Pedobiologia - Journal of Soil Ecology 89 (2021) 150768

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Pedobiologia - Journal of Soil Ecology

journal homepage: www.elsevier.com/locate/pedobi

Native arbuscular mycorrhizal fungi respond to rehabilitation in iron ore

mining areas from the Eastern Brazilian Amazon
Raquel Milagros Rodríguez-Rodríguez a, b, Karl Kemmelmeier b, c, Daniela de Fátima Pedroso b,
Flávio Araújo Pinto b, Jessé Valentim dos Santos b, Markus Gastauer d, Cecílio Fróis Caldeira d,
Silvio Junior Ramos d, Jose Oswaldo Siqueira b, d, Marco Aurélio Carbone Carneiro b, *
a
Departamento de Biofertilizantes y Nutrición de las plantas, Instituto Nacional de Ciencias Agrícolas, Carretera Tapaste km 3½, Gaveta Postal no.1, 32700, San José de
las Layas, Mayabeque, Cuba
b
Departamento de Ciências do Solo, Universidade Federal de Lavras, Caixa Postal 3037, 37200-900, Lavras, MG, Brazil
c
Departamento de Ciências Naturais, Universidade Regional de Blumenau, Blumenau, SC89030-903, Brazil
d
Instituto Tecnológico Vale Desenvolvimento Sustentável, Rua Boaventura da Silva, 955, Nazar, 66055-200, Belém, PA, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Rehabilitation of mining areas is a relevant and current subject, in which the response of microbial communities
Arbuscular mycorrhiza is an important evaluation tool regarding their progress. In this context, our study had the objective of evaluating
Environmental recovery the effect of rehabilitation on the community composition and activity of arbuscular mycorrhizal fungi, and their
Iron ore deposit
relation with main soil properties, in three iron ore mining sites from Serra Norte of Carajás in the Eastern
Soil properties
Tropical rainforest
Brazilian Amazon. For that, non-rehabilitated, rehabilitated, and reference areas within each site were evaluated
for spore density and diversity, mycorrhizal colonization, glomalin contents, as well as soil chemical and physical
attributes. In general, all mycorrhizal variables responded positively to the rehabilitation processes in the three
mining sites. Over time, an increase of similarity of the mycorrhizal communities between rehabilitated and
reference areas from each site was observed, as well as, a decrease of the easily extractable glomalin/total
extractable glomalin ratio. High ratios of easily extractable glomalin/total extractable glomalin indicated high
degree of disturbance and/or disequilibrium of ecosystems, whereas low ratios indicated their equilibrium and/
or recovery. This relation can be an interesting parameter, easy to interpret and compare for assessing the degree
of disturbance, equilibrium and/or recovery of environments, regardless of absolute values of these variables.
Main soil properties related to mycorrhizal response were pH, texture, organic matter content, and cation ex­
change capacity. Briefly, our results showed the resilience of native mycorrhizal communities and the success of
the rehabilitation actions carried out in this Amazonian region.

1. Introduction
The Brazilian Amazon encompasses nine of the twenty seven Bra­
zilian states, covering more than 60 % of the continental Amazon region
and about 59 % of the country’s territory. Although its natural heritage
is only partially known, the Amazon is recognized as one of the most
biodiverse regions in the world, with a high degree of plant and animal
endemism (IBGE, 2003). Likewise, it presents several geological envi­
ronments with a large variety of mineral deposits, such as iron, man­
ganese, aluminum, copper, zinc, nickel, chromium, titanium, phosphate,
gold, silver, platinum, palladium, rhodium, tin, tungsten, niobium,
tantalum, zirconium, rare earths, uranium, and diamonds (dos Santos,
2002).
The Serra of Carajás mountain complex, located in the domains of
the homonymous geomorphological unit of the southeast of Pará State,
Eastern Brazilian Amazon, is considered the richest mineral region in
Brazil and one of the most important on the planet. This region possesses
major high content iron ore deposits (around 64.8 %) in the world and
they represent 10.7 % of national reserves. Concurrently, this mineral
province lies almost entirely within the limits of the Carajás National
Forest (FLONA of Carajás), an important unit for conservation and
sustainable use (Carmo and Kamino, 2015; ICMBio, 2017).
Although mining is an important source of commodities (such as
metals, mineral industry, as well as fuels) that are essential to consumer

* Corresponding author.
E-mail addresses: raquelm@inca.edu.cu (R.M. Rodríguez-Rodríguez), marcocarbone@ufla.br (M.A.C. Carneiro).

