Evolution of nitrogen cycling in regrowing Amazonian rainforest

Abstract

Extensive regions of tropical forests are subjected to high rates of deforestation and forest regrowth and both are strongly affect soil nutrient cycling. Nitrogen (N) dynamics changes during forest regrowth and the recovery of forests and functioning similar to pristine conditions depends on sufficient N availability. We show that, in a chronosequence of Amazonian forests, gross nitrification and, as a result, nitrate-to-ammonium (NO3−: NH4+) ratio were lower in all stages of regrowing forests (10 to 40 years) compared to pristine forest. This indicates the evolution of a more conservative and closed N cycle with reduced risk for N leaking out of the ecosystem in regrowing forests. Furthermore, our results indicate that mineralization and nitrification are decoupled in young regrowing forests (10 years), such as that high gross mineralization is accompanied by low gross nitrification, demonstrating a closed N cycle that at the same time maintains N supply for forest regrowth. We conclude that the status of gross nitrification in disturbed soil is a key process to understand the mechanisms of and time needed for tropical forest recovery.

Full text

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Evolution of nitrogen cycling in
regrowing Amazonian rainforest
Viviane Figueiredo1,2,3, Alex Enrich-Prast1,2,3,4 & Tobias Rütting5
Extensive regions of tropical forests are subjected to high rates of deforestation and forest regrowth
and both are strongly aect soil nutrient cycling. Nitrogen (N) dynamics changes during forest regrowth
and the recovery of forests and functioning similar to pristine conditions depends on sucient N
availability. We show that, in a chronosequence of Amazonian forests, gross nitrication and, as a
result, nitrate-to-ammonium (NO3−: NH4+) ratio were lower in all stages of regrowing forests (10 to
40 years) compared to pristine forest. This indicates the evolution of a more conservative and closed N
cycle with reduced risk for N leaking out of the ecosystem in regrowing forests. Furthermore, our results
indicate that mineralization and nitrication are decoupled in young regrowing forests (10 years), such
as that high gross mineralization is accompanied by low gross nitrication, demonstrating a closed N
cycle that at the same time maintains N supply for forest regrowth. We conclude that the status of gross
nitrication in disturbed soil is a key process to understand the mechanisms of and time needed for
tropical forest recovery.
In the Brazilian Amazon region, almost 800 000 km2 of land has been deforested, mainly for soya bean cultiva-
tion, logging and cattle ranching1. e high rate of tropical deforestation led to global concern since these areas
are a hot spot of biodiversity and have direct inuence on the global climate through hydrology and exchange
of greenhouse gases2–5. However, a large area of approximately 167 000 km2 previously deforested land has been
abandoned aer exploitation6 and secondary forests have established on that land7. e regrowth area in the
Amazon is increasing6, but our current knowledge about nutrient availability, biogeochemical processes, and how
the post-disturbance regeneration inuences these processes is poorly understood8. Likewise, nutrient shortage
in deforested areas is expected9, but the inuence and magnitude of limitation, which can drive the recovery tra-
jectory, on regrowth forest are still uncertain10.
Early secondary forests have high growth rates with rapidly increasing forest biomass11, even when N is appar-
ently limited12. is indicates that feedback mechanisms on soil N availability exist, providing sucient plant
available N to maintain forest regrowth. Microbial processes, such as mineralization and nitrication, drive the
soil N cycle and thereby control the amount of organic and inorganic N forms in soil13,14. Mineralization of soil
organic matter (SOM) is responsible for inorganic N production in terrestrial ecosystems, which is important
for plant N uptake that occurs mainly in inorganic form. e NH4+ released by mineralization also supports
nitrication15, the oxidation of NH4+ to NO3−. ese two inorganic N forms may have dierent fates in soils, as
immobilization in biomass, leaching and gas losses16, and the occurrence and magnitude of these pathways might
inuence the forest growth17.
Davidson et al.8 investigated the N cycling recovery in secondary forest age chronosequences aer agricul-
tural abandonment in the Amazon region. ese authors found indications for a conservative N cycling in soils
of young successional tropical forests based on N and phosphorus (P) contents in leaves, litterfall and soils, low
NO3−: NH4+ ratios as well as low nitrous oxide (N2O) emissions. However, the mechanistic changes in the soil N
cycleduring forest regrowth have not been studied in the Amazon Region. e actual dynamic of labile N in soils
is best represented by gross soil N cycle dynamics, such as gross N mineralization and nitrication, since the gross
transformations directly control the inorganic N availability for plants growth. erefore, quantifying the gross
N transformations in tropical regrowth forest soils is an important step in managing and enhancing abandoned
managed areas, which also provides valuable information for model implementation.
