diverse
and productive plant communities (Silva and Anand
2011). Indeed additional inputs from native N-xers led
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LUCAS C. R. SILVA ET AL.
FIG. 6. Conceptual scheme of alternate stable states
expected as a result of disturbance and resource inputs.
Directional responses from low (open) to high (dense) energy
stable states are expected under disturbance suppression and/or
increased resource availability. The gray circles show vegetation
and soil carbon conditions after four decades of abandonment
after mining activities. The white circles show the expected
trajectory. The black circles show observed outcomes, which
match expectations in (A) vegetation cover, but not in (B) soil
carbon accumulation. The expected levels of carbon accumu-
lation at each of these co-existing ecosystems are based on
values found in undisturbed ecosystems in the same region
(Silva et al. 2008, 2010a, b). All broken lines represent
disturbance effects of various intensities, the most severe being
mining activities that would lead to stable bare soils.
to lower leaf d15N (,4%) in invasive grasses, which until
then relied mostly on N from the applied biosolids
(.8%). Atmospheric d15N is dened as 0 and for this
reason plants that rely on symbiotic N uptake have
depleted signatures (Michener and Lajtha 2007). Here,
low d15N values reect the highest contribution of
nutrients from N-xers between years 3 and 6 after
restoration, but further d15N enrichment was observed
as exotic species excluded native species. The arrival of
native woody species in some of our sites is far from
characterizing a transition to savanna, but it is
reasonable to assume that the introduction of such
species would bring the system closer to its original state.
However, it is difcult to predict whether soil carbon
accumulation could be maintained without the exotic
vegetation cover. When better understood, the mecha-
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nisms of SOC stabilization in exposed regoliths could be
used in combination with species introduction, adding to
the benets of resource-based restoration the mainte-
nance of species diversity.
Final considerations
Based on our observations we conclude that the
synergistic effects of resource input and species invasion
can be benecial, restoring soil productivity and
vegetation cover in severely disturbed areas, or delete-
rious, resulting in resilient low-diversity systems. Sum-
marizing our results, we have elaborated a conceptual
scheme that illustrates the effect of disturbance and
resource input in driving the development of alternative
stable equilibria at degraded sites. The various states of
stability presented in this scheme correspond to several
ecosystems that coexist in the study region and one
unvegetated state caused by mining impacts (Fig. 6A).
Each state corresponds to a stable conguration, but
negative or positive perturbations could cause transi-
tions to a different stable state. Using SOC values
typically found in each of these regional ecosystems, we
represented the expected trajectory of soil restoration,
measured as SOC accumulation (Fig. 6B). As with
vegetation, directional responses in SOC from low to
high energy levels are expected under disturbance
suppression or increased resource availability, but
unexpected responses may also occur. Here this was
evidenced by an unprecedented SOC accumulation.
These ndings show that ecosystems undergoing resto-
ration are moving targets, with multiple possible futures
dened by converging soil and vegetation processes.
Management strategies must be exible, adaptive, and
prepared to deal with unexpected outcomes. Soilplant
feedbacks determine tipping points beyond which
stability is gained or lost. Thus, integrative analysis of
soils and vegetation are essential to assess and mitigate
adverse impacts, while keeping the benets of species
invasion on soil formation and restoration of degraded
lands.
ACKNOWLEDGMENTS
We thank Mark Leithead from the Laboratory of
Quantitative Ecology UFRGS (Brazil), Madhur Anand from
the Global Ecological Change Laboratory and Paul Voroney
from the School of Environmental Sciences University of
Guelph (Canada), and other participants and organizers of the
Kinross CanadaBrazil Research Network Symposium (2012,
Sao Paulo, Brazil) for valuable discussion. We also thank the
landowners for allowing research activities and collection of
material in their properties and the Catholic University of
Brasilia for logistic support during eld activities, sample
storage and preparation. Support for this study was provided
by J. G. Boswell Endowed Chair in Soil Science.
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SUPPLEMENTAL MATERIAL
Appendix A
Vegetation census at all study sites (Ecological Archives XXXXX).

Appendix B
Soil C to N ratios at each physical fraction in surface and deep layers (Ecological Archives XXXXX).

Appendix C
Average C to P ratios in soil proles (Ecological Archives XXXXX).

Appendix D
Average d15N in invasive grass leaves across sites (Ecological Archives XXXXX).

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