https://doi.org/10.1016/j.pedobi.2021.150768
Received 17 December 2020; Received in revised form 14 August 2021; Accepted 17 August 2021
Available online 20 August 2021
0031-4056/© 2021 Elsevier GmbH. All rights reserved.
R.M. Rodríguez-Rodríguez et al.
goods and services supply, employment generation, and foreign ex­
change (NRC, 2002), it brings considerable environmental impacts,
causing direct damage to the soil and vegetation (Palmer et al., 2010;
Huang et al., 2015). These impacts result in loss of biodiversity and
adverse environmental conditions which hamper natural regeneration
of the affected ecosystems (Mansourian et al., 2005). Therefore, reha­
bilitation of mine lands is essential for conservation of biodiversity and
recovery of ecosystem functions and services.
Soil and its biodiversity are mainly responsible for sustaining goods
and services linked to well being of humans and sustainability in
terrestrial systems, directly or indirectly related to soil functions and
processes. Soil microorganisms are involved in the decomposition of
organic matter, carbon and nutrient cycling, disease control, regulation
of plant growth, as well as primary productivity (Millennium Ecosystem
Assessment, 2005; Wall et al., 2012). Thus, soil microorganisms and
their relationship with plants are important driving forces in different
environmental recovery processes (Wildman, 2015).
Arbuscular mycorrhizal (AM) fungi are one of the most important
components of the soil microbiota. They are widespread in most diverse
environments (Stürmer et al., 2018) and in mutualistic symbiosis with
most plants (Brundrett, 2009). This association allows plants to sup­
plement nutrient uptakes, such as phosphorus and cationic micro­
nutrients (Johnson et al., 2017), and to increase their tolerance to heavy
metal excess (Hildebrandt et al., 2007) and water stress (Augé et al.,
2015), as well as their resistance to pathogen attack and herbivory (Sikes
et al., 2009; Kempel et al., 2010). AM fungi are also involved in the
development of soil physical structure (Rillig, 2004; Daynes et al.,
2013), related to soil fertility, water content, and erosion reduction
(Johnson et al., 2017). In summary, AM fungi are a determining factor in
soil quality, due to their effect on plant community ecology and
ecosystem processes (Rillig, 2004).
The role of AM fungi in the rehabilitation of degraded areas has been
pointed out, with emphasis on their support in colonization and estab­
lishment of plants in new environments (Maltz and Treseder, 2015;
Cortese and Bunn, 2017; Wang, 2017; Neuenkamp et al., 2019), as well
as their contribution to improvement of structure and soil quality
(Wang, 2017; Kumar et al., 2018). Similarly, rehabilitation actions can
promote the reestablishment of local mycorrhizal communities. In
Brazil, several studies regarding AM fungi in iron ore mining areas have
been carried out previously, however, they are concentrated in the
Fig. 1. Mining sites Sandstone II, Northwest II and South IV, and sampled areas: non-rehabilitated areas (NR), rehabilitated areas (R) with their respective years of
intervention and management, and rainforest reference areas (REF), located in the Serra Norte of FLONA of Carajás, Parauapebas municipality, Pará State, Brazil.
2
Pedobiologia - Journal of Soil Ecology 89 (2021) 150768
Quadrilátero Ferrífero, Minas Gerais (Trindade et al., 2000; Matias et al.,
2009; Teixeira et al., 2017; Vieira et al., 2018). Therefore, an extension
of these studies to other ferruginous systems of the country are neces­
sary, especially in the Brazilian Amazon. Thus, the objective of this study
was to evaluate the effect of rehabilitation on community composition
and activity of arbuscular mycorrhizal fungi and their relation with the
main chemical and physical soil properties in three iron ore mining sites
of the Serra Norte of Carajás-PA in the Eastern Brazilian Amazon. In
order to do that, we assessed the mycorrhizal colonization rate, density
and diversity of AM fungal spores, glomalin contents and main soil at­
tributes in non-rehabilitated, rehabilitated and rainforest reference
areas at each mining site. Main questions posed were: Are the rehabil­
itation efforts favoring the recovery of native arbuscular mycorrhizal
communities? If yes, and knowing that the AM fungi are mediator of soil
fertility and indicator of soil quality, what soil properties were related
with the mycorrhizal response? We expected that rehabilitation would
promote the recovery of native arbuscular mycorrhizal communities at
the three mining sites.
2. Material and methods
2.1. Characterization of the study site
This study was developed at the iron ore mining sites: Sandstone II,
Northwest II, and South IV of the Carajás Mineral Complex in Serra
Norte, region of FLONA of Carajás, Parauapebas municipality, Pará State
(Fig. 1). The climate of the region is tropical type Aw according to the
classification of Köppen-Geiger, with an average temperature of 26.2 ◦ C
and accumulated rainfall of up to 1900 mm year.
Within each mining site we selected four to five habitat conditions:
non rehabilitated (NR) areas, rehabilitated (R) areas with different times
of intervention and management, and rainforest reference areas (REF).
The distance between sites ranged from six to 8 km and the size of areas
ranged from 0.8 to 3.12 ha in Sandstone II, from 0.48 to 1.66 in
Northwest II and from 0.32 to 9.5 ha in South IV. Mining areas of
Sandstone II were pits filled with granite waste, coming from a nearby
mine, and later covered by a topsoil layer from vegetation suppression
areas. For their revegetation, seedlings of native species were planted,
and a commercial and native seed cocktail was applied. Meanwhile,
mining areas of Northwest II and South IV sites were waste piles filled
R.M. Rodríguez-Rodríguez et al. Pedobiologia - Journal of Soil Ecology 89 (2021) 150768

with heterogeneous material from several mine sites of the Carajás Sandstone II the distance between areas ranged from 150 to 350 m, in
Mineral Complex. For their rehabilitation, liming and fertilization were the Northwest II from the 200 to 800 m and in South IV from 200 to 1000
carried out, followed by seeding of commercial and native species. A m. The distance between sampling plots was of at least 30 m. In each
functional ecology approach was considered for species selection for the plot, a composited soil sample was collected in the first 20 cm of soil
revegetation of the three mining sites, with species being used as per layer. Five sampling points were systematically distributed (X-shaped)
Gastauer et al. (2020). Main characteristics of the different areas under within plots of 10 × 20 m to form each composited soil sample. Part of
study are summarized on Table 1. the soil was air-dried and sieved through a 2 mm mesh for its chemical
and physical characterization, and another part was separated and
stored at 4 ◦ C for mycorrhizal evaluations.
2.2. Soil sampling
2.3. Chemical and physical soil analysis
Soil sampling was carried out in October 2016 (dry season). For
sampling, three 10 × 20 m plots were installed in each selected area, Soil chemical and physical characterization was performed accord­
adding up to 42 plots for all three mining sites. The distance between ing to the methods described and standardized by EMBRAPA (2017).
areas varied according to the particularities of each mining site. In The methods included pH in H2O (1: 2.5 v/v), P, K and Fe available
(Mehlich 1 extractor), Ca2+, Mg2+ and Al3+ available (KCl 1 mol L− 1
Table 1 extractor), H + Al (SMP extractor), organic carbon (Walkley and Black,
Identification and characterization of study areas from iron ore mining sites. 1934), and granulometry by the pipette method (Bouyoucos, 1927).
Non-rehabilitated areas (NR), rehabilitated areas (R) with their respective years
of intervention and management, and the rainforest reference areas (REF).
2.4. Mycorrhizal variables
SITE AREA INTERVENTION SIZE CHARACTERIZATION
YEAR (ha) All samples were evaluated for AM fungal spores density and di­
NR – 0.9 Only deposition of topsoil versity, mycorrhizal colonization, easily extractable glomalin, and total
Deposition of topsoil extractable glomalin.
Seeding of native species
Density and diversity of spores were determined from 10 g of soil.
(cocktail). Later enrichment
R3 2013 1.45 with seedlings and with Spore extraction was carried out by the wet sieving and decanting
cover fertilization technique of Gerdemann and Nicolson (1963), followed by the sucrose
Open Vegetation with Small centrifugation (Jenkins, 1964). Then, intact spores were counted and
Trees and Shrubs separated by morphotypes under a stereomicroscope and placed on
Deposition of topsoil
Seeding of native species
slides with the reagents PVLG (polyvinyl alcohol-lactoglycerol) and
(cocktail). Later enrichment PVGL/Melzer 1:1 (v/v) for their morphological identification.
Sandstone with seedlings and with The morphotype identification was performed under a light micro­
R10 2006 0.80
II cover fertilization scope following the classification system of Redecker et al. (2013) with
Arboreal and shrub
additions of genera accepted by Wijayawardene et al. (2018). Identifi­
vegetation, with continuous
canopy cation was also made by comparison to the original species descriptions
Deposition of topsoil and the online reference collections of INVAM from West Virginia
Seeding of native species University, USA (https://invam.wvu.edu) and the Department of Plant
(cocktail). Later enrichment Pathology, University of Agriculture, Szczecin, Poland (http://www.zor.
R12 2004 3.12 with seedlings and with
cover fertilization
zut.edu.pl/Mycota).
Shrub and tree vegetation For mycorrhizal colonization quantification, roots smaller than 2
with a very closed canopy mm in diameter were separated from the soil and washed, and then cut
REF – – Rain Forest into segments with approximately 1 cm in length. These roots were
NR 1.03 Exposed soil
–
cleared and stained with Trypan Blue (0.05 %) according to the method
Liming and fertilization
Seeding of commercial and of Phillips and Hayman (1970) modified by Herrera-Peraza et al. (2004).
R3 2013/14 1.66 native species Mycorrhizal colonization rate of 300 root segments per sample was
Northwest
Predominantly herbaceous quantified by the method of intersections from Giovannetti and Mosse
vegetation (1980).
II
Seeding of commercial and
native species
Glomalin, a protein secreted (or otherwise released) into the soil by
R5 2011 0.65 AM fungi is used as an indicator of AM fungal activity in soils (Wright
Arboreal vegetation with
canopy closed et al., 1996; Wright and Upadhyaya, 1996) and extra-radical hyphal
REF – – Rain Forest growth (Lovelock et al., 2004a; Driver et al., 2005). This is operationally
NR 0.48 Exposed soil
–
quantified in soils as glomalin-related soil protein (Rillig et al. 2004) and
Liming and fertilization
Seeding of commercial and their pools are also operationally defined as easily extractable glomalin,
R0.6 2016 0.32 native species total extractable glomalin, and non-extractable, residual glomalin, only
Predominantly herbaceous the first two were measurable (Janos et al., 2008). Here, easily
vegetation, with few shrubs extractable glomalin (EEG) and total extractable glomalin (TEG) con­
Liming and fertilization
Seeding of commercial and
tents were determined from 0.5 g of dry soil. We utilized solvent/soil
R4 2012 6.63 native species ratio of 16: 1 (v / w) according to the results of Janos et al. (2008) and
South IV
Shrub vegetation with previous tests (data not shown); other conditions for extracting and
canopy still open separating glomalin fractions followed Wright and Upadhyaya (1996,
Liming and fertilization
1998). Quantification of both glomalin fractions was performed by the
Seeding of commercial and
native species Bradford assay (Brandford, 1976), using the same matrix for pattern and
R8 2008/09 9.45 samples. Patterns of EEG and TEG were constructed using bovine serum
Shrub and tree vegetation
with relatively closed albumin (0− 500 μg ml− 1) in 20 mM sodium citrate adjusted to pH 7 and
canopy 50 mM sodium citrate adjusted to pH 8, respectively. All determinations
REF Rain Forest
were performed in duplicate with the absorbance measurement
– –