1Department of Botany, University Federal of Rio de Janeiro, 21941-971 Avenida Carlos Chagas Filho, Rio de Janeiro,
Brazil. 2Postgraduate Program in Geochemistry, University Federal Fluminense, 24020-007 Outeiro de São João
Batista, Niterói, Brazil. 3Postgraduate Program in Biotechnology, University Federal of Rio de Janeiro, 21941-971
Avenida Carlos Chagas Filho, Rio de Janeiro, Brazil. 4Department of Environmental Change, Linköping University,
58183, Linköping, Sweden. 5Department of Earth Sciences, University of Gothenburg, 405 30, Gothenburg, Sweden.
Correspondence and requests for materials should be addressed to A.E.-P. (email: alex.enrich.prast@liu.se)
Received: 6 August 2018
Accepted: 12 April 2019
Published: xx xx xxxx
OPEN
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We evaluated the gross soil N cycling in four forests, including one pristine forest and one regrowth forest
(40 years old) located inside and two regrowth forests (10 and 20 years old) near the Ecological Station of Cuniã
in the state of Rondônia, Western Amazonia, with focus on gross N mineralization and gross nitrication. e
slash-and-burn practice was applied in all three regrowth areas studied here. e 15N pool dilution technique
using the “virtual soil core” approach18 was used to quantify in situ gross N processes rates. Predominant soil
type of the investigated forests is Plinthosol19, soil texture in the pristine forest is sandy loam with 55.4% (±4.4)
sand, 39.1% (±4.8) silt and 4.9% (±0.7) clay (mean ± SD; N = 7). e vegetation is dominated by hardwood with
abundance of palms20.
Results and Discussion
Sustained production of plant available N in tropical regrowth forests. Changes in the internal
soil N cycle as consequence of reforestation reect alterations in the microbial and plant community during
regrowth stage21. Rates of gross mineralization in the pristine forest at Cuniã (7.8 ± 4.7 µg N g−1 d−1; Fig.1) are
within the range of gross mineralization reported in other pristine tropical forests22,23. In a study in Eastern
Amazon forest24 during the dry season, gross mineralization was measured in situ with a rate of 13.9 ± 3.8 and
7.2 ± 1.8 µg N g−1 d−1 from clay and sandy soils24 respectively, similar to the gross rate in the pristine forests in
our study.
Along the chronosequence of forest regrowth gross N mineralization was nearly doubled in the youngest
forest (10 years old; 14.8 ± 6.5 µg N g−1 d−1) but only half in the older regrowth forests (3.8 ± 2.1 µg N g−1 d−1)
compared to the pristine forest (Fig.1). A similar pattern was also observed in subtropical Australia, where gross
N mineralization was 2 to 3 times higher in early monospecic forest plantation (5 years) than pristine forest and
older (53 years) plantation25. In general, the main pattern seen in early successional forests is high rate of NH4+
release through mineralization25,26, although fewer contrasting results have also been reported27–29.
e observed change in gross N mineralization is not caused by SOM content. Across the chronosequence,
the SOM content in pristine forest was signicantly higher (P < 0.05) than in 10 years old regrowth, but not dif-
ferent from 20 and 40 years old regrowth forest (Table1). is pattern is exactly the opposite as found for gross
mineralization, hence the lowest SOM content was found in the forest with the highest gross N mineralization
rate (10 years old regrowth forest). Instead, the quality of SOM30,31 might be more important for controlling N
mineralization24,32,33. e C: N ratio, an indicator of the SOM quality and its degradation rate34, conrmed that,
since the 10 years old regrowth had the lowest C: N ratio of 17.7 of the investigated forest soils (Table1). Gross
mineralization in early regrowth forests can be high due to the recent disturbance that redistribute SOM stored
in deeper soil layers to soil surface26. Furthermore, the previous management, as slash-and-burn, degraded the
SOM, releasing labile compounds that are easier to mineralize21,35. Subsequently, gross mineralization decreases
over time, possibly due to depleting in labile SOM and are lower than in pristine forests due to reduced root
exudation and rhizosphere priming36–38. Aer a re event, soil texture might change, usually showing a decrease
of clay and increase of sand content39. In addition, clay aggregates can change in terms of size and distribution in
the soil. We only measured soil texture in the pristine forest, which had low clay content (4.9 ± 0.7%). erefore,
Figure 1. Gross N mineralization (gray bars) and nitrication (white bars) rates (µg N g−1 SDW d−1;
Mean ± SE) in four forest soils at Cuniã Ecological Station, Rondônia, with one pristine forest (set to t = 0
years) and three regrowth forests (10, 20 and 40 years old). (a) For gross N mineralization, the 10 years old
regrowth forest was signicantly higher than the 40 years old forest (One way ANOVA with Tukey’s post hoc
test P < 0.05) and slightly higher than the 20 years old forest (One way ANOVA with Tukey’s post hoc test
P = 0.055). F value was 1.327 with degree of freedom of 3. (b) For gross nitrication, the pristine forest was
signicantly higher than all three regrowth forests (One way ANOVA with Tukey’s post hoc test P < 0.05). F
value was 1.629 with degree of freedom of 3.
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we expect small changes in soil texture along the chronosequence caused by the slash-and-burn practice, conse-
quently hence having minimal eects on the measured microbial processes rates.
Our results indicate that plant available N is sustained during forest regrowth due to enhanced gross N miner-
alization. High N mineralization in the early successional stage provides plant available N, overcoming a potential
N limitation of forest regrowth. With time, N demand will decrease, which is also reected in the decrease in
gross N mineralization over time of forest regrowth found by us (Fig.1) and others25.
Conservative N cycling in tropical regrowth forests through decreased nitrication. Secondary
forests exhibit a more conservative N cycle compared to pristine forests in the Amazon region, indicated by the
shi in the dominant inorganic N form in the soil towards NO3− (refs8,40), which is also observed at the chronose-
quence at Cuniã (Fig.2). We show here that the underlying process is a change in gross nitrication, which was
signicantly lower in all stages of forest regrowth than the pristine forest in our chronosequence (Fig.1). Gross
nitrication rates of 3.27 ± 1.14 µg N g−1 d−1 in the pristine forest at Cuniã are in accordance with rates reported
in earlier studies, which reported in situ gross nitrication in the range of 0.5 to 5.2 µg N g−1 d−1 (e.g. refs22,24,41).
Gross nitrication rates were lower in all regrowth stages, than in the pristine forest (Fig.1), justifying the
measured low soil NO3− content (Fig.2), which conrms the idea of N retention and conservation during ecosys-
tems succession8. is could be related to the alteration of the soil microbial community. In the Amazon region a
higher abundance of nitriers was found in a pristine forest in comparison to regrowth forest soils42, explaining
the higher rates in pristine forest. In addition, an enhanced plant N demand, competing with the nitriers for
NH4+, could also contribute to a low gross nitrication in regrowing forests.
Pristine 10 yrs. 20 yrs. 40 yrs. F, degrees of freedom
pH 3.7 ± 0.04a
N = 14 3.4 ± 0.1b
N = 6 3.9 ± 0.04c
N = 6 3.8 ± 0.05a,c
N = 6 12.82, 3
GWC (%) 35.1 ± 0.8a
N = 51 22.0 ± 0.9b
N = 17 35.8 ± 1.5a
N = 15 30.8 ± 1.2a
N = 14 Non-parametric data
SOM (%) 7.5 ± 0.4a
N = 50 5.1 ± 0.7b
N = 11 8.0 ± 1.8a,b
N = 10 6.8 ± 1.2a,b
N = 11 6.676, 3
TC (%) 4.4 ± 0.3a
N = 51 2.9 ± 0.4b
N = 12 4.7 ± 1.a,b
N = 10 3.9 ± 0.7a,b
N = 11 4.074, 3
TN (%) 0.19 ± 0.01a
N = 51 0.17 ± 0.02a
N = 12 0.20 ± 0.01a
N = 12 0.17 ± 0.02a
N = 11 0.7086, 3
C: N 24., 8 ± 1, 6a
N = 51 17., 7 ± 2, 1a
N = 12 28. ± 3.7a
N = 9 25.0 ± 4.8a
N = 11 Non-parametric data
Table 1. Soil properties (mean ± SE) of pristine forest and three regrowth forests (10, 20 and 40 years old)
at/near the Ecological Station of Cuniã, Rondônia (Brazil). e letters a, b and c represent the values that are
statistically signicantly dierent in the four studied sites One way ANOVA with Tukey’s post hoc test (P < 0.05)
was used for parametric soil properties (pH, SOM, TC, TN) and Kruskal-Wallis test with Dunn’s post hoc test,
P < 0.05 for non-parametric (GWC and C: N). e F values and degrees of freedom were provide for parametric
data.