3
R.M. Rodríguez-Rodríguez et al.
performed in a spectrophotometer at 595 nm. Results were extrapolated
to mg g− 1 soil. It is important to point out that the Bradford assay is not
specific for glomalin. However, the behaviour of the Bradford mea­
surements has been observed to be consistent with predictions for glo­
malin behaviour suggesting that its response can be related to AM fungal
activity in some cases (Koide and Peoples, 2013).
2.5. Data analysis
We carried out univariate analyses in order to evaluate mycorrhizal
response to rehabilitation, and differences in soil properties among areas
(habitat conditions) within each mining site. Previously, normality and
homoscedasticity of the data were verified by the Shapiro-Wilk and
Levene tests, respectively. When necessary, transformations were car­
ried out to fulfill the ANOVA assumption. Variables that fulfilled both
premises were analyzed by one-way ANOVA and, subsequently, the
Scott-Knott test was applied to identify the differences between areas,
when detected. For the variables with normal distribution, but which
did not reach the homogeneity of variance, the Welch test for unequal
variances was performed, also followed by the Scott-Knott test.
A presence-absence matrix was generated from the identified AM
fungi. This was used to determine species richness, expressed as the total
number of species per area; estimated richness, calculated by the first-
order Jackknife estimator (Heltshe and Forrester, 1983) which uses
100 randomized runs without replacement; and the frequency of
occurrence (FO) of the species per site, expressed as the ratio between
the number of areas that contain the species and the total number of
areas per mining site. Whittaker’s beta diversity (Whittaker, 1960) was
also calculated using the formula: βW = S/α − 1, where S is the regional
diversity, in our case it is equivalent to total species at each site
(Sandstone II, Northwest II e South IV), and α the mean alpha diversity,
here defined as the number of species per area. To compare the degree of
similarity between the areas of each site, the measure re-expressed of
a+b+c
βw: (2a+b+c)/2 − 1 was used according to Koleff et al. (2003), where the a
component corresponds to the total number of species shared between
both areas, and b and c correspond to the total number of species unique
to each area. The βW can take values from 0 (when all species present on
the site are present in all areas) to a maximum of n-1 (being n the total
number of areas) when there is no overlap between areas. On the other
hand, the degree of similarity between the areas can take values between
zero (maximum similarity) and one (maximum dissimilarity).
We also carried out a non-metric multidimensional scaling (NMDS)
ordination using the Gower similarity index to elucidate the mycorrhizal
response among mining sites and habitat conditions. In order to do that,
we considered the response variables spore density, species richness,
mycorrhizal colonization, and glomalin fractions. Soil properties were
fitted as vectors in the ordination to explore the correlation between
environmental variables and the arrangement of the areas. The dimen­
sionality was selected from analysis of stress values generated after
ordination, in which the two-dimensional solution was satisfactory.
Finally, relationship between mycorrhizal and soil variables were tested
by Spearman correlation. Statistical analyses were performed using the
software PAST version 3.25 (Hammer et al., 2001) and InfoStat version
2018 (Di Rienzo et al., 2018), and richness analyses using the BioDi­
versity Professional version 9.1.0 (Colwell, 2013).
3. Results
3.1. Soil analysis
Results of soil analysis are summarized on Table 2. Overall, mining
affected soil chemical and physical attributes, especially pH, SOM,
available Fe, %V, CEC and soil texture. At all three sites, SOM and CEC
increased with rehabilitation time, while pH and %V varied differen­
tially within each site. Unusually, low available Fe content and high
4
Pedobiologia - Journal of Soil Ecology 89 (2021) 150768
available Cu content were observed in R8 area of South IV and NR area
of Sandstone II, respectively.
3.2. Density and diversity of AM fungal spores
Rehabilitation processes stimulated the AM fungal sporulation,
evidently depressed by mining. In all three sites, significant differences
were detected (Fig. 2a) and always observed between non-rehabilitated
and reference areas (Fig. 2a). The lowest and highest spore density was
also observed in the non-rehabilitated and rehabilitated areas,
respectively.
A total of 66 morphotypes of AM fungi distributed in 20 genera
belonging to nine families, plus the incertae-sedis, Entrophospora infre­
quens were recovered (Table 3). The total observed richness per mining
site was 44, 48 and 40 species for Sandstone II, Northwest II, and South
IV, respectively, while the total estimated richness was 58, 68 and 60
species for the respective sites. In general, Glomeraceae was the family
with the largest number of species found, followed by Acaulosporaceae
and Gigasporaceae. Reference areas of the three sites had similar values
of specific richness, sharing more than 50 % of the species. In the
Sandstone II, 16 species had high FO (≥ 60 %), while in the Northwest II
and Southern IV there were only eight and six species, respectively. The
species Acaulospora mellea, Glomus sp.1, Rhizophagus clarus, and Oehlia
diaphana were the most common species shared at these three mining
sites.
In general, differentiation for composition of the AM fungal com­
munities was lower in the Sandstone II site, compared to Northwest II
and South IV (Sandstone II βW(n=5) = 1.2917, Northwest II βW(n=4) =
1.4304, South IV βW(n=5) = 2.125). Within each site, the smallest
dissimilarity was observed between the rehabilitated areas with simi­
larly management time, and between the references and rehabilitated
areas with longer management time (Table 4).
3.3. Mycorrhizal colonization
High rates of mycorrhizal colonization (>50 %) were observed in the
assessed areas of the three mining sites (Fig. 2b). Only non-rehabilitated
areas (exposed soil) of the Northwest II and South IV sites were not
evaluated because they had no roots. In Sandstone II significant differ­
ences were detected between non-rehabilitated and remaining areas,
while in Northwest II and South IV no significant differences were
detected between rehabilitated and reference areas (Fig. 2b).
3.4. Soil glomalin
Soil glomalin content and the relation between their fractions varied
within each mining site (Table 5). The easily extractable fraction (EEG)
varied between 0.29 and 2.94 mg g− 1 of soil in Sandstone II, between
0.40 and 3.50 mg g− 1 of soil in Northwest II, and between 0.24 and 2.41
mg g-1 of soil in Southern IV. On the other hand, TEG ranged between
2.04 and 10.97 mg g-1 of soil in Sandstone II, between 0.64 and 15.39 mg
g-1 of soil in Northwest II, and between 0.38 and 16.47 mg g-1 of soil in
Southern IV. Overall, there was an increase in glomalin content in the
direction non-rehabilitated – rehabilitated –reference area and TEG
distinguished best between environments.
Analyzing the EEG/TEG ratio was possible to distinguish two main
situations: (i) the predominance of labile fraction of glomalin in non-
rehabilitated areas and rehabilitated areas with up to three years of
management and (ii) the predominance of the stable fraction in reha­
bilitated areas with more than four years of management and reference
areas (Table 5). Exceptionally, the EEG/TEG ratio of non-rehabilitated
area of Sandstone II was low, probably due to the topsoil addition.
High EEG/TEG ratios indicated greater degree of disturbance and/or
disequilibrium of the ecosystems, whereas low EEG/TEG ratios indi­
cated the opposite, as well as, the recovery of the disturbed ecosystems.
 R.M. Rodríguez-Rodríguez et al.
Table 2
Chemical and physical properties of the non-rehabilitated areas (NR), rehabilitated (R) with their respective years of intervention and management, and the rainforest reference areas (REF) from the iron ore mining sites.
SITE AREA PHH2O SOM† P K Ca Mg CEC‡ V§ Zn Fe Mn Cu Sand Silt Clay
– g Kg− 1 mg dm− 3
mg dm− 3
cmolc cmolc cmolc % mg dm− 3
mg dm− 3
mg dm− 3
mg dm− 3
% % %
dm− 3 dm− 3 dm− 3