Figure 2. Content of soil NH4+ and NO3− as well as NO3−: NH4+ ratio in pristine forest (set to t = 0 years) and
three regrowth forests (10, 20 and 40 years) at the Ecological Station of Cuniã, Rondônia (Brazil). e contents
were calculated from the rst extraction aer 15N labelling by subtracting the amount of tracer recovered (based
on 15N enrichment). e black circle represents NH4+ content, the empty circle represents NO3− content and the
symbol X represents the NO3−: NH4+ ratio. e unit of the N contents is µg N g−1 SDW and the values represent
mean ± standard e rror.
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e pattern of gross nitrication observed in this study, based on pseudo-replicated eld experiment, is con-
sistent and in agreement with studies from other chronosequences of tropical and sub-tropical forests22,25,28,29,43,44
(Fig.3), which enables generalizations. Although the magnitude of gross nitrication varies between the dierent
studies, consistently higher gross nitrication rate in pristine than secondary forests has been found, corroborat-
ing gross nitrication as the most suitable N process to evaluate recovery of tropical forest ecosystems.
Nitrogen cycling along a tropical forest chronosequence. Tropical regrowing forests are character-
ized by a closed N cycle with low risk for N losses, indicated by decreased NO3−: NH4+ ratios7,8, which was also
observed in this study (Fig.2). Here, we provide a mechanistic understanding of the biogeochemical processes
responsible for the evolution of the N cycle under forest regrowth. e consistent pattern reported for gross nitri-
cation (Figs1 and 3) explains the observed pattern of low NO3−: NH4+ ratios in regrowing forests. e relative
excess in NO3− in pristine forests promotes N losses by leaching and gaseous emission23,24,45,46. Regrowing forests,
on the other hand, have a tighter N cycle with decreased N losses and enhanced N retention47.
Particularly the results from an investigation in a pristine sub-tropical forest and two forest plantations of
dierent age in Australia are strikingly similar to our study25 (Fig.3). Early regrowth forests are in both studies
characterized by high rates of gross N mineralization and low rates of gross nitrication (Fig.2), showing a decou-
pling of these two processes. As a consequence, inorganic N in the young forests is mainly in the form of NH4+,
which leads to reduced N losses24,48, but maintains availability of N for plant uptake. Older regrowing forests, have
a lower N demand49 and not only nitrication but also mineralization rates are low25 (Fig.1).
Although we did not directly investigate this, our results infer that plants are probably crucial in regulating the
observed pattern of dominant N pathways during the forest regrowth in this part of the Amazon (Fig.4). Root
exudation and plant N uptake control the availability of inorganic N by aecting N cycling processes. e root
exudation of labile organic compounds in the pristine forest provides not only a substrate for N mineralization
but can stimulate gross mineralization further by rhizosphere priming37,38,50 (Fig.4c). In regrowing forests with
lower tree biomass, root exudation is lower, thereby reducing the eects on gross mineralization (Fig.4a,b). In
the youngest regrowth forest, this negative eect is though more than compensated for by the presence of labile
SOM from the slash-and-burn practices21,35. e N assimilation in biomass is larger in regrowth in comparison
to pristine forests, which have more N loss from litterfall than regrowth forests40. Because of that, the net uptake
(gross N uptake minus N loss) is higher in regrowth (Fig.4a,b) in comparison to pristine51 (Fig.4c), decreasing
the availability of NH4+ for nitriers, leading to a decrease in nitrication. As a consequence, the NO3−: NH4+
ratio will vary according to the forest status.
Relevance of gross nitrication as an indicator of forest recovery. Gross N mineralization and gross
nitrication are sensitive to environmental changes and ecosystem disturbance. Our ndings suggest that 20
years aer slash-and-burn disturbance gross N mineralization process decreases to closer rates to pristine stage.
On the other hand, gross nitrication did not recovery even aer 40 years (Fig.1). is result indicates that the
time for gross nitrication to recover to pristine conditions is much longer, suggesting that this process is more
sensitive to disturbances. As a consequence, N is retained in the soil as NH4+, a plant available form, which is less
prone to leaching processes. It is important to highlight the small range of environmental conditions evaluated
here, such as season of the year associated to the precipitation variability, limited spatial replication, and absence
of some characteristics of soil (i.e. soil texture), which can be a source of variation and spatial limitations. In the
Eastern Amazon, Sotta et al.24 did not nd dierences in gross soil N cycling between clay and sand soil, neither
between seasons. Moreover, the environmental factors probably have stronger inuence on the magnitude than
the patterns of the N cycle rates. Our ndings have shown similar gross nitrication patterns as other tropical
chronosequence forests (Fig.3), which gives condence in the robustness of the observed pattern. ese ndings
combined demonstrate the sensibility of nitrication to disturbances in dierent tropical forests around the world
and highlights the importance of gross nitrication as being the best mechanism to evaluate the evolution and
recovery of N cycling in soils of secondary succession forests.
Figure 3. Relative gross nitrication rates in dierent tropical and subtropical pristine and secondary forests
around the world. e gure compiles relative nitrication rates from pristine forest soil (black bar representing
the highest nitrication rate in percentage) and secondary forests (plantation or regrowth) of dierent age (bars
with dierent tones of gray). Data from refs22,25,28,43,44 and the present study.
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Methods
Study area. e study was carried out at and near the Ecological Station of Cuniã, Porto Velho municipal-
ity, Rondônia, Brazil (08°06′23″ S and 63°28′59″ W). e station was established for conservation and scientic
research in 2001 in one of the Brazilian states with highest deforestation rate in the Amazonian region between
1980 and 199052. e area of the station corresponds to 125,849.23 hectares of open rainforest dominated by
hardwood with abundance of palms20. e soils studied were classied as Plinthosols, iron-rich and humus-
poor and predominance of kaolinitic clay19. Inside the station, there is an area of 2500 ha previously dened for
sampling and used in long-term monitoring. e mean annual precipitation in this region is 2500 mm, the rain
season occurs from October to April, and the dry season from June to August. e mean annual temperature is
around 26 °C53.
To investigate the in situ gross N transformations in intact soils of pristine and regrowth forest, one pristine
forest and three regrowth forests with an age of 10, 20 and 40 years aer slash-and-burn practice were chosen.
e pristine forest was inside a grid of 1 km2 and was within the long-term monitoring site, as was the 40 years old
regrowth forest (3–4 km from the pristine plot). e other two regrowth forests (approximately 10 and 20 years
old; personal communication by local farmer) were located in the surrounding area, 10–12 km away.
In situ 15N labelling. To investigate the in situ gross N transformations in intact soil, with an intact rhizos-
phere, a 15N labelling using the “virtual soil core” approach18 was conducted at the beginning of the dry season in
April 2013. Earlier studies on tropical forest soils24,41 found no dierences in gross N rates between dry and wet
season.
In the pristine forest seven plots in two straight lines, 1 km apart, were established with 10 m distance between
plots. In the regrowing forests, three plots were randomly chosen with a distance of 10 m either in a straight line
(40 years old) or in a triangle (10 and 20 years old), which was mainly governed by accessibility. Each plot was
a pseudoreplication and, in each of them, two sets of a paired labelling spots were establish receiving a solution
containing NH4+ and NO3− with one of the N species enriched with 15N at 99% (Supplementary Fig.1a).
Each spot received eleven 1 mL injections of 15N solution in a circular area of 7 cm in diameter, homogenously
distributed into the soil underneath the litter to a depth of 9 cm using a 1 mL syringe and 9 cm spinal needle18
Figure 4. Conceptual model of N cycling along a forest chronosequence in Amazon region, (a,b). Nitrogen
pathways in regrowing forest soils of dierent ages aer one time disturbance by slash-and-burn. In the
early regrowth forest (10 years; a) a new source of labile N from the burning of biomass stimulates gross
mineralization and, as consequence of investment in forest growth, higher N uptake by plants. Nitrication and
N uptake receiving support from N mineralization in 20 and 40 years old regrowth forests (b). In pristine forest
(c) root exudations stimulates mineralization, which supports nitrication. See text for more details.