Sandstone NR 5.17 ± 2.67 ± 4.9 ± 0.9 140.4 ± 1.55 ± 0.3 0.2 ± 0 b 3.46 ± 60.28 ± 0.63 ± 114.03 ± 35.07 ± 13.93 ± 70.9 ± 3.6 12.77 ± 16.33 ±
0.1 a 0.7 b a 45.4 a ns 0.4 c 4.7 a 0.03 b 11.3 b 0.8 a 5.8 a a 3.5 a 0.7 c
II R3 5 ± 0.1 a 34.33 ± 2.43 ± 95.33 ± 1.13 ± 0.2 0.33 ± 7.01 ± 24.59 ± 0.73 ± 226.7 ± 78.17 ± 0.73 ± 55.93 ± 9.07 ± 4.1 35 ± 3.1 b
1.8 a 0.2 b 3.7 a ns 0.03 a 0.5 b 3.4 b 0.1 b 32.1 a 27.5 a 0.5 b 2.9 b a
R10 4.8 ± 0 a 32 ± 6.8 9.53 ± 62.2 ± 3.3 1.3 ± 0.5 0.37 ± 8.42 ± 20.85 ± 2.5 ± 0.7 101.37 ± 35.1 ± 6 a 0.7 ± 0.3 68.8 ± 2.9 5.2 ± 1.07 26 ± 2.1 c
a 2.4 a b ns 0.1 a 0.7 a 5.1 b a 17.2 b b a b
R12 4.97 ± 29.66 ± 1.9 ± 0.2 49.37 ± 0.67 ± 0.2 0.17 ± 6.63 ± 14.56 ± 0.5 ± 0 c 207.77 ± 14.53 ± 0.33 ± 75.8 ± 1 a 2.53 ± 1 b 21.67 ±
0.2 a 0.9 a b 2.9 b ns 0.03 b 0.1 b 4.2 b 25.8 a 3.1 b 0.03 b 0.7 c
REF 4.47 ± 40 ± 2.08 1.83 ± 67.4 ± 1.87 ± 0.5 0.4 ± 0.1 10.81 ± 22.04 ± 0.73 ± 198.87 ± 33.43 ± 0.23 ± 32.77 ± 12.23 ± 55 ± 3.06
0.2 b a 0.2 b 13.2 b ns a 0.8 a 3.7 b 0.03 b 28 a 2.9 a 0.03 b 2.9 c 0.2 a a

Northwest NR 5.2 ± 0 a 2±0c 1.6 ± 0.2 10.8 ± 1.5 0.65 ± 0.3 0.3 ± 0.1 1.79 ± 39.27 ± 1.73 ± 49.37 ± 3.1 38.27 ± 3.87 ± 43.73 ± 49.6 ± 7±6c
ns c ns ns 0.6 b 8.9 ns 0.9 ns b 7.4 b 1.5 a 12.9 ns 19.1 ns
II R3 5.33 ± 10.67 ± 3.93 ± 41.57 ± 0.43 ± 0.2 ± 0 ns 3.01 ± 24.76 ± 0.97 ± 54.37 ± 4.6 134.57 ± 0.77 ± 33.63 ± 32.7 ± 1.4 33.67 ±
0.03 a 1.5 b 1.3 ns 1.1 b 0.03 ns 0.2 b 1.8 ns 0.1 ns b 1.2 a 0.1 b 2.6 ns ns 1.8 b
5

R5 5.3 ± 0.1 12 ± 2.08 3.63 ± 36.93 ± 1.17 ± 0.5 0.4 ± 0.2 3.43 ± 46.08 ± 1.33 ± 50.43 ± 126.93 ± 0.8 ± 0.1 39.57 ± 28.1 ± 1.3 32.33 ±
a b 0.9 ns 9.9 b ns ns 0.3 b 15.3 ns 0.2 ns 6.07 b 6.4 a b 6.2 ns ns 5.2 b
REF 4.17 ± 45.33 ± 2.07 ± 90.73 ± 0.67 ± 0.4 0.3 ± 0.1 12.37 ± 10.6 ± 4.7 1.07 ± 191.1 ± 40 77 ± 30.3 2.23 ± 20.9 ± 4.2 18.1 ± 1.5 61 ± 3.1 a
0.1 b 1.8 a 0.3 ns 4.7 a ns ns 1.3 a ns 0.3 ns a b 0.03 a ns ns

South NR 5.67 ± 2±0b 4 ± 0.7 b 17.97 ± 1 ± 0.2 b 0.7 ± 0.3 2.78 ± 63.78 ± 3.03 ± 46.97 ± 5.1 32.53 ± 0.93 ± 25.6 ± 2.5 60.73 ± 13.67 ±
0.2 a 5.1 b b 0.3 b 4.9 a 0.7 b b 1.9 b 0.2 b a 13.8 a 12.7 a
IV R0.6 5.87 ± 2±0b 2.9 ± 0.2 81.1 ± 21 2.8 ± 0.3 6.7 ± 3.5 10.61 ± 89.45 ± 21.27 ± 50.03 ± 9.5 51 ± 11.4 2.8 ± 0.7 31.3 ± 6.9 63.7 ± 4.6 5.33 ± 2.6
0.2 a b a a a 3.7 a 3.6 a 9.5 a b b a a a a
R4 6 ± 0.3 a 8.66 ± 4.27 ± 58.33 ± 1.1 ± 0.3 0.97 ± 3.78 ± 57.55 ± 2.1 ± 0.4 54 ± 10.8 b 119.73 ± 9 1.63 ± 37.37 ± 5 30.97 ± 31.67 ±
4.06 b 0.5 b 11.8 a b 0.3 b 0.6 b 7.1 a b a 0.5 b a 1.7 b 3.3 a
R8 6.23 ± 41.66 ± 8.6 ± 1.7 117.8 ± 5.57 ± 2.1 1.57 ± 9.4 ± 1.8 75.56 ± 4.9 ± 1.4 10.1 ± 6.2 c 131.43 ± 0.97 ± 51.03 ± 25.97 ± 23 ± 1.2 a

Pedobiologia - Journal of Soil Ecology 89 (2021) 150768

0.3 a 5.4 a a 28.4 a a 0.1 b a 9.7 a b 1.1 a 0.3 b 2.2 a 1.9 b
REF 4.3 ± 0.1 43.33 ± 2.37 ± 76.63 ± 1.5 ± 0.2 0.53 ± 9.53 ± 23.26 ± 2.13 ± 169.23 ± 107.87 ± 3.2 ± 0.2 11.7 ± 1.5 23.97 ± 64.33 ±
b 1.3 a 0.2 b 13.6 a b 0.1 b 0.5 a 2.1 b 0.2 b 27.1 a 7.3 a a b 0.2 b 1.3 b

Different letters represent significant differences at p < 0.05 for the Scott-Knott test.
†
SOM = Soil organic matter.
‡
CEC = Potential cation exchange capacity.
§
V = Base saturation b.
R.M. Rodríguez-Rodríguez et al.
Fig. 2. Mean values ± standard error of spore density (a) and mycorrhizal colonization (b) of non-rehabilitated areas (NR), rehabilitated areas (R) with their
respective years of intervention and management, and rainforest reference areas (REF) of the mining sites Sandstone II (ST), Northwest II (NW) and South IV (S).
Means with different letters represent significant differences at p < 0.05 for the Scott-Knott test, ns - non-significant differences, * - not evaluated (due to the absence
of roots).
3.5. Relationship between mycorrhizal and soil variables
The two-dimensional representation using NMDS (Fig. 3) showed the
arrangement of the areas of the three iron mining sites evaluated ac­
cording to the mycorrhizal variables and their correlation with soil
properties. Spatial separation between non-rehabilitated and R0.6 of
South IV areas (with less than one year of management) from the rain­
forest reference areas was observed, while the rehabilitated areas were
heterogeneously distributed between both extremes. Rehabilitated areas
with longer management time (especially those with ≥ 10 years) were
arranged closer to reference areas. Areas with lower mycorrhizal attri­
bute values were positively correlated with pH, V, silt, and Cu contents,
while areas with higher values of mycorrhizal attributes were positively
correlated with CEC and SOM, Fe, and clay contents.
As shown in the biplot (Fig. 3), main soil properties correlated with
mycorrhizal variables were pH, silt and clay content, V, CEC, Fe, Cu, and
SOM (Table 6). SOM and V were significantly correlated with most of
mycorrhizal variables, while glomalin fractions were significantly
correlated with highest number of soil properties.
4. Discussion
Our study verified the positive effect of rehabilitation processes on
the recovery of native arbuscular mycorrhizal communities in the iron
mining sites of Sandstone II, Northwest II, and South IV of Serra Norte of
Carajás-PA, in the Eastern Brazilian Amazon. The functional ecology
approach applied to species selection and the use of native plants for the
revegetation may have been a key to the results.
Prior to the rehabilitation actions, there was an evident decrease of
infective propagules (spores and roots colonized), as well as, a very low
or no production and accumulation of soil glomalin at the three mining
sites. Among non-rehabilitated areas, colonized roots were found only in
Sandstone, probably arising from topsoil added. Topsoil addition
6
Pedobiologia - Journal of Soil Ecology 89 (2021) 150768
represented an input of AM fungal propagules when compared to the
exposed soil areas of Northwest II and South IV.
Spore density increased significantly after rehabilitation. It was only
in R0.6 of South IV that the spore number was very low, probably due to
the short time of intervention in the area (less than one year). At
Northwest II and South IV sites, it was not possible to detect statistical
differences between some data rehabilitated areas and references.
Despite the evident difference in their mean values, there was a large
data dispersion in rehabilitated areas, probably due to the greater soil
heterogeneity of these environments. Similarly, previous works, both of
mycorrhizal infectivity assays with soil of disturbed and undisturbed
environments (Boerner et al., 1996) and quantification of spore density
in different stages of tropical succession (Zangaro et al., 2008; Stürmer
and Siqueira, 2011), observed a high variance in disturbed areas
compared to mature forests.
In general, spore density values in our study were far superior to
most previous reports in Brazilian ecosystems (Aidar et al., 2004;
Stürmer and Siqueira, 2011; Teixeira et al., 2017; Vieira et al., 2018),
although similar to those of Zangaro et al. (2008, 2013) in different
tropical successional chronosequences in the south of the country. In all
cases, the behavior of this variable was similar, observing the sporula­
tion stimulation with succession/rehabilitation, which tends to decrease
in later stages, but with values always higher than reference ecosystem.
This behavior is attributed to environmental stress conditions such as
high luminous intensity and low water and nutrient content, which
promote higher rates of sporulation in successional stages that have not
reached equilibrium yet (Oliveira and Oliveira, 2010; Zangaro et al.,
2013).
AM fungal diversity studies in the Brazilian Amazon, including nat­
ural, reforested, agricultural, and pasture ecosystems, have been carried
out, both from field samples and trap cultures (Caproni et al., 2003; Leal
et al., 2009; Stürmer and Siqueira, 2011; Freitas et al., 2014; Reyes et al.,
2019). The number of morphotypes identified in our study (66)
R.M. Rodríguez-Rodríguez et al. Pedobiologia - Journal of Soil Ecology 89 (2021) 150768

Table 3
Recorded arbuscular mycorrhizal fungi, observed species richness, and estimated species richness in non-rehabilitated areas (NR), rehabilitated areas (R) with their
respective years of intervention and management, and rainforest reference areas (REF). Frequency of species occurrence (FO) per site.
Sandstone II Northwest II South IV