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(Supplementary Fig.1b). e total amount added corresponded to 1.73 µg NH4+-N and 0.86 µg NO3−-N per
gram dry soil. One of the paired labelling spots was sampled immediately aer labelling (t0) and the second one
24 hours (t24) aer labelling. Soil sampling was conducted in the inner 4 cm labeling spot. e larger labeling area
provides a buer zone around the sampling18.
e intact soil samples were immediately transported to the eld laboratory, where they were gently broken by
hand to remove stones, leaves and large roots by tweezers. Aer sieving, 50 grams of each soil sample was added
to a brown plastic bottle together with 100 mL of 1 M KCl, placed on a shaker for 1 hour, and lastly ltered through
MN 615 lter paper (Macherey-Nagel).
e remaining soil was dried later in the laboratory to measured physicochemical soil properties; gravi-
metric water content (GWC) by drying at 100 °C, the soil organic matter content (SOM) by loss-on-ignition,
and the total C and N (TC and TN) was measured on an elemental analyser coupled to an Isotope Ratio Mass
Spectrometer (IRMS) (20–22, Sercon Ltd., Cheshire, UK). e pH was measured in the 1 M KCl extracts with a
pH meter (691, Metrohm AG, Herisau, CH). Concentrations of NH4+ and NO3− in KCl extracts were measured
on ow injection analyser (FIAstar 5000, Foss Tecator AB, Brazil). e soil texture was determined in the pristine
soil using a laser type granulometer (Malvern Mastersizer 2000, Malvern Instruments SA, Orsay cedex, France).
e soil properties are showed in Table1.
For analysis of 15N abundance, NO3− in extracts was measured using the automatic measuring method Sample
Preparation of Inorganic N compounds Mass Spectrometry (SPINMAS)54 at UFZ Halle. e 15N abundance of
NH4+ was analysed using the micro-diusion technique55, in which NH4+ is trapped in acidied glass bre lters
and analysed using an elemental analyser (ANCA-GSL, PDZ Europa, UK) coupled to the same IRMS as above,
conducted at the Stable Isotope Facility at the University of California, Davis.
Data analysis. Gross N mineralization and nitrication rates were calculated for each plot using the analyt-
ical 15N tracing model56, using data from the 15NH4+ labelling for gross mineralization and 15NO3− labeling for
gross nitrication:
=−×
′
′
()
NNN
tNN
transformation rate log
log( /)
,
(1)
t
a
a
t
010
10 0
t
0
where N0 and Nt are soil NH4+ or NO3− content at time zero and t, respectively, t is the time in days. e a′0 and a′t
are the excess 15N fractions of NH4+ or NO3− at time zero and t, respectively. All raw data used in the equation1
to calculate gross rates are presented Supplementary Table1. Average gross rates were calculated per forest type
and are presented on soil dry weight (SDW). A one-way analysis of variance (ANOVA) with Tukey’s post-test
(P < 0.05) was carried out to examine the dierences between the four forest sites.
e Normality test (Shapiro-Wilk) was used to examine the normality of soil properties. As some of our data,
such as GWC, TN, soil NH4+ and NO3− content were not normally distributed, the Kruskal-Wallis test with
Dunn’s post-test (P < 0.05) was conducted to examine the dierence between the four forest sites. Data of pH,
SOM and TC showed a normal distribution and one-way analysis of variance (ANOVA) was conducted. All the
analyses were conducted using GraphPad Prism (Version 5.01, GraphPad Soware, Inc.).
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Acknowledgements
We thank Karina Tôsto, Silvia Rivera and Anders Primé for assistance in the eld, Ângelo Manzatto, Márcio
Miranda and Wanderlay Bastos for logistic helping, and Ricardo Pollery for soil texture measurement. V.F.
and A.E.P. thanks CNPq, CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), FAPERJ
(Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro) and STINT (The Swedish Foundation for
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International Cooperation in Research and Higher Education) for travel support from Brazil to Sweden for
sample analyses. V.F. has a post doctoral fellowship from FAPERJ NOTA 10 program.A.E.P. is a research fellow
from CNPq (Conselho Nacional de Desenvolvimento Cientíco e Tecnológico) and Cientista do Estado from
FAPERJ (Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro). T.R. is supported by the Strategic
Research Area BECC (Biodiversity and Ecosystems services in a Changing Climate; www.becc.lu.se).
Author Contributions
is work was originally conceived by V.F., A.E.P. and T.R.; eldwork was done by V.F., T.R. and A.E.P.; laboratory
analyses were conducted by V.F. and T.R.; data analyses were performed by V.F. and T.R. All authors contributed
to the writing.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-43963-4.
Competing Interests: e authors declare no competing interests.
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