Family/Species NR R3 R10 R12 REF FO NR R3 R5 REF FO NR R0.6 R4 R8 REF FO

Acaulosporaceae
Acaulospora colombiana (Spain & N.C. Schenck) + + – – – 0.40 – + – – 0.25 – – – – – 0
Kaonongbua, J.B. Morton & Bever
Acaulospora foveata Trappe & Janos – – + + – 0.40 – – – – 0 – – – – – 0
Acaulospora lacunosa J.B. Morton – – – – + 0.20 – – – – 0 – – – – – 0
Acaulospora mellea Spain & N.C. Schenck – + + + + 0.80 + + + + 1.00 – + + + + 0.80
Acaulospora morrowiae Spain & N.C. Schenck + – – – – 0.20 + + + – 0.75 – – + – – 0.20
Acaulospora punctata Oehl, Palenz, Sánchez-Castro, – – – – – 0 – – + – 0.25 – – – – – 0
G.A.Silva, C.Castillo & Sieverd.
Acaulospora scrobiculataTrappe – + – + – 0.40 + – + – 0.50 – – + + – 0.40
Acaulospora spinosissima Oehl, Palenz, Sánchez- – + + + – 0.60 – + – – 0.25 – – – – – 0
Castro, Tchabi, Hount. & G. A. Silva
Acaulospora tuberculata Janos & Trappe – + + – – 0.40 – + + – 0.50 – – – – – 0
Acaulospora sp.1 – – – + + 0.40 – – – + 0.25 – – – – + 0.20
Acaulospora sp.2 + – + + + 0.80 – – + – 0.25 – – – + – 0.20
Acaulospora sp.3 – – – + – 0.20 – – – – 0 – – – – – 0
Acaulospora sp.4 – – + – – 0.20 – – – – 0 – – – – – 0
Acaulospora sp.5 – – – – + 0.20 – – – – 0 – – – – – 0
Ambisporaceae
Ambispora cf. nicolsonii (C. Walker, L.E. Reed & F.E. – – + – – 0.20 – – – – 0 – – – – – 0
Sanders) Oehl, G.A. Silva, B.T. Goto & Sieverd.
Ambispora leptoticha (N.C. Schenck & G.S. Sm.) C. + + – + – 0.60 – – + – 0.25 – – – – – 0
Walker, Vestberg & A. Schüssler
Archaeosporaceae
Archaeospora myriocarpa (Spain, Sieverd. & N.C. + + + – – 0.60 – + + – 0.50 – – – + + 0.40
Schenck) Oehl, G.A. Silva, B.T. Goto & Sieverd.
Archaeospora trappei (R.N. Ames & Linderman) J.B. + – – – – 0.20 – – + – 0.25 – – – – + 0.20
Morton & D. Redecker
Claroideoglomeraceae
Claroideoglomus etunicatum (W.N. Becker & Gerd.) – – – – – 0 – – – – 0 – – + + – 0.40
C. Walker & A. Schüßler
Corymbiglomus tortuosum (N.C. Schenck & G.S. Sm.) – – – – – 0 – + – – 0.25 – – – – – 0
Błaszk. & Chwat
Diversisporaceae
Diversispora epigaea (B.A. Daniels & Trappe) C. – – – – – 0 – – + + 0.50 – – – – + 0.20
Walker & A. Schüßler
Diversispora sp.1 – – + – – 0.20 – + – – 0.25 – – + + – 0.40
Diversispora sp.2 – – – – – 0 – – – – 0 – – – + – 0.20
Diversispora sp.3 – – – – – 0 – – – – 0 – + – – – 0.20
Gigasporaceae
Cetraspora pellucida (T.H. Nicolson & N.C. Schenck) + + – + – 0.60 + – + – 0.50 – – + + – 0.40
Oehl, F.A. Souza & Sieverd
Dentiscutata biornata (Spain, Sieverd. & S. Toro) – + + + – 0.60 – – – – 0 – – – – – 0
Sieverd.
Dentiscutata cerradensis (Spain & J. Miranda) – – – – – 0 – – + – 0.25 – – – – – 0
Sieverd., F.A. Souza & Oehl
Dentiscutata savannicola (R.A. Herrera & Ferrer) C. – – – – – 0 – + + – 0.50 – – – – – 0
Walker & A. Schüßler
Fuscutata aurea Oehl, C.M. Mello & G.A. Silva – + – – – 0.20 + – + – 0.50 – – – – – 0
Gigaspora albida N.C. Schenck & G.S + + – – – 0.40 + + – – 0.50 – – – – – 0
Gigaspora cf. gigantea (T.H. Nicolson & Gerd.) Gerd. – – – – – 0 – + + – 0.50 – – – – – 0
& Trappe
Gigaspora margarita W.N. Becker & I.R. Hall – – + – – 0.20 + – – + 0.50 – + + + – 0.60
Racocetra fulgida (Koske & C. Walker) Oehl, F.A. – – – – – 0 – – – + 0.25 – – – – + 0.20
Souza & Sieverd.
Scutellospora cf. arenicola Koske & Halvorson – + – – – 0.20 – – – – 0 – – – – – 0
Scutellospora dipurpurescens J.B. Morton & Koske – – – – – 0 – – + – 0.25 – – – – – 0
Scutellospora sp.1 – – – – – 0 – – – – 0 – – – – + 0.20
Glomeraceae
Dominikia minuta (Błaszk., Tadych & Madej) Błaszk. – – + – – 0.20 – – – – 0 – + – – – 0.20
Funneliformis cf. badium (Oehl, D. Redecker & – – – + – 0.20 – – – + 0.25 – – – – + 0.20
Sieverd.) C. Walker & A. Schüßler
Funneliformis mosseae (T.H. Nicolson & Gerd.) C. – – – – – 0 – – – – 0 – – – + – 0.20
Walker & A. Schüßler
Glomus cf. ambisporum G.S. Sm. & N.C. Schenck – – + – – 0.20 – + + – 0.50 – – – + – 0.20
Glomus glomerulatum Sieverd. – + + + + 0.80 – – + + 0.50 – – – + + 0.40
Glomus microaggregatum Koske, Gemma & P.D. – – – – – 0 – – + – 0.25 – – + – – 0.20
Olexia
Glomus spinuliferum Sieverd. & Oehl – – – + – 0.20 – – + – 0.25 – – – – – 0
Glomus sp.1 + + + + + 1.00 + + + + 1.00 + + + + + 1.00
Glomus sp.2 + + + + + 1.00 – + + + 0.75 – – + – – 0.20
Glomus sp.3 – – + + – 0.40 – – + – 0.25 – – + + – 0.40
(continued on next page)

7
R.M. Rodríguez-Rodríguez et al. Pedobiologia - Journal of Soil Ecology 89 (2021) 150768

Table 3 (continued )
Sandstone II Northwest II South IV

Family/Species NR R3 R10 R12 REF FO NR R3 R5 REF FO NR R0.6 R4 R8 REF FO

Glomus sp.4 – – – – + 0.20 – – + – 0.25 – – – – – 0

Glomus sp.5 – – – – – 0 + – + + 0.75 – – – – + 0.20
Glomus sp.6 – – – – + 0.20 – – + – 0.25 – – – – – 0
Glomus sp.7 – – – – – 0 – + – – 0.25 – – – – – 0
Oehlia diaphana (J.B.Morton & C.Walker) Błaszk., – + + + + 0.80 – + + + 0.75 – – + + + 0.60
Kozłowska & Dalpé
Rhizophagus clarus (T.H. Nicolson & N.C. Schenck) + + + – – 0.60 + + + – 0.75 – + + + – 0.60
C. Walker & A. Schüßler
Rhizophagus fasciculatus (Thaxt.) C. Walker & A. – + + + + 0.80 – – – + 0.25 – – – + + 0.40
Schüßler
Rhizophagus intraradices (N.C. Schenck & G.S. Sm.) – – – – – 0 – – – – 0 + – – – – 0.20
C. Walker & A. Schüßler
Rhizophagus cf. proliferus (Dalpé & Declerck) C. + + – – – 0.40 + – – – 0.25 – – + – – 0.20
Walker & A. Schüßler
Rhizophagus sp.1 + – – + + 0.60 – – – – 0 – – – – – 0
Rhizophagus sp.2 – – – – – 0 – – + – 0.25 – – – – – 0
Sclerocystis coremioides Berk. & Broome – – + + + 0.60 – + + + 0.75 – – + – + 0.40
Sclerocystis rubiformis Gerd. & Trappe – + + + + 0.80 – – + + 0.50 + – + – + 0.60
Sclerocystis sinuosa Gerd. & B.K. Bakshi – – + + – 0.40 – – – + 0.25 – – – + – 0.20
Sclerocystis taiwanensis C.G. Wu & Z.C. Chen – – – – + 0.20 – – – + 0.25 – – – – + 0.20
Sclerocystis sp.1 – – – + + 0.40 – – – + 0.25 – – – – + 0.20
Septoglomus cf. constrictum (Trappe) Sieverd., G.A. – – – – – 0 – – – – 0 – – – + – 0.20
Silva & Oehl.
Pacisporaceae
Pacispora cf. franciscana Sieverd. & Oehl – – – – – 0 – – – – 0 – – + – – 0.20
Paraglomeraceae
Paraglomus sp.1 – – – – – 0 – – + – 0.25 – – + – – 0.20
Incertae-sedis
Entrophospora infrequens (I.R. Hall) R.N. Ames & R. – – – – – 0 – – + – 0.25 – – + – – 0.20
W. Schneid.
OBSERVED RICHNESS 13 20 23 23 17 —— 11 18 33 17 —— 3 6 19 19 17 ——
ESTIMATED RICHNESS 20 29 32 34 25 —— 16 25 47 23 —— 4 9 26 28 23 ——

Table 4
Pairwise comparison of the degree of dissimilarity of AM fungal communities (beta diversity) between non-rehabilitated areas (NR), rehabilitated areas (R) with their
respective years of intervention and management, and rainforest reference areas (REF) of each mining site.
SANDSTONE II NORTHWEST II SOUTH IV

NR R3 R10 R12 NR R3 R5 NR R0.6 R4 R8

R3 0.45 R3 0.66 R0.6 0.78

R10 0.72 0.44 R5 0.64 0.53 R4 0.82 0.68
R12 0.67 0.44 0.39 REF 0.71 0.71 0.64 R8 0.91 0.68 0.47
REF 0.73 0.62 0.55 0.40 REF 0.80 0.83 0.72 0.67

0 = maximum similarity; 1 = maximum dissimilarity.

exceeded previous reports, where only Stürmer and Siqueira (2011)
obtained values close to ours (61) in the upper Solimões River region in
western Amazon. Similarly, and compared to studies by Teixeira et al.
(2017) and Vieira et al. (2018) in ferruginous ecosystems of the Quad­
rilátero Ferrífero, AM fungal richness from field samples in our study
was 2.13 and 0.85 times higher, respectively. In general, the sampling
effort allowed recovery between 65 and 75 % of the estimated AM
fungal richness in the areas and sites studied. In addition, analysis of
beta diversity revealed less differentiation in the composition of AM
fungal communities in Sandstone II than in Northwest II and South IV,
indicating a better recovery. This may be related to the longer man­
agement time, topography of waste deposits and topsoil addition in
Sandstone II. In the three mining sites, the dissimilarity between AM
fungal communities of references and rehabilitated areas decreased with
the age increase in the rehabilitated areas. Besides this, dissimilarity
between rehabilitated areas was lower among areas with close ages. This
behavior indicated the positive effect of rehabilitation on recovery of
AM fungal communities, and shows that the ferruginous ecosystems of
FLONA of Carajás harbors a high AM fungal diversity, which are highly
resilient to drastic modifications of the environments.
High mycorrhizal colonization rates found in all areas, along with a
considerable increase in spore density with rehabilitation suggested an
introduction of mycotrophic plants (intentional or not) and stimulation
of sporulation, thus contributing to the increase of propagules avail­
ability in the soil. Availability of propagules is a key factor in the
establishment of mycrotrophic species in new substrates (Cázares et al.,
2005), and consequently in the recovery of degraded areas (Maltz and
Treseder, 2015). Similarly, plants influence mycorrhizal communities
(Zobel and Opik, 2014); therefore, presence of mycotrophic species can
also facilitate the recovery of mycorrhizal communities. Oliveira and
Oliveira (2010) reviewed literature reference publications, from 1980
until 2010, of the mycorrhizal status of plants in the Brazilian Amazon
and verified that from 207 species evaluated, 87 % were found to be
mycorrhizal, reporting in many occasions, rates of mycorrhizal coloni­
zation higher than 50 % or qualified as heavily colonized. Both results
obtained here from the mycorrhizal colonization at the ecosystem level
(since individual plants were not evaluated but an aliquot of roots from
soil samples), as the previous records of mycorrhizal plants from
Amazon, suggested a high level of arbuscular mycorrhizal association in
this important Brazilian biome. Most species used for the revegetation of
the three mining sites studied were native.
Glomalin production and accumulation also responded positively to
the rehabilitation progress. Increases in glomalin contents when
conservationist land management or rehabilitation actions are carried

8
R.M. Rodríguez-Rodríguez et al. Pedobiologia - Journal of Soil Ecology 89 (2021) 150768

Table 5 Added topsoil was previously removed and stored in piles until it was
Mean ± standard error (SE) of easily extractable glomalin (EEG), total extract­ used to fill pits and tailing piles to be rehabilitated. Intense minerali­
able glomalin (TEG), and EEG/TEG ratio in the non-rehabilitated areas (NR), zation of organic matter from this removed and relocated soil could have
rehabilitated areas (R) with their respective years of intervention and manage­ been the main cause of this glomalin reduction, but it also allowed the
ment, and rainforest reference areas (REF) of the mining sites. maintenance of its microbial activity. Several experimental studies, both
SITE AREA EEG ± SE TEG ± SE EEG/TEG in situ and ex situ, have observed glomalin decomposition (Steinberg and
mg g− 1 mg g− 1 (%) Rillig, 2003; Rillig et al., 2003; Wu et al., 2014). It is also recognized that
NR 0.29 ± 0.05b 2.04 ± 0.06b 14.43 ± 2.12b the EEG is not only the newly deposited glomalin in the soil, but also the
R3 1.92 ± 0.20a 3.71 ± 1.02b 60.15 ± 15.57a fraction recently modified by its decomposition (Steinberg and Rillig,
Sandstone II R10 2.08 ± 0.28a 9.28 ± 1.12a 22.30 ± 0.30b
2003; Rillig, 2004), which also justifies its detection in the tailings piles
R12 1.91 ± 0.19a 7.51 ± 1.36a 26.49 ± 4.19b
REF 2.94 ± 0.47a 10.97 ± 1.34a 26.85 ± 3.71b without vegetation from Northwest II and South IV sites.
From the ordination of areas (habitat conditions) and sites according
NR 0.41 ± 0. 08b 0.64 ± 0.04c 62.83 ± 11.37a to their mycorrhizal attributes, it was possible to distinguish the non-
Northwest II
R3 1.34 ± 0.28b 3.27 ± 0.56b 45.15 ± 13.55a rehabilitated areas and newly rehabilitated areas from the reference
R5 1.13 ± 0.28b 5.92 ± 2.35b 24.61 ± 8.55b areas and rehabilitated areas with longer management time. Areas with
REF 3.50 ± 0.27a 15.39 ± 2.72a 24.27 ± 4.57b
lower values of mycorrhizal attributes were positively correlated to pH,
NR 0.27 ± 0.00b 0.38 ± 0.01c 72.66 ± 1.45a V, and silt content, probably due to the chemical and physical modifi­
R0.6 0.47 ± 0.19b 0.78 ± 0.32c 60.48 ± 2.15b cations of substrate product from ore extraction. Likewise, their negative
South IV R4 0.24 ± 0.06b 1.21 ± 0.29c 19.42 ± 1.55c correlation with attributes such as SOM, CEC, available Fe, and clay
R8 1.67 ± 0.43a 8.49 ± 1.64b 19.10 ± 1.94c contents revealed an intense degradation of these environments. Several
REF 2.41 ± 0.30a 16.47 ± 0.73a 14.82 ± 2.42c
studies (Wang et al., 2015; Kumar et al., 2018; Reyes et al., 2019; Vieira
Different letters represent significant differences at p < 0.05 for the Scott-Knott et al., 2018) have also shown the correlation of these soil variables with
test. AM behavior. Especially, pH, Fe, SOM, and CEC which have been
recorded as key soil properties influencing AM fungal communities.
out is widely recognized, being identified as an important indicator of Overall, this analysis showed that arbuscular mycorrhizal communities
land use changes (Rillig et al., 2003; Wang et al., 2015; Gottshall et al., of the three mining sites responded positive and similarly to rehabili­
2017). Glomalin is an important component of SOM, contributing to the tation, improving their recovery over time.
sequestration and stocking of soil C and N (Lovelock et al., 2004b;
Schindler et al., 2007; Kumar et al., 2018). High positive correlation of 5. Conclusions
both glomalin fractions with SOM and CEC in the current study showed
this important relation, as well as its possible contribution to soil CEC. Our study provides important knowledge regarding communities of
Another relevant aspect of this variable in response to rehabilitation arbuscular mycorrhizal fungi in natural, rehabilitated, and degraded
was shown by the relation between its fractions (EEG/TEG ratio). A low ecosystems of the Eastern Brazilian Amazon, under pressure from the
and similar ratio observed in reference areas and rehabilitated areas iron mining industry. In general, evaluated ferruginous areas presented
with more than four years of management shows a tendency to equi­ a high diversity of AM fungi, as well as a high degree of mycorrhizal
librium of the rehabilitated areas with the predominance of the most association, being highly resilient to drastic modifications of the envi­
stable fraction, even if the absolute values of each one are far from the ronments. The EEG/TEG ratio turned out to be an interesting, easily
reference. A singular case was observed in the non-rehabilitated area of interpretable, and comparable parameter of the AM response, showing
Sandstone. It was dominated by the most stable fraction of glomalin, the degree of equilibrium and/or recovery of new rehabilitated envi­
even though the EEG contents, and other soil attributes were similar to ronments. The rehabilitation processes carried out in this Amazonian
those of other non-rehabilitated areas. This shows that, although the region are succeeding in the recovery of their native mycorrhizal
proposed relation seems to be a good indicator of disturbed and/or re­ communities.
covery status of environments, its analysis is not simplistic. The absence,
or non consideration of the history from the environments, may lead to Funding
erroneous interpretation. The non-rehabilitated area of Sandstone II
received a topsoil layer, which could be related to the results obtained. This work was sponsored by PEC-PG CAPES (grant number

Fig. 3. Two-dimensional ordination of the different areas of the mining sites Sandstone II, Northwest II and South IV using NMDS, considering the mycorrhizal
variables. Correlation between NMDS scores and the chemical and physical properties is also represented.

9
R.M. Rodríguez-Rodríguez et al. Pedobiologia - Journal of Soil Ecology 89 (2021) 150768

23038.005401/2016-08), the Foundation for Research Support of the

Variables: SD = Spore density, MC = Mycorrhizal colonization, EEG = Easily extractable glomalin, TEG = Total extractable glomalin, SOM = Soil organic matter, V = Base saturation, CEC = Potential cation exchange
0.60***
0.65***
State of Minas Gerais (FAPEMIG) and the National Council for Scientific

0.43**

0.47**
0.13
Clay
and Technological Development (CNPq - 313222/2020-7).

Data availability

0.50***
0.56***
0.34*

0.34*
All raw data generated from this study are available. Data inquiries

0.26
Silt should be addressed to raquelm@inca.edu.cu.
−
−
−
−
−
Author contributions

− 0.05
− 0.1
Sand

0.24
0.26

0.13
MACC, JOS, MG and SJR conceived and designed this research. MG,
SJR and CFC performed soil sampling and chemical and physical anal­
ysis. RMRR, KK and DFP performed the biological analysis with MACC,
¡0.65***
− 0.54***

− 0.39**

− 0.41**

FAP and JVS advice. RMRR analyzed data and wrote manuscript,
− 0.22

advised by MACC and JOS. All authors revised and contributed to the
Cu

manuscript.

Declaration of Competing Interest

− 0.01
0.27

0.01
0.09
0.28
Mn

The authors report no declarations of interest.

0.55***
0.49***

Acknowledgments
− 0.03
0.16

0.21
Fe

The authors would like to thank the PEC-PG CAPES (Programa de

Estudantes-Convênio de Pós-Graduação da Coordenação de Aperfei­
0.28
0.14
0.21
0.22
0.19

çoamento de Pessoal de Nível Superior) for the scholarship granted to

Zn

the first author RMRR and CNPq (Conselho Nacional de Desenvolvi­

−
−
−
−
−

mento Científico e Tecnológico) for productivity scholarships for MACC

and JOS. The authors also would like to thank the DCS-UFLA (Depar­
0.73***
0.69***

tamento de Ciências do Solo da Universidade Federal de Lavras) and

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partnership and availability of equipment and resources for conducting